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BIOTECHNOLOGY (227 journals)                  1 2 | Last

Showing 1 - 200 of 227 Journals sorted alphabetically
3 Biotech     Open Access   (Followers: 7)
Advances in Bioscience and Biotechnology     Open Access   (Followers: 14)
Advances in Genetic Engineering & Biotechnology     Hybrid Journal   (Followers: 7)
African Journal of Biotechnology     Open Access   (Followers: 6)
Algal Research     Partially Free   (Followers: 9)
American Journal of Biochemistry and Biotechnology     Open Access   (Followers: 68)
American Journal of Bioinformatics Research     Open Access   (Followers: 8)
American Journal of Polymer Science     Open Access   (Followers: 29)
Animal Biotechnology     Hybrid Journal   (Followers: 9)
Annales des Sciences Agronomiques     Full-text available via subscription  
Applied Biochemistry and Biotechnology     Hybrid Journal   (Followers: 42)
Applied Bioenergy     Open Access  
Applied Biosafety     Hybrid Journal  
Applied Microbiology and Biotechnology     Hybrid Journal   (Followers: 62)
Applied Mycology and Biotechnology     Full-text available via subscription   (Followers: 5)
Arthroplasty Today     Open Access   (Followers: 1)
Artificial Cells, Nanomedicine and Biotechnology     Hybrid Journal   (Followers: 2)
Asia Pacific Biotech News     Hybrid Journal   (Followers: 2)
Asian Journal of Biotechnology     Open Access   (Followers: 8)
Asian Pacific Journal of Tropical Biomedicine     Open Access   (Followers: 2)
Australasian Biotechnology     Full-text available via subscription   (Followers: 1)
Banat's Journal of Biotechnology     Open Access  
BBR : Biochemistry and Biotechnology Reports     Open Access   (Followers: 4)
Bio-Algorithms and Med-Systems     Hybrid Journal   (Followers: 1)
Bio-Research     Full-text available via subscription   (Followers: 2)
Bioactive Materials     Open Access   (Followers: 1)
Biocatalysis and Agricultural Biotechnology     Hybrid Journal   (Followers: 4)
Biocybernetics and Biological Engineering     Full-text available via subscription   (Followers: 5)
Bioethics UPdate     Hybrid Journal  
Biofuels     Hybrid Journal   (Followers: 11)
Biofuels Engineering     Open Access   (Followers: 1)
Biological & Pharmaceutical Bulletin     Full-text available via subscription   (Followers: 5)
Biological Cybernetics     Hybrid Journal   (Followers: 10)
Biomarkers and Genomic Medicine     Open Access   (Followers: 5)
Biomarkers in Drug Development     Partially Free   (Followers: 1)
Biomaterials Research     Open Access   (Followers: 4)
BioMed Research International     Open Access   (Followers: 6)
Biomédica     Open Access  
Biomedical Engineering Research     Open Access   (Followers: 7)
Biomedical glasses     Open Access  
Biomedical Reports     Full-text available via subscription  
BioMedicine     Open Access  
Bioprinting     Hybrid Journal  
Bioresource Technology Reports     Hybrid Journal  
Bioscience, Biotechnology, and Biochemistry     Hybrid Journal   (Followers: 22)
Biosimilars     Open Access   (Followers: 1)
Biosurface and Biotribology     Open Access  
Biotechnic and Histochemistry     Hybrid Journal   (Followers: 2)
BioTechniques : The International Journal of Life Science Methods     Full-text available via subscription   (Followers: 28)
Biotechnologia Acta     Open Access   (Followers: 1)
Biotechnologie, Agronomie, Société et Environnement     Open Access   (Followers: 2)
Biotechnology     Open Access   (Followers: 6)
Biotechnology & Biotechnological Equipment     Open Access   (Followers: 5)
Biotechnology Advances     Hybrid Journal   (Followers: 33)
Biotechnology and Applied Biochemistry     Hybrid Journal   (Followers: 44)
Biotechnology and Bioengineering     Hybrid Journal   (Followers: 159)
Biotechnology and Bioprocess Engineering     Hybrid Journal   (Followers: 6)
Biotechnology and Genetic Engineering Reviews     Hybrid Journal   (Followers: 14)
Biotechnology and Health Sciences     Open Access   (Followers: 1)
Biotechnology and Molecular Biology Reviews     Open Access   (Followers: 1)
Biotechnology Annual Review     Full-text available via subscription   (Followers: 7)
Biotechnology for Biofuels     Open Access   (Followers: 10)
Biotechnology Frontier     Open Access   (Followers: 2)
Biotechnology Journal     Hybrid Journal   (Followers: 15)
Biotechnology Law Report     Hybrid Journal   (Followers: 4)
Biotechnology Letters     Hybrid Journal   (Followers: 33)
Biotechnology Progress     Hybrid Journal   (Followers: 39)
Biotechnology Reports     Open Access  
Biotechnology Research International     Open Access   (Followers: 2)
Biotechnology Techniques     Hybrid Journal   (Followers: 10)
Biotecnología Aplicada     Open Access  
Biotribology     Hybrid Journal  
BMC Biotechnology     Open Access   (Followers: 15)
Chinese Journal of Agricultural Biotechnology     Full-text available via subscription   (Followers: 3)
Communications in Mathematical Biology and Neuroscience     Open Access  
Computational and Structural Biotechnology Journal     Open Access   (Followers: 2)
Computer Methods and Programs in Biomedicine     Hybrid Journal   (Followers: 8)
Contributions to Tobacco Research     Open Access   (Followers: 3)
Copernican Letters     Open Access   (Followers: 1)
Critical Reviews in Biotechnology     Hybrid Journal   (Followers: 20)
Crop Breeding and Applied Biotechnology     Open Access   (Followers: 4)
Current Bionanotechnology     Hybrid Journal  
Current Biotechnology     Hybrid Journal   (Followers: 3)
Current Opinion in Biomedical Engineering     Hybrid Journal   (Followers: 1)
Current Opinion in Biotechnology     Hybrid Journal   (Followers: 55)
Current Pharmaceutical Biotechnology     Hybrid Journal   (Followers: 9)
Current Research in Bioinformatics     Open Access   (Followers: 14)
Current trends in Biotechnology and Pharmacy     Open Access   (Followers: 9)
EBioMedicine     Open Access  
Electronic Journal of Biotechnology     Open Access   (Followers: 1)
Entomologia Generalis     Full-text available via subscription  
Environmental Science : Processes & Impacts     Full-text available via subscription   (Followers: 4)
Experimental Biology and Medicine     Hybrid Journal   (Followers: 3)
Folia Medica Indonesiana     Open Access  
Food Bioscience     Hybrid Journal  
Food Biotechnology     Hybrid Journal   (Followers: 12)
Food Science and Biotechnology     Hybrid Journal   (Followers: 9)
Frontiers in Bioengineering and Biotechnology     Open Access   (Followers: 6)
Frontiers in Systems Biology     Open Access   (Followers: 2)
Fungal Biology and Biotechnology     Open Access   (Followers: 1)
GM Crops and Food: Biotechnology in Agriculture and the Food Chain     Full-text available via subscription   (Followers: 1)
GSTF Journal of BioSciences     Open Access  
HAYATI Journal of Biosciences     Open Access  
Horticulture, Environment, and Biotechnology     Hybrid Journal   (Followers: 11)
IEEE Transactions on Molecular, Biological and Multi-Scale Communications     Hybrid Journal   (Followers: 1)
IET Nanobiotechnology     Hybrid Journal   (Followers: 2)
IIOAB Letters     Open Access  
IN VIVO     Full-text available via subscription   (Followers: 4)
Indian Journal of Biotechnology (IJBT)     Open Access   (Followers: 2)
Indonesia Journal of Biomedical Science     Open Access   (Followers: 1)
Indonesian Journal of Biotechnology     Open Access   (Followers: 1)
Industrial Biotechnology     Hybrid Journal   (Followers: 18)
International Biomechanics     Open Access  
International Journal of Bioinformatics Research and Applications     Hybrid Journal   (Followers: 15)
International Journal of Biomechatronics and Biomedical Robotics     Hybrid Journal   (Followers: 4)
International Journal of Biomedical Research     Open Access   (Followers: 2)
International Journal of Biotechnology     Hybrid Journal   (Followers: 5)
International Journal of Biotechnology and Molecular Biology Research     Open Access   (Followers: 2)
International Journal of Biotechnology for Wellness Industries     Partially Free   (Followers: 1)
International Journal of Environment, Agriculture and Biotechnology     Open Access   (Followers: 5)
International Journal of Functional Informatics and Personalised Medicine     Hybrid Journal   (Followers: 4)
International Journal of Medicine and Biomedical Research     Open Access   (Followers: 1)
International Journal of Nanotechnology and Molecular Computation     Full-text available via subscription   (Followers: 3)
International Journal of Radiation Biology     Hybrid Journal   (Followers: 4)
Iranian Journal of Biotechnology     Open Access  
ISABB Journal of Biotechnology and Bioinformatics     Open Access  
Italian Journal of Food Science     Open Access   (Followers: 1)
Journal of Biometrics & Biostatistics     Open Access   (Followers: 3)
Journal of Bioterrorism & Biodefense     Open Access   (Followers: 6)
Journal of Petroleum & Environmental Biotechnology     Open Access   (Followers: 2)
Journal of Advanced Therapies and Medical Innovation Sciences     Open Access  
Journal of Advances in Biotechnology     Open Access   (Followers: 5)
Journal Of Agrobiotechnology     Open Access  
Journal of Analytical & Bioanalytical Techniques     Open Access   (Followers: 7)
Journal of Animal Science and Biotechnology     Open Access   (Followers: 6)
Journal of Applied Biomedicine     Open Access   (Followers: 3)
Journal of Applied Biotechnology     Open Access   (Followers: 2)
Journal of Applied Biotechnology Reports     Open Access   (Followers: 2)
Journal of Applied Mathematics & Bioinformatics     Open Access   (Followers: 5)
Journal of Biologically Active Products from Nature     Hybrid Journal   (Followers: 1)
Journal of Biomaterials and Nanobiotechnology     Open Access   (Followers: 6)
Journal of Biomedical Photonics & Engineering     Open Access  
Journal of Biomedical Practitioners     Open Access  
Journal of Bioprocess Engineering and Biorefinery     Full-text available via subscription  
Journal of Bioprocessing & Biotechniques     Open Access  
Journal of Biosecurity, Biosafety and Biodefense Law     Hybrid Journal   (Followers: 3)
Journal of Biotechnology     Hybrid Journal   (Followers: 68)
Journal of Chemical and Biological Interfaces     Full-text available via subscription   (Followers: 1)
Journal of Chemical Technology & Biotechnology     Hybrid Journal   (Followers: 10)
Journal of Chitin and Chitosan Science     Full-text available via subscription  
Journal of Colloid Science and Biotechnology     Full-text available via subscription  
Journal of Commercial Biotechnology     Full-text available via subscription   (Followers: 6)
Journal of Crop Science and Biotechnology     Hybrid Journal   (Followers: 7)
Journal of Essential Oil Research     Hybrid Journal   (Followers: 3)
Journal of Experimental Biology     Full-text available via subscription   (Followers: 25)
Journal of Genetic Engineering and Biotechnology     Open Access   (Followers: 5)
Journal of Ginseng Research     Open Access  
Journal of Industrial Microbiology and Biotechnology     Hybrid Journal   (Followers: 16)
Journal of Integrative Bioinformatics     Open Access  
Journal of International Biotechnology Law     Hybrid Journal   (Followers: 3)
Journal of Medical Imaging and Health Informatics     Full-text available via subscription  
Journal of Molecular Microbiology and Biotechnology     Full-text available via subscription   (Followers: 14)
Journal of Nano Education     Full-text available via subscription  
Journal of Nanobiotechnology     Open Access   (Followers: 4)
Journal of Nanofluids     Full-text available via subscription   (Followers: 2)
Journal of Organic and Biomolecular Simulations     Open Access  
Journal of Plant Biochemistry and Biotechnology     Hybrid Journal   (Followers: 6)
Journal of Science and Applications : Biomedicine     Open Access  
Journal of the Mechanical Behavior of Biomedical Materials     Hybrid Journal   (Followers: 11)
Journal of Trace Elements in Medicine and Biology     Hybrid Journal   (Followers: 1)
Journal of Tropical Microbiology and Biotechnology     Full-text available via subscription  
Journal of Yeast and Fungal Research     Open Access   (Followers: 1)
Marine Biotechnology     Hybrid Journal   (Followers: 5)
Messenger     Full-text available via subscription  
Metabolic Engineering Communications     Open Access   (Followers: 4)
Metalloproteinases In Medicine     Open Access  
Microalgae Biotechnology     Open Access   (Followers: 2)
Microbial Biotechnology     Open Access   (Followers: 9)
MicroMedicine     Open Access   (Followers: 3)
Molecular and Cellular Biomedical Sciences     Open Access  
Molecular Biotechnology     Hybrid Journal   (Followers: 16)
Molecular Genetics and Metabolism Reports     Open Access   (Followers: 3)
Nanobiomedicine     Open Access  
Nanobiotechnology     Hybrid Journal   (Followers: 3)
Nanomaterials and Nanotechnology     Open Access  
Nanomaterials and Tissue Regeneration     Open Access  
Nanomedicine and Nanobiology     Full-text available via subscription  
Nanomedicine Research Journal     Open Access  
Nanotechnology Reviews     Hybrid Journal   (Followers: 5)
Nature Biotechnology     Full-text available via subscription   (Followers: 520)
Network Modeling and Analysis in Health Informatics and Bioinformatics     Hybrid Journal   (Followers: 3)
New Biotechnology     Hybrid Journal   (Followers: 4)
Nigerian Journal of Biotechnology     Open Access  
Nova Biotechnologica et Chimica     Open Access  
NPG Asia Materials     Open Access  
npj Biofilms and Microbiomes     Open Access  
OA Biotechnology     Open Access  
Plant Biotechnology Journal     Open Access   (Followers: 10)
Plant Biotechnology Reports     Hybrid Journal   (Followers: 4)
Preparative Biochemistry and Biotechnology     Hybrid Journal   (Followers: 4)

        1 2 | Last

Journal Cover Plant Biotechnology Journal
  [SJR: 2.591]   [H-I: 77]   [10 followers]  Follow
  This is an Open Access Journal Open Access journal
   ISSN (Print) 1467-7644 - ISSN (Online) 1467-7652
   Published by John Wiley and Sons Homepage  [1597 journals]
  • Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers
           high-efficiency multiplex genome editing in kiwifruit

    • Authors: Zupeng Wang; Shuaibin Wang, Dawei Li, Qiong Zhang, Li Li, Caihong Zhong, Yifei Liu, Hongwen Huang
      Abstract: Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In the present study, we developed a new cloning strategy for generating paired-sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired-sgRNA cloning, our strategy only requires the synthesis of two gRNA-containing primers which largely reduces the cost. We further compared efficiencies of paired-sgRNA/Cas9 vectors containing different sgRNA-expression devices, including both the polycistronic tRNA-sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system were 10-fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418-resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-13T09:30:31.828758-05:
      DOI: 10.1111/pbi.12884
  • Verification of DNA Motifs in Arabidopsis using CRISPR/Cas9 Mediated

    • Authors: Chenlong Li; Chen Chen, Huhui Chen, Suikang Wang, Xuemei Chen, Yuhai Cui
      Abstract: Transcription factors (TFs) and chromatin-modifying factors (CMFs) access chromatin by recognizing specific DNA motifs in their target genes. Chromatin Immunoprecipitation followed by next generation sequencing (ChIP-seq) has been widely used to discover the potential DNA binding motifs for both TFs and CMFs. Yet, an in vivo method for verifying DNA motifs captured by ChIP-seq is lacking in plants. Here, we describe the use of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to verify DNA motifs in their native genomic context in Arabidopsis. Using a single guide RNA (sgRNA) targeting the DNA motif bound by REF6, a DNA-sequence-specific H3K27 demethylase in plants, we generated stable transgenic plants where the motif was disrupted in a REF6 target gene. We also deleted a cluster of multiple motifs from another REF6 target gene using a pair of sgRNAs, targeting upstream and downstream regions of the cluster, respectively. We demonstrated that endogenous genes with motifs disrupted and/or deleted become inaccessible to REF6. This strategy should be widely applicable for in vivo verification of DNA motifs identified by ChIP-seq in plants.This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-13T09:30:26.71186-05:0
      DOI: 10.1111/pbi.12886
  • Modification of chrysanthemum odour and taste with chrysanthemol synthase
           induces strong dual resistance against cotton aphids

    • Authors: Hao Hu; Jinjin Li, Thierry Delatte, Jacques Vervoort, Liping Gao, Francel Verstappen, Wei Xiong, Jianping Gan, Maarten A. Jongsma, Caiyun Wang
      Abstract: Aphids are pests of chrysanthemum that employ plant volatiles to select host plants and ingest cell contents to probe host quality before engaging in prolonged feeding and reproduction. Changes in volatile and non-volatile metabolite profiles can disrupt aphid-plant interactions and provide new methods of pest control. Chrysanthemol synthase (CHS) from Tanacetum cinerariifolium, represents the first committed step in the biosynthesis of pyrethrin ester insecticides, but no biological role for the chrysanthemol product alone has yet been documented. In this study, the TcCHS gene was over-expressed in Chrysanthemum morifolium, and resulted in both the emission of volatile chrysanthemol (ca. 47 pmol/h/gFW) and accumulation of a chrysanthemol glycoside derivative, identified by NMR as chrysanthemyl-6-O-malonyl-β-D-glucopyranoside (ca. 1.1 mM), with no detrimental phenotypic effects. Dual-choice assays separately assaying these compounds in pure form and as part of the headspace and extract demonstrated independent bioactivity of both components against the cotton aphid (Aphis gossypii). Performance assays showed that the TcCHS plants significantly reduced aphid reproduction, consistent with disturbance of aphid probing activities on these plants as revealed by electropenetrogram (EPG) studies. In open-field trials, aphid population development was very strongly impaired demonstrating the robustness and high impact of the trait. The results suggest that expression of the TcCHS gene induces a dual defense system, with both repellence by chrysanthemol odour and deterrence by its non-volatile glycoside, introducing a promising new option for engineering aphid control into plants.This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-13T09:30:22.655537-05:
      DOI: 10.1111/pbi.12885
  • Biogenesis and regulatory hierarchy of phased small interfering RNAs in

    • Authors: Pingchuan Deng; Sajid Muhammad, Min Cao, Liang Wu
      Abstract: Several varieties of small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs) are generated in plants to regulate development, genome stability, and response to adverse environments. Phased siRNA (phasiRNA) is a type of secondary siRNA that is processed from a miRNA-mediated cleavage of RNA transcripts, increasing silencing efficiency or simultaneously suppressing multiple target genes. Trans-acting siRNAs (ta-siRNAs) are a particular class of phasiRNA produced from non-coding transcripts that silence targets in trans. It was originally thought that “one hit” and “two hit” models were essential for processing distinct TAS precursors; however, a single hit event was recently shown to be sufficient at triggering all types of ta-siRNAs. This review discusses the findings about biogenesis, targeting modes, and regulatory networks of plant ta-siRNAs. We also summarize recent advances in the generation of other phasiRNAs and their possible biological benefits to plants.This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-11T20:40:48.946329-05:
      DOI: 10.1111/pbi.12882
  • Establishing RNA virus resistance in plants by harnessing CRISPR immune

    • Authors: Tong Zhang; Qiufeng Zheng, Xin Yi, Hong An, Yaling Zhao, Siqi Ma, Guohui Zhou
      Abstract: Recently, CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR-Cas system to target the viral genome directly. Here we reprogrammed the CRISPR-Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus-targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveals that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-11T20:30:34.699724-05:
      DOI: 10.1111/pbi.12881
  • ZmSTK1 and ZmSTK2, Encoding Receptor-like Cytoplasmic Kinase, are Involved
           in Maize Pollen Development with Additive Effect

    • Authors: Mingxia Fan; Chunyu Zhang, Lei Shi, Chen Liu, Wenjuan Ma, Meiming Chen, Kuichen Liu, Fengchun Cai, Guohong Wang, Zhengyi Wei, Min Jiang, Zaochang Liu, Ansar Javeed, Feng Lin
      Abstract: Pollen germination and pollen tube growth are important physiological processes of sexual reproduction of plants, and also are involved in signal transduction. Our previous study reveals that ZmSTK1 and ZmSTK2 are two receptor-like cytoplasmic kinases (RLCK) homologs in Zea mays as members of receptor-like protein kinase (RLK) subfamily, sharing 86% identity at the amino acid level. Here, we report that ZmSTK1 and ZmSTK2, expressed at late stages of pollen development, regulate maize pollen development with additive effect. ZmSTK1 or ZmSTK2 mutation exhibited severe pollen transmission deficiency, which thus influenced pollen fertility. Moreover, the kinase domains of ZmSTKs were cross-interacted with C-terminus of enolases detected by Co-Immunoprecipitation (Co-IP) and yeast two-hybrid system (Y2H), respectively. Further, the detective ZmSTK1 or ZmSTK2 was associated with decreased activity of enolases, and also reduced downstream metabolite contents, which enolases are involved in glycolytic pathway, such as phosphoenolpyruvate (PEP), pyruvate, ADP/ATP, starch, glucose, sucrose, and fructose. This study reveals that ZmSTK1 and ZmSTK2 regulate maize pollen development and indirectly participate in glycolytic pathway.This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-11T20:30:27.778705-05:
      DOI: 10.1111/pbi.12880
  • A Maize phytochrome-interacting factors Protein ZmPIF1 Enhances Drought
           Tolerance by Inducing Stomatal Closure and Improves Grain Yield in Oryza

    • Authors: Yong Gao; Meiqin Wu, Mengjiao Zhang, Wei Jiang, Xiaoyun Ren, Enxing Liang, Dongping Zhang, Changquan Zhang, Ning Xiao, Yan Li, Yi Dai, Jianmin Chen
      Abstract: Phytochrome-interacting factors (PIFs) play major roles in regulating plant growth and development, but their roles in drought stress remain elusive. Here, we cloned and characterized a maize (Zea mays) PIF transcription factor, ZmPIF1. The expression level of ZmPIF1 was significantly induced by independent drought and abscisic acid (ABA) treatments. The ZmPIF1 transgenic rice and Arabidopsis displayed water-saving and drought-resistance, which were associated with reduced a stomatal aperture and transpiration rate. Moreover, the ZmPIF1 transgenic rice were hypersensitive to exogenous ABA, while the endogenous ABA level was not significantly changed, suggesting that ZmPIF1 was a positive regulator of the ABA signaling pathway. Digital gene expression (DGE) results further indicated that ZmPIF1 participated in ABA signaling pathway and regulated the stomatal aperture in rice. In addition, grain yield and agronomic traits analysis over four years showed that ZmPIF1 was able to increase the grain yield through an increase in tiller and panicle numbers in transgenic rice. Overall, ZmPIF1 plays an important role in the ABA-mediated regulation of stomatal closure to control water loss. ZmPIF1 can enhance water-saving and drought-resistance and improve the crop yield in rice, illustrating the capacity of ZmPIF1 for crop improvement.This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-11T20:20:31.197703-05:
      DOI: 10.1111/pbi.12878
  • Developing a flexible, High-efficiency Agrobacterium-mediated Sorghum
           Transformation System with Broad Application

    • Authors: Ping Che; Ajith Anand, Emily Wu, Jeffry D. Sander, Marissa K. Simon, Weiwei Zhu, Amy L. Sigmund, Gina Zastrow-Hayes, Michael Miller, Donglong Liu, Shai J. Lawit, Zuo-Yu Zhao, Marc C. Albertsen, Todd J. Jones
      Abstract: Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here we report a ternary vector (also known as co-habitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum. We report regeneration frequencies ranging from 6-29% in Tx430 using different selectable markers and single copy, backbone free “quality events” ranging from 45-66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84-7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9 mediated gene knockouts in Tx430.This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-11T20:20:24.772799-05:
      DOI: 10.1111/pbi.12879
  • A guide to the contained use of plant virus infectious clones

    • Authors: H C Brewer; D L Hird, A M Bailey, S E Seal, G D Foster
      Abstract: Plant virus infectious clones are important tools with wide ranging applications in different areas of biology and medicine. Their uses in plant pathology include the study of plant‐virus interactions, and screening of germplasm as part of pre‐breeding programmes for virus resistance. They can also be modified to induce transient plant gene silencing (Virus Induced Gene Silencing ‐ VIGS) and as expression vectors for plant or exogenous proteins, with applications in both plant pathology and more generally for the study of plant gene function. Plant viruses are also increasingly being investigated as expression vectors for in planta production of pharmaceutical products, known as molecular farming. However, plant virus infectious clones may pose a risk to the environment due to their ability to reconstitute fully functional, transmissible viruses. These risks arise both from their inherent pathogenicity and the effect of any introduced genetic modifications. Effective containment measures are therefore required. There has been no single comprehensive review of the biosafety considerations for the contained use of genetically modified plant viruses, despite their increasing importance across many biological fields. This review therefore explores the biosafety considerations for working with genetically modified plant viruses in contained environments, with focus on plant growth facilities. It includes regulatory frameworks, risk assessment, assignment of biosafety levels, facility features and working practices. The review is based on international guidance together with information provided by plant virus researchers.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-22T02:52:31.42042-05:0
      DOI: 10.1111/pbi.12876
  • The pomegranate (Punica granatum L.) genome provides insights into fruit
           quality and ovule developmental biology

    • Authors: Zhaohe Yuan; Yanming Fang, Taikui Zhang, Zhangjun Fei, Fengming Han, Cuiyu Liu, Min Liu, Wei Xiao, Wenjing Zhang, Shan Wu, Mengwei Zhang, Youhui Ju, Huili Xu, He Dai, Yujun Liu, Yanhui Chen, Lili Wang, Jianqing Zhou, Dian Guan, Ming Yan, Yanhua Xia, Xianbin Huang, Dongyuan Liu, Hongmin Wei, Hongkun Zheng
      Abstract: Pomegranate (Punica granatum L.) has an ancient cultivation history, and has become an emerging profitable fruit crop due to its attractive features such as the bright red appearance and the high abundance of medicinally valuable ellagitannin‐based compounds in its peel and aril. However, the limited genomic resources have restricted further elucidation of genetics and evolution of these interesting traits. Here we report a 274‐Mb high‐quality draft pomegranate genome sequence, which covers approximately 81.5% of the estimated 336‐Mb genome, consists of 2,177 scaffolds with an N50 size of 1.7 Mb, and contains 30,903 genes. Phylogenomic analysis supported that pomegranate belongs to the Lythraceae family rather than the monogeneric Punicaceae family, and comparative analyses showed that pomegranate and Eucalyptus grandis shares the paleotetraploidy event. Integrated genomic and transcriptomic analyses provided insights into the molecular mechanisms underlying the biosynthesis of ellagitannin‐based compounds, the color formation in both peels and arils during pomegranate fruit development, and the unique ovule development processes that are characteristic of pomegranate. This genome sequence provides an important resource to expand our understanding of some unique biological processes and to facilitate both comparative biology studies and crop breeding.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-22T02:47:34.675368-05:
      DOI: 10.1111/pbi.12875
  • The Ralstonia solanacearum csp22 peptide, but not flagellin‐derived
           peptides, is perceived by plants from the Solanaceae family

    • Authors: Yali Wei; Carlos Caceres-Moreno, Tamara Jimenez-Gongora, Keke Wang, Yuying Sang, Rosa Lozano-Duran, Alberto P. Macho
      Abstract: Ralstonia solanacearum, the causal agent of bacterial wilt disease, is considered one of the most destructive bacterial pathogens due to its lethality, unusually wide host range, persistence, and broad geographical distribution. In spite of the extensive research on plant immunity over the last years, the perception of molecular patterns from R. solanacearum that activate immunity in plants is still poorly understood, which hinders the development of strategies to generate resistance against bacterial wilt disease. The perception of a conserved peptide of bacterial flagellin, flg22, is regarded as paradigm of plant perception of invading bacteria; however, no elicitor activity has been detected for R. solanacearum flg22. Recent reports have shown that other epitopes from flagellin are able to elicit immune responses in specific species from the Solanaceae family, yet our results show that these plants do not perceive any epitope from R. solanacearum flagellin. Searching for elicitor peptides from R. solanacearum, we found several protein sequences similar to the consensus of the elicitor peptide csp22, reported to elicit immunity in specific Solanaceae plants. A R. solanacearum csp22 peptide (csp22Rsol) was indeed able to trigger immune responses in N. benthamiana and tomato, but not in Arabidopsis thaliana. Additionally, csp22Rsol treatment conferred increased resistance to R. solanacearum in tomato. Transgenic A. thaliana plants expressing the tomato csp22 receptor (SlCORE) gained the ability to respond to csp22Rsol and became more resistant to R. solanacearum infection. Our results shed light on the mechanisms for perception of R. solanacearum by plants, paving the way for improving current approaches to generate resistance against R. solanacearum.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-19T02:05:39.984387-05:
      DOI: 10.1111/pbi.12874
  • Dissection of the genetic architecture of three seed‐quality traits and
           consequences for breeding in Brassica napus

    • Authors: Bo Wang; Zhikun Wu, Zhaohong Li, Qinghua Zhang, Jianlin Hu, Yingjie Xiao, Dongfang Cai, Jiangsheng Wu, Graham J King, Haitao Li, Kede Liu
      Abstract: Genome‐wide association studies (GWASs) combining high‐throughput genome resequencing and phenotyping can accelerate the dissection of genetic architecture and identification of genes for plant complex traits. In this study, we developed a rapeseed genomic variation map consisting of 4,542,011 SNPs and 628,666 INDELs. GWAS was performed for three seed‐quality traits, including erucic acid content (EAC), glucosinolate content (GSC) and seed oil content (SOC) using 3.82 million polymorphisms in an association panel. Six, 49 and 17 loci were detected to be associated with EAC, GSC and SOC in multiple environments, respectively. The mean total contribution of these loci in each environment was 94.1% for EAC and 87.9% for GSC, notably higher than that for SOC (40.1%). A high correlation was observed between phenotypic variance and number of favorable alleles for associated loci, which will contribute to breeding improvement by pyramiding these loci. Furthermore, candidate genes were detected underlying associated loci, based on functional polymorphisms in gene regions where sequence variation was found to correlate with phenotypic variation. Our approach was validated by detection of well characterized FAE1 genes at each of two major loci for EAC on chromosomes A8 and C3, along with MYB28 genes at each of three major loci for GSC on chromosomes A9, C2 and C9. Four novel candidate genes were detected by correlation between GSC and SOC and observed sequence variation, respectively. The present study provides insights into the genetic architecture of three seed‐quality traits, which would be useful for genetic improvement of B. napus.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-19T00:40:45.305532-05:
      DOI: 10.1111/pbi.12873
  • Precise editing of CLAVATA genes in Brassica napus L. regulates
           multilocular silique development

    • Authors: Yang Yang; Kaiyu Zhu, Huailin Li, Shaoqing Han, Qingwei Meng, Shahid Ullah Khan, Chuchuan Fan, Kabin Xie, Yongming Zhou
      Abstract: Multilocular silique is a desirable agricultural trait with great potential for the development of high‐yield varieties of Brassica. To date, no spontaneous or induced multilocular mutants have been reported in Brassica napus, which likely reflects its allotetraploid nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed homologues of CLAVATA3 (CLV3) for a secreted peptide and its related receptors CLV1 and CLV2 in the CLV signalling pathway using the CRISPR/Cas9 system and achieved stable transmission of the mutations across three generations. Each BnCLV gene has two copies located in two sub‐genomes. The multilocular phenotype can be recovered only in knockout mutations of both copies of each BnCLV gene, illustrating that the simultaneous alteration of multiple gene copies by CRISPR/Cas9 mutagenesis has great potential in generating agronomically important mutations in rapeseed. The mutagenesis efficiency varied widely from 0% to 48.65% in T0 with different single‐guide RNAs (sgRNAs), indicating that the appropriate selection of the sgRNA is important for effectively generating indels in rapeseed. The double mutation of BnCLV3 produced more leaves and multilocular siliques with a significantly higher number of seeds per silique and a higher seed weight than the wild‐type and single mutant plants, potentially contributing to increased seed production. We also assessed the efficiency of the horizontal transfer of Cas9/gRNA cassettes by pollination. Our findings reveal the potential for plant breeding strategies to improve yield traits in currently cultivated rapeseed varieties.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-18T00:30:48.290355-05:
      DOI: 10.1111/pbi.12872
  • Chrysanthemum CmHSFA4 gene positively regulates salt stress tolerance in
           transgenic chrysanthemum

    • Authors: Li Fei; Huanru Zhang, Husheng Zhao, Tianwei Gao, Aiping Song, Jiafu Jiang, Fadi Chen, Sumei Chen
      Abstract: Salinity‐induced Na+ toxicity and oxidative stress hamper plant growth. Here, we showed that expression of the chrysanthemum CmHSFA4, a homologue of the heat shock factor AtHSFA4a, is inducible by salt, and localizes to the nucleus. It is a transcription activator binding with HSE. Chrysanthemum overexpressing CmHSFA4 displayed enhanced salinity tolerance by limiting Na+ accumulation while maintaining K+ concentration, which is consistent with the up‐regulation of ion‐transporters CmSOS1 and CmHKT2. Additionally, the transgenic plants reduced H2O2 and O2∙‐ accumulation under salinity, which could be due to up‐regulation of ROS‐scavenger activities such as SOD, APX and CAT as well as CmHSP70, CmHSP90. Together, these results suggest that CmHSFA4 conferred salinity tolerance in chrysanthemum as a consequence of Na+/K+ ion and ROS homeostasis.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-12T02:50:08.5263-05:00
      DOI: 10.1111/pbi.12871
  • Application of protoplast technology to CRISPR/Cas9 mutagenesis: From
           single cell mutation detection to mutant plant regeneration

    • Authors: Choun-Sea Lin; Chen-Tran Hsu, Ling-Hung Yang, Lan-Ying Lee, Jin-Yuan Fu, Qiao-Wei Cheng, Fu-Hui Wu, Han Chen-Wei Hsiao, Yesheng Zhang, Ru Zhang, Wan-Jung Chang, Chen-Ting Yu, Wen Wang, Li-Jen Liao, Stanton B. Gelvin, Ming-Che Shih
      Abstract: Plant protoplasts are useful for assessing the efficiency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-12T00:35:52.914854-05:
      DOI: 10.1111/pbi.12870
  • Plant‐expressed Fc‐fusion protein tetravalent dengue vaccine with
           inherent adjuvant properties

    • Authors: Mi Young Kim; Alastair Copland, Kaustuv Nayak, Anmol Chandele, Muhammad Shamsher Ahmed, Qibo Zhang, Gil Reynolds Diogo, Matthew John Paul, Sven Hofmann, Moon-Sik Yang, Yong-Suk Jang, Julian K-C. Ma, Rajko Reljic
      Abstract: Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi‐Pasteur Denvaxia vaccine does not protect children under the age of nine and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant‐expression to produce a humanised and highly immunogenic Poly‐Immunoglobulin G Scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralising antibodies and evidence of cell‐mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen‐specific CD4+ and CD8+ T cell proliferation, IFN‐γ and antibody production. The purified polymeric fraction of dengue PIGS (D‐PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low affinity Fcγ receptors on antigen‐presenting cells. These results show that the plant‐expressed D‐PIGS have the potential for translation towards a safe and easily scalable single antigen based tetravalent dengue vaccine.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-09T19:40:27.503826-05:
      DOI: 10.1111/pbi.12869
  • Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava

    • Authors: Aaron W. Hummel; Raj Deepika Chauhan, Tomas Cermak, Andrew M. Mutka, Anupama Vijayaraghavan, Adam Boyher, Colby G. Starker, Rebecca Bart, Daniel F. Voytas, Nigel J. Taylor
      Abstract: Effective weed control can protect yields of cassava (Manihot esculenta) storage roots (Doll and Piedrahita Cañola, 1978). Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and non‐homologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-09T19:35:26.759373-05:
      DOI: 10.1111/pbi.12868
  • Genetic‐based dissection of arsenic accumulation in maize using a
           genome‐wide association analysis method

    • Authors: Zhan Zhao; Huaisheng Zhang, Zhongjun Fu, Hao Chen, Yanan Lin, Pengshuai Yan, Weihua Li, Huiling Xie, Zhanyong Guo, Xuehai Zhang, Jihua Tang
      Abstract: Understanding the mechanism of arsenic (As) accumulation in plants is important in reducing As's toxicity to plants and its potential risks to human health. Here, we performed a genome‐wide association study to dissect the genetic basis of the As contents of different maize tissues in Xixian, which was irrigated with As‐rich surface water, and Changge using an association population consisting of 230 representative inbred maize lines. Phenotypic data revealed a wide normal distribution and high repeatability for the As contents in maize tissues. The As concentrations in maize tissues followed the same trend in the two locations: kernels < axes < stems < bracts < leaves. In total, 15, 16 and 15 non‐redundant quantitative trait loci (QTLs) associated with As concentrations were identified (p < 2.04 × 10−6) in five tissues from Xixian, Changge and the combination of the locations, respectively, explaining 9.70% to 24.65% of the phenotypic variation for each QTL, on average. Additionally, four QTLs [involving 15 single nucleotide polymorphisms (SNPs)], were detected in the single and the combined locations, indicating that these loci/SNPs might be stable across different environments. The candidate genes associated with these four loci were predicted. In addition, four non‐redundant QTLs (6 SNPs), including a QTL that was detected in multiple locations according to the genome‐wide association study, were found to co‐localize with four previously reported QTL intervals. These results are valuable to understand the genetic architecture of As mechanism in maize and facilitate the genetic improvement of varieties without As toxicity.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-04T23:00:34.238051-05:
      DOI: 10.1111/pbi.12853
  • De novo assembly of the zucchini genome reveals a whole genome duplication
           associated with the origin of the Cucurbita genus

    • Authors: Javier Montero-Pau; José Blanca, Aureliano Bombarely, Peio Ziarsolo, Cristina Esteras, Carlos Martí-Gómez, María Ferriol, Pedro Gómez, Manuel Jamilena, Lukas Mueller, Belén Picó, Joaquín Cañizares
      Abstract: The Cucurbita genus (squashes, pumpkins and gourds) includes important domesticated species such as C. pepo, C. maxima and C. moschata. In this study, we present a high‐quality draft of the zucchini (C. pepo) genome. The assembly has a size of 263 Mb, a scaffold N50 of 1.8 Mb, and 34,240 gene models. It includes 92% of the conserved BUSCO core gene set, and it is estimated to cover 93.0% of the genome. The genome is organized in 20 pseudomolecules that represent 81.4% of the assembly, and it is integrated with a genetic map of 7,718 SNPs. Despite the small genome size, three independent lines of evidence support that the C. pepo genome is the result of a Whole Genome Duplication: the topology of the gene family phylogenies, the karyotype organization, and the distribution of 4DTv distances. Additionally, 40 transcriptomes of 12 species of the genus were assembled and analyzed together with all the other published genomes of the Cucurbitaceae family. The duplication was detected in all the Cucurbita species analyzed, including C. maxima and C. moschata, but not in the more distant cucurbits belonging to the Cucumis and Citrullus genera, and it is likely to have occurred 30 ± 4 Mya in the ancestral species that gave rise to the genus.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-04T21:36:08.292824-05:
      DOI: 10.1111/pbi.12860
  • Homoeologous exchange is a major cause of gene presence/absence variation
           in the amphidiploid Brassica napus

    • Authors: Bhavna Hurgobin; Agnieszka A. Golicz, Philipp E. Bayer, Chon-Kit Kenneth Chan, Soodeh Tirnaz, Aria Dolatabadian, Sarah V. Schiessl, Birgit Samans, Juan D. Montenegro, Isobel A. P. Parkin, J. Chris Pires, Boulos Chalhoub, Graham J. King, Rod Snowdon, Jacqueline Batley, David Edwards
      Abstract: Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In the present study we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterisation of the association between HEs and PAVs in B. napus at the pangenome level.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-04T09:24:52.585692-05:
      DOI: 10.1111/pbi.12867
  • CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in
           small RNA processing of Glycine max and Medicago truncatula

    • Authors: Shaun J. Curtin; Yer Xiong, Jean-Michel Michno, Benjamin W. Campbell, Adrian O. Stec, Tomas Čermák, Colby Starker, Daniel F. Voytas, Andrew L. Eamens, Robert M. Stupar
      Abstract: Processing of double‐stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soybean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL‐effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi‐allelic double‐mutant for the two soybean paralogous Double‐stranded RNA‐binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9‐generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ‐line transmissible. Furthermore, TALENs were used to generate a mutation within the soybean Dicer‐like2 gene. CRISPR/Cas9 mutagenesis of the soybean Dicer‐like3 gene and the GmHen1a gene were observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole genome sequencing to reveal a spectrum of non‐germline targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-04T04:26:48.097036-05:
      DOI: 10.1111/pbi.12857
  • Overexpression of Serine Acetyltransferase in Maize Leaves Increases
           Seed‐Specific Methionine‐Rich Zeins

    • Authors: Xiaoli Xiang; Yongrui Wu, José Planta, Joachim Messing, Thomas Leustek
      Abstract: Maize kernels do not contain enough of the essential sulfur‐amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulfur (S)‐amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S‐assimilation leading to Cys and Met biosynthesis and SAT overexpression is known to enhance S‐assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle‐sheath‐cell‐specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12‐fold higher SAT activity without negative impact on growth. S‐assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10‐kDa δ‐zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40 fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high‐Met zein accumulation. Moreover, SAT overcomes the shortage of S‐amino acids that limits the expression and accumulation of high Met‐zeins during kernel development.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-29T05:41:02.915631-05:
      DOI: 10.1111/pbi.12851
  • Provitamin A Biofortification of Cassava Enhances Shelf‐Life but Reduces
           Dry Matter Content of Storage Roots Due to Altered Carbon Partitioning
           into Starch

    • Authors: Getu Beyene; Felix R. Solomon, Raj Deepika Chauhan, Eliana Gaitán-Solis, Narayanan Narayanan, Jackson Gehan, Dimuth Siritunga, Robyn L. Stevens, John Jifon, Joyce Van Eck, Edward Linsler, Malia Gehan, Muhammad Ilyas, Martin Fregene, Richard T. Sayre, Paul Anderson, Nigel J. Taylor, Edgar B. Cahoon
      Abstract: Storage roots of cassava, a major subsistence crop of sub‐Saharan Africa, are calorie‐rich but deficient in essential micronutrients, including provitamin A β‐carotene. In this study, β‐carotene concentrations in cassava storage roots were enhanced by co‐expression of transgenes for deoxy‐D‐xylulose‐5‐phosphate synthase (DXS) and bacterial phytoene synthase (crtB), mediated by the patatin type‐1 promoter. Storage roots harvested from field‐grown plants accumulated carotenoids to ≤50 μg/g DW, 15‐ to 20‐fold increases relative to roots from non‐transgenic plants. Approximately 85‐90% of these carotenoids accumulated as all‐trans‐β‐carotene, the most nutritionally efficacious carotenoid. β‐Carotene‐accumulating storage roots displayed delayed onset of post‐harvest physiological deterioration, a major constraint limiting utilization of cassava products. Large metabolite changes were detected in β‐carotene enhanced storage roots. Most significantly, an inverse correlation was observed between β‐carotene and dry matter content, with reductions of 50% to 60% of dry matter content in the highest carotenoid accumulating storage roots of different cultivars. Further analysis confirmed concomitant reduction in starch content, and increased levels of total fatty acids, triacylglycerols, soluble sugars, and abscisic acid. Potato engineered to co‐express DXS and crtB displayed a similar correlation between β‐carotene accumulation, reduced dry matter and starch content, and elevated oil and soluble sugars in tubers. Transcriptome analyses revealed reduced expression of genes involved in starch biosynthesis including ADP‐glucose pyrophosphorylase genes in transgenic, carotene‐accumulating cassava roots relative to non‐transgenic roots. These findings highlight unintended metabolic consequences of provitamin A biofortification of starch‐rich organs and point to strategies for redirecting metabolic flux to restore starch production.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-28T04:43:09.292743-05:
      DOI: 10.1111/pbi.12862
  • The C2H2 zinc finger‐protein SlZF3 regulates AsA synthesis and salt
           tolerance by interacting with CSN5B

    • Authors: Ying Li; Zhuannan Chu, Jinying Luo, Yuhong Zhou, Yujing Cai, Yongen Lu, Junhui Xia, Hanhui Kuang, Zhibiao Ye, Bo Ouyang
      Abstract: Abiotic stresses are a major cause of crop loss. Ascorbic acid (AsA) promotes stress tolerance by scavenging reactive oxygen species (ROS), which accumulate when plants experience abiotic stress. Although the biosynthesis and metabolism of AsA are well established, the genes that regulate these pathways remain largely unexplored. Here, we report on a novel regulatory gene from tomato (Solanum lycopersicum) named SlZF3 that encodes a Cys2/His2‐type zinc‐finger protein with an EAR repression domain. The expression of SlZF3 was rapidly induced by NaCl treatments. The overexpression of SlZF3 significantly increased the levels of AsA in tomato and Arabidopsis. Consequently, the AsA‐mediated ROS scavenging capacity of the SlZF3‐overexpressing plants was increased, which enhanced the salt tolerance of these plants. Protein‐protein interaction assays demonstrated that SlZF3 directly binds CSN5B, a key component of the COP9 signalosome. This interaction inhibited the binding of CSN5B to VTC1, a GDP‐Man pyrophosphorylase that contributes to AsA biosynthesis. We found that the EAR domain promoted the stability of SlZF3 but was not required for the interaction between SlZF3 and CSN5B. Our findings indicate that SlZF3 simultaneously promotes the accumulation of AsA and enhances plant salt‐stress tolerance.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-28T04:43:05.562167-05:
      DOI: 10.1111/pbi.12863
  • Nitrogen use efficiency is regulated by interacting proteins relevant to
           development in wheat

    • Authors: Lei Lei; Genqiao Li, Hailin Zhang, Carol Powers, Tilin Fang, Yihua Chen, Shuwen Wang, Xinkai Zhu, Brett F. Carver, Liuling Yan
      Abstract: Wheat (Triticum aestivum) has low nitrogen use efficiency (NUE). The genetic mechanisms controlling NUE are unknown. Positional cloning of a major quantitative trait locus (QTL) for N‐related agronomic traits showed that the vernalization gene TaVRN‐A1 was tightly linked with TaNUE1, the gene shown to influence NUE in wheat. Because of an Ala180/Val180 substitution, TaVRN‐A1a and TaVRN‐A1b proteins interact differentially with TaANR1, a protein encoded by a wheat orthologue of Arabidopsis nitrate regulated 1 (ANR1). The transcripts of both TaVRN‐A1 and TaANR1 were down‐regulated by nitrogen. TaANR1 was functionally characterized in TaANR1::RNAi transgenic wheat, and in a natural mutant with a 23‐bp deletion including 10 bp at the 5′ end of intron 5 and 13 bp exon 6 in gDNA sequence in its gDNA sequence, which produced transcript that lacked the full 84‐bp exon 6. Both TaANR1 and TaHOX1 bound to the Ala180/Val180 position of TaVRN‐A1. Genetically incorporating favorable alleles from TaVRN‐A1, TaANR1, and TaHOX1 increased grain yield from 9.84% to 11.58% in the field. Molecular markers for allelic variation of the genes that regulate nitrogen can be used in breeding programs aimed at improving NUE and yield in novel wheat cultivars.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-28T04:42:59.01721-05:0
      DOI: 10.1111/pbi.12864
  • Overexpression of the class I homeodomain transcription factor
           TaHDZipI‐5 increases drought and frost tolerance in transgenic wheat

    • Authors: Yunfei Yang; Sukanya Luang, John Harris, Matteo Riboni, Yuan Li, Natalia Bazanova, Maria Hrmova, Stephan Haefele, Nataliya Kovalchuk, Sergiy Lopato
      Abstract: Characterisation of the function of stress‐related genes helps to define mechanisms of plant responses to environmental conditions. The findings of this work defined the role of the wheat TaHDZipI‐5 gene, encoding a stress‐responsive homeodomain‐leucine zipper class I (HD‐Zip I) transcription factor, during the development of plant tolerance to frost and drought. Strong induction of TaHDZipI‐5 expression by low temperatures, and the elevated TaHDZipI‐5 levels of expression in flowers and early developing grains in the absence of stress, suggest that TaHDZipI‐5 is involved in the regulation of frost tolerance at flowering. In the absence of stress, TaHDZipI‐5 was expressed in The TaHDZipI‐5 protein behaved as an activator in a yeast trans‐activation assay, and the TaHDZipI‐5 activation domain was localised to its C‐terminus. The TaHDZipI‐5 protein homo‐ and hetero‐dimerises with related TaHDZipI‐3, and differences between DNA interactions in both dimers were specified at 3D molecular levels. The constitutive overexpression of TaHDZipI‐5 in bread wheat significantly enhanced frost and drought tolerance of transgenic wheat lines with the appearance of undesired phenotypic features, which included a reduced plant size and biomass, delayed flowering and a grain yield decrease. An attempt to improve the phenotype of transgenic wheat by the application of stress‐inducible promoters with contrasting properties did not lead to the elimination of undesired phenotype, apparently due to strict spatial requirements for TaHDZipI‐5 overexpression.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-28T04:42:25.631052-05:
      DOI: 10.1111/pbi.12865
  • Genome‐wide discovery of tissue‐specific miRNAs in clusterbean
           (Cyamopsis tetragonoloba) indicates their association with galactomannan

    • Authors: Anshika Tyagi; Deepti Nigam, S. V. Amitha Mithra, Amolkumar U. Solanke, Nagendra Kumar Singh, Tilak Raj Sharma, Kishor Gaikwad
      Abstract: Owing to the presence of 80% soluble dietary fiber, high protein content and high value gum, clusterbean (Cyamopsis tetragonoloba) has recently emerged as an economically important legume. The developing clusterbean seeds accumulate 90% galactomannans in the endosperm, and therefore, can be used as a model crop to understand galactomannan biosynthesis and its regulation. miRNAs are tiny master regulators of their corresponding target genes, resulting in variations in the amounts of their metabolic end products. To understand the role of these regulators in galactomannan biosynthetic regulation, small RNA libraries were prepared and sequenced from five tissues of clusterbean genotype RGC‐936, and miRanalyzer and DSAP programs were used to identify conserved miRNAs and novel small RNAs. A total of 187 known and 171 novel miRNAs were found to be differentially expressed, out of which 10 miRNAs were validated in vivo. A complicated network topology and 35% sharing of the target mRNAs between known and novel miRNAs suggest random evolution of novel miRNAs. The gene ontology (GO) annotation of potential target genes revealed the genes coding for signaling and carbohydrate metabolism (50.10%), kinases and other enzymes (20.75%), transcription factors (10.20%), transporters (8.35%) and other targets (10.6%). Two novel unigenes were annotated as ManS(Mannosyltransferase/Mannan synthase) and UGE (UDP‐ D‐Glucose 4‐epimerase) and validated as targets for three novel miRNAsi.e.,Ct‐miR3130, Ct‐miR3135, andCt‐miR3157. Our findings reveal that these novel miRNAs could play an important role in the regulation of the galactomannan pathway in C. tetragonoloba and possibly other galactomannan producing species.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-28T04:42:19.154691-05:
      DOI: 10.1111/pbi.12866
  • Isolation of five rice non‐endosperm tissue‐expressed promoters and
           evaluation of their activities in transgenic rice

    • Authors: Hao Li; Juan Li, Rongfang Xu, Ruiying Qin, Fengshun Song, Li Li, Pengcheng Wei, Jianbo Yang
      Abstract: Using promoters expressed in non‐endosperm tissues to activate target genes in specific plant tissues or organs with very limited expression in the endosperm is an attractive approach in crop transgenic engineering. In this paper, five putative non‐endosperm tissue expressed promoters were cloned from the rice genome and designated POsNETE1, POsNETE2, POsNETE3, POsNETE4 and POsNETE5. By qualitatively and quantitatively examining GUSplus reporter gene expression in transgenic rice plants, POsNETE1‐POsNETE5 were all found to be active in the roots, leaves, stems, sheaths and panicles but not in the endosperm of plants at different developmental stages. In addition, POsNETE2, POsNETE4 and POsNETE5 were also inactive in rice embryos. Among these promoters, POsNETE4 and POsNETE5 exhibited higher activities in all of the tested tissues, and their activities in stems, leaves, roots and sheaths were higher than or comparable to those of the rice Actin1 promoter. We also progressively monitored the activities of POsNETE1‐POsNETE5 in two generations of single‐copy lines and found that these promoters were stably expressed between generations. Transgenic rice was produced using POsNETE4 and POsNETE5 to drive a modified Bt gene, mCry1Ab. Bt protein expressed in the tested plants ranged from 1769.4 to 4428.8 ng/g fresh leaves, whereas Bt protein was barely detected in the endosperm. Overall, our study identified five novel non‐endosperm tissue‐expressed promoters that might be suitable for rice genetic engineering and might reduce potential social concern regarding the safety of GMO crops.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-28T01:20:41.316699-05:
      DOI: 10.1111/pbi.12858
  • Low‐gluten, non‐transgenic wheat engineered with CRISPR/Cas9

    • Authors: Susana Sánchez-León; Javier Gil-Humanes, Carmen V. Ozuna, María J. Giménez, Carolina Sousa, Daniel F. Voytas, Francisco Barro
      Abstract: Celiac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley, and rye. The ‐gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33‐mer is the main immunodominant peptide in celiac patients. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33‐mer in the ‐gliadin genes. Twenty‐one mutant lines were generated, all showing strong reduction in ‐gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene‐free lines were identified, and no off‐target mutations have been detected in any of the potential targets. The low‐gluten, transgene‐free wheat lines described here could be used to produce low gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-24T00:06:28.836324-05:
      DOI: 10.1111/pbi.12837
  • PpNAC1, a main regulator of phenylalanine biosynthesis and utilization in
           maritime pine

    • Authors: María Belén Pascual; María-Teresa Llebrés, Blanca Craven-Bartle, Rafael A. Cañas, Francisco M. Cánovas, Concepción Ávila
      Abstract: The transcriptional regulation of phenylalanine metabolism is particularly important in conifers, long‐lived species that use large amounts of carbon in wood. Here, we show that the Pinus pinaster transcription factor, PpNAC1, is a main regulator of phenylalanine biosynthesis and utilization. A phylogenetic analysis classified PpNAC1 in the NST proteins group, and was selected for functional characterization. PpNAC1 is predominantly expressed in the secondary xylem and compression wood of adult trees. Silencing of PpNAC1 in P. pinaster results in the alteration of stem vascular radial patterning and the downregulation of several genes associated with cell wall biogenesis, and secondary metabolism. Furthermore, trans‐activation and EMSA analyses showed that PpNAC1 is able to activate its own expression and PpMyb4 promoter, while PpMyb4 is able to activate PpMyb8, a transcriptional regulator of phenylalanine and lignin biosynthesis in maritime pine. Together, these results suggest that PpNAC1 is a functional ortholog of the Arabidopsis SND1 and NST1 genes and support the idea that key regulators governing secondary cell wall formation could be conserved between gymnosperms and angiosperms. Understanding the molecular switches controlling wood formation is of paramount importance for fundamental tree biology and pave the way for applications in conifer biotechnology.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-23T04:41:53.243455-05:
      DOI: 10.1111/pbi.12854
  • Engineering Camelina sativa (L.) Crantz for Enhanced Oil and Seed Yields
           by Combining Diacylglycerol Acyltransferase1 and Glycerol‐3‐Phosphate
           Dehydrogenase Expression

    • Authors: Sudesh Chhikara; Hesham M. Abdullah, Parisa Akbari, Danny Schnell, Om Parkash Dhankher
      Abstract: Plant seed oils based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum‐derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproducts applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by co‐expressing the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol‐3‐phosphate dehydrogenase (GPD1) genes under the control of seed‐specific promoters. Plants co‐expressing DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild type plants. Further, DGAT1 and GDP1 co‐expressing lines showed significantly higher seed and oil yields on a dry‐weight basis than the wild type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1 and GPD1 co‐expressing lines was almost two‐folds higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-19T21:20:36.634762-05:
      DOI: 10.1111/pbi.12847
  • A HD‐ZIP III gene, PtrHB4, is required for interfascicular cambium
           development in Populus

    • Authors: Yingying Zhu; Dongliang Song, Peng Xu, Jiayan Sun, Laigeng Li
      Abstract: Wood production is dependent on the activity of the vascular cambium which develops from the fascicular and interfascicular cambium. However, little is known about the mechanisms controlling how the vascular cambium is developed in woody species. Here we show that PtrHB4, belonging to the Populus HD‐ZIP III family, plays a critical role in the process of vascular cambium development. PtrHB4 was specifically expressed in shoot tip and stem vascular tissue at an early developmental stage. Repression of PtrHB4 caused defects in the development of the secondary vascular system due to failures in interfascicular cambium formation. By contrast, overexpression of PtrHB4 induced cambium activity and xylem differentiation during secondary vascular development. Transcriptional analysis of PtrHB4 repressed plants indicated that auxin response and cell proliferation were affected in the formation of the interfascicular cambium. Taken together, these results suggest that PtrHB4 is required for interfascicular cambium formation to develop the vascular cambium in woody species.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-18T00:08:22.065688-05:
      DOI: 10.1111/pbi.12830
  • TALEN mediated targeted mutagenesis of more than 100 COMT copies/alleles
           in highly polyploid sugarcane improves saccharification efficiency without
           compromising biomass yield

    • Authors: Baskaran Kannan; Je Hyeong Jung, Geoffrey W. Moxley, Sun-Mi Lee, Fredy Altpeter
      Abstract: Sugarcane is the world's most efficient feedstock for commercial production of bioethanol due to its superior biomass production and accumulation of sucrose in stems. Integrating first and second generation ethanol conversion processes will enhance the biofuel yield per unit area by utilizing both sucrose as well as cell wall bound sugars for fermentation. RNAi suppression of the lignin biosynthetic gene caffeic acid O‐methyltransferase (COMT) has been demonstrated to improve bioethanol production from lignocellulosic biomass. Genome editing has been used in a number of crops for creation of loss of function phenotypes but is very challenging in sugarcane due to its highly polyploid genome. In this study, a conserved region of COMT was targeted with a single Transcription Activator‐Like Effector Nuclease (TALEN) pair for multi‐allelic mutagenesis to modify lignin biosynthesis in sugarcane. Field grown TALEN mediated COMT mutants showed up to 19.7% lignin reduction and significantly decreased syringyl to guaiacyl (S/G) ratio resulting in an up to 43.8% improved saccharification efficiency. Biomass production of COMT mutant lines with superior saccharification efficiency did not differ significantly from the original cultivar under replicated field conditions. Sanger sequencing of cloned COMT amplicons (1351‐1657 bp) revealed co‐editing of 107 of the 109 unique COMT copies/alleles in vegetative progeny of line CB6 using a single TALEN pair. Line CB6 combined altered cell wall composition and drastically improved saccharification efficiency with good agronomic performance. These findings confirm the feasibility of co‐mutagenesis of a very large number of target alleles/copies for improvement of crops with complex genomes.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-18T00:07:05.259558-05:
      DOI: 10.1111/pbi.12833
  • Long non‐coding RNAs involve in resistance to Verticillium dahliae,
           a fungal disease in cotton

    • Authors: Lin Zhang; Maojun Wang, Nannan Li, Honglei Wang, Ping Qiu, Liuling Pei, Zheng Xu, Tianyi Wang, Erlin Gao, Junxia Liu, Shiming Liu, Qin Hu, Yuhuan Miao, Keith Lindsey, Lili Tu, Longfu Zhu, Xianlong Zhang
      Abstract: Long non‐coding RNAs (lncRNAs) have several known functions in plant development, but their possible roles in responding to plant disease remain largely unresolved. In this study, we described a comprehensive disease‐responding lncRNA profiles in defense against a cotton fungal disease Verticillium dahliae. We further revealed the conserved and specific characters of disease responding process between two cotton species. Conservatively for two cotton species, we found the expression dominance of induced lncRNAs in the Dt subgenome, indicating a biased induction pattern in the co‐existing subgenomes of allotetraploid cotton. Comparative analysis of lncRNA expression and their proposed functions in resistant Gossypium barbadense cv. ‘7124′ versus susceptible G. hirsutum cv. ‘YZ1′ revealed their distinct disease response mechanisms. Species‐specific (LS) lncRNAs containing more SNPs displayed a fiercer inducing level post infection than the species‐conserved (core) lncRNAs. Gene Ontology enrichment of LS lncRNAs and core lncRNAs indicates distinct roles in the process of biotic stimulus. Further functional analysis showed that two core lncRNAs GhlncNAT‐ANX2‐ and GhlncNAT‐RLP7‐ silenced seedlings displayed an enhanced resistance towards V. dahliae and Botrytis cinerea, possibly associated with the increased expression of LOX1 and LOX2. This study represents the first characterization of lncRNAs involved in resistance to fungal disease and provides new clues to elucidate cotton disease response mechanism.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-17T09:10:37.20596-05:0
      DOI: 10.1111/pbi.12861
  • High‐resolution mapping of the of the peri‐centromeric region on wheat
           chromosome arm 5AS harboring the Fusarium head blight resistance QTL

    • Authors: Maria Buerstmayr; Barbara Steiner, Christian Wagner, Petra Schwarz, Klaus Brugger, Delfina Barabaschi, Andrea Volante, Giampiero Valè, Luigi Cattivelli, Hermann Buerstmayr
      Abstract: The Qfhs.ifa‐5A allele, contributing to enhanced Fusarium head blight resistance in wheat, resides in a low recombinogenic region of chromosome 5A close to the centromere. A near isogenic RIL population segregating for the Qfhs.ifa‐5A resistance allele was developed and among 3650 lines as few as four recombined within the pericentromeric C‐5AS1‐0.40 bin, yielding only a single recombination point. Genetic mapping of the pericentromeric region using a recombination dependent approach was thus not successful. To facilitate fine‐mapping the physically large Qfhs.ifa‐5A interval, two gamma‐irradiated deletion panels were generated: (1) Seeds of line NIL3 carrying the Qfhs.ifa‐5A resistance allele in an otherwise susceptible background were irradiated and plants thereof were selfed to obtain deletions in homozygous state. (2) A radiation hybrid panel was produced using irradiated pollen of the wheat line Chinese Spring (CS) for pollinating the CS‐nullisomic5Atetrasomic5B. In total 5157 radiation selfing and 276 radiation hybrid plants were screened for deletions on 5AS and plants containing deletions were analysed using 102 5AS specific markers. Combining genotypic information of both panels yielded an 817 fold map improvement (cR/cM) for the centromeric bin and was 389 fold increased across the Qfhs.ifa‐5A interval compared to the genetic map, with an average map resolution of 0.77 Mb/cR. We successfully proved that the RH mapping technique can effectively resolve marker order in low‐recombining regions, including pericentromeric intervals, and simultaneously allow developing an in vivo panel of sister lines differing for induced deletions across the Qfhs.ifa‐5A interval that can be used for phenotyping.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-10T05:05:47.476204-05:
      DOI: 10.1111/pbi.12850
  • CRISPR/Cas9‐mediated efficient targeted mutagenesis in grape in the
           first generation

    • Authors: Xianhang Wang; Mingxing Tu, Dejun Wang, Jianwei Liu, Yajuan Li, Zhi Li, Yuejin Wang, Xiping Wang
      Abstract: The clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR/Cas9) system is a powerful tool for editing plant genomes. Efficient genome editing of grape (Vitis vinifera) suspension cells using the type II CRISPR/Cas9 system has been demonstrated; however, it has not been established whether this system can be applied to get biallelic mutations in the first generation of grape. In this current study, we designed four guide RNAs for the VvWRKY52 transcription factor gene for using with the CRISPR/Cas9 system, and obtained transgenic plants via Agrobacterium‐mediated transformation, using somatic embryos of the Thompson Seedless cultivar. Analysis of the first generation transgenic plants verified 22 mutant plants out of the 72 T‐DNA inserted plants. Of these, 15 lines carried biallelic mutations and 7 were heterozygous. A range of RNA‐guided editing events, including large deletions, were found in the mutant plants, while smaller deletions comprised the majority of the detected mutations. Sequencing of potential off‐target sites for all four targets revealed no off‐target events. In addition, knock out of VvWRKY52 in grape increased the resistance to Botrytis cinerea. We conclude that the CRISPR/Cas9 system allows precise genome editing in the first generation of grape and represents a useful tool for gene functional analysis and grape molecular breeding.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-10T04:57:00.716239-05:
      DOI: 10.1111/pbi.12832
  • Interactions of WRKY15 and WRKY33 transcription factors and their roles in
           the resistance of oilseed rape to Sclerotinia infection

    • Authors: Fei Liu; Xiaoxia Li, Meirong Wang, Jing Wen, Bin Yi, Jinxiong Shen, Chaozhi Ma, Tingdong Fu, Jinxing Tu
      Abstract: WRKY transcription factors are known to participate in the defense responses of higher plants. However, little is known about the roles of such proteins, especially regarding their functions in the resistance of oilseed rape (Brassica napus) to Sclerotinia sclerotiorum, a necrotrophic fungal pathogen that causes stem rot. In the present study, we identified BnWRKY33 as a S. sclerotiorum‐responsive gene that positively regulates resistance to this pathogen by enhancing the expression of genes involved in camalexin synthesis and genes regulated by salicylic acid (SA) and jasmonic acid (JA). We also identified a S. sclerotiorum‐responsive region in the promoter of BnWRKY33, which we revealed to be a relatively conserved W‐box region in the promoters of homologous genes in different species. By using this S. sclerotiorum‐responsive region as bait in a yeast one‐hybrid assay, we identified another WRKY transcription factor, BnWRKY15, and observed that both BnWRKY15 and BnWRKY33 could bind to this region. In addition, BnWRKY15 overexpression simultaneously increased the susceptibility of B. napus to S. sclerotiorum and down‐regulated BnWRKY33 after different durations of infection. Furthermore, BnWRKY15, which contains a transcriptional repression domain, exhibited reduced transactivation ability and could reduce the transactivation ability of BnWRKY33 in Arabidopsis protoplast assays. Therefore, we suggest that the increased susceptibility of BnWRKY15‐overexpressing plants results from reduced BnWRKY33 expression, which is due to the inhibition of BnWRKY33 transcriptional activation by BnWRKY15.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-09T00:06:51.232121-05:
      DOI: 10.1111/pbi.12838
  • Expression and assembly of largest foreign protein in chloroplasts: Oral
           delivery of human FVIII made in lettuce chloroplasts robustly suppresses
           inhibitor formation in hemophilia A mice

    • Authors: Kwang-chul Kwon; Alexandra Sherman, Wan-Jung Chang, Aditya Kamesh, Moanaro Biswas, Roland W. Herzog, Henry Daniell
      Abstract: Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X‐linked bleeding disorder hemophilia A and occurs in 20‐30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full‐length CTB‐FVIII sequence to cover the entire patient population, regardless of individual CD4+ T cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15‐42 fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII HC (99.3 kDa), LC (91.8 kDa), C2 (31kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3,321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB‐FVIII‐SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulfide bonds were maintained upon lyophilization and long‐term storage as revealed by GM1‐ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB‐FVIII‐HC/CTB‐FVIII‐LC reduced inhibitor titers ~10‐fold (average 44 BU/ml to 4.7 BU/ml) in hemophilia A mice. Most importantly, increase in the frequency of circulating LAP‐expressing CD4+CD25+FoxP3+ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant‐based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect hemophilia A patients receiving FVIII injections.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-06T10:40:29.155829-05:
      DOI: 10.1111/pbi.12859
  • Comprehensive Description of Genome‐Wide Nucleotide and Structural
           Variation in Short‐Season Soybean

    • Authors: Davoud Torkamaneh; Jérôme Laroche, Aurélie Tardivel, Louise O'Donoughue, Elroy Cober, Istvan Rajcan, François Belzile
      Abstract: Next‐generation sequencing (NGS) and bioinformatics tools have greatly facilitated the characterization of nucleotide variation; nonetheless, an exhaustive description of both SNP haplotype diversity and of structural variation remains elusive in most species. In this study, we sequenced a representative set of 102 short‐season soybeans and achieved an extensive coverage of both nucleotide diversity and structural variation (SV). We called close to 5M sequence variants (SNPs, MNPs, and Indels) and noticed that the number of unique haplotypes had plateaued within this set of germplasm (1.7M tag SNPs). This dataset proved highly accurate (98.6%) based on a comparison of called genotypes at loci shared with a SNP array. We used this catalogue of SNPs as a reference panel to impute missing genotypes at untyped loci in datasets derived from lower density genotyping tools (150K GBS‐derived SNPs/530 samples). After imputation, 96.4% of the missing genotypes imputed in this fashion proved to be accurate. Using a combination of three bioinformatics pipelines, we uncovered ~92K SVs (deletions, insertions, inversions, duplications, CNVs, and translocations), and estimated that over 90% of these were accurate. Finally, we noticed that the duplication of certain genomic regions explained much of the residual heterozygosity at SNP loci in otherwise highly inbred soybean accessions. This is the first time that a comprehensive description of both SNP haplotype diversity and SV has been achieved within a regionally relevant subset of a major crop.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-03T23:06:29.059846-05:
      DOI: 10.1111/pbi.12825
  • MuMADS1 and MaOFP1 regulate fruit quality in a tomato ovate mutant

    • Authors: Juhua Liu; Jing Zhang, Jingyi Wang, Jianbin Zhang, Hongxia Miao, Caihong Jia, Zhuo Wang, Biyu Xu, Zhiqiang Jin
      Abstract: Fruit ripening and quality are common botanical phenomena that are closely linked and strictly regulated by transcription factors. It was previously discovered that a banana MADS‐box protein named MuMADS1 interacted with an ovate family protein named MaOFP1 to regulate banana fruit ripening. In order to further investigate the role of MuMADS1 and MaOFP1 in the regulation of fruit quality, a combination of genetic transformation and transcriptional characterization was used. The results indicated that the co‐expression of MuMADS1 and MaOFP1 in the ovate mutant could compensate for fruit shape and inferior qualities relating to fruit firmness, soluble solids and sugar content. The number of differentially expressed genes (DEGs) was 1,395 in WT vs. ovate, with 883 up‐regulated and 512 down‐regulated genes, while the numbers of DEGs gradually decreased with the transformation of MuMADS1 and MaOFP1 into ovate. “Starch and sucrose metabolism” constituted the primary metabolic pathway, and the gene numbers in this pathway were obviously different when MuMADS1 and MaOFP1 were integrated into ovate. A series of metabolic genes involved in cell wall biosynthesis were up‐regulated in the WT vs. ovate, which probably resulted in the firmer texture and lower sugar contents in the ovate fruit. These results demonstrate that MuMADS1 and MaOFP1 are co‐regulators of fruit quality, facilitating the dissection of the molecular mechanisms underlying fruit quality formation.This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-02T21:16:11.556254-05:
      DOI: 10.1111/pbi.12843
  • Development and evaluation of high density SNP array
           (Axiom®CicerSNP Array) for high resolution genetic mapping and breeding
           applications in chickpea

    • Authors: Manish Roorkiwal; Ankit Jain, Sandip M. Kale, Dadakhalandar Doddamani, Annapurna Chitikineni, Mahendar Thudi, Rajeev K. Varshney
      Abstract: To accelerate genomics research and molecular breeding applications in chickpea, a high‐throughput SNP genotyping platform ‘Axiom®CicerSNP array’ has been designed, developed and validated. Screening of whole genome re‐sequencing data from 429 chickpea lines identified 4.9 million SNPs from which a sub‐set of 70,463 high quality non redundant SNPs was selected using different stringent filter criteria's. This was further narrowed down to 61,174 SNPs based on p‐convert score ≥ 0.3, of which 50,590 SNPs could be tiled on array. Among these tiled SNPs, a total of 11,245 SNPs (22.23%) were from the coding regions of 3,673 different genes. The developed Axiom®CicerSNP array was used for genotyping two recombinant inbred line populations namely ICCRIL03 (ICC 4958 × ICC 1882) and ICCRIL04 (ICC 283 × ICC 8261). Genotyping data reflected high success and polymorphic rate, with 15,140 (29.93%; ICCRIL03) and 20,018 (39.57%; ICCRIL04) polymorphic SNPs. High‐density genetic maps comprising 13,679 SNPs spanning 1033.67 cM and 7,769 SNPs spanning 1076.35 cM were developed for ICCRIL03 and ICCRIL04 populations, respectively. Multi‐location, multi‐season phenotyping data on these RILs identified 70 (ICCRIL03) and 120 (ICCRIL04) main effect QTLs on genetic map. Higher precision and potential of this array is expected to advance chickpea genetics and breeding applications.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-31T23:05:55.860178-05:
      DOI: 10.1111/pbi.12836
  • Agronomic and chemical performance of field grown tobacco engineered for
           triterpene and methylated triterpene metabolism

    • Authors: Zuodong Jiang; Chase Kempinski, Santosh Kumar, Scott Kinison, Kristin Linscott, Eric Nybo, Sarah Janze, Connie Wood, Joe Chappell
      Abstract: Squalene is a linear intermediate to nearly all classes of triterpenes and sterols and is itself highly valued for its use in wide range of industrial applications. Another unique linear triterpene is botryococcene and its methylated derivatives generated by the alga Botryococcus braunii race B, which are progenitors to fossil fuel deposits. Production of these linear triterpenes was previously engineered into transgenic tobacco by introducing the key steps of triterpene metabolism into the particular subcellular compartments. In this study, the agronomic characteristics (height, biomass accumulation, leaf area), the photosynthetic capacity (photosynthesis rate, conductance, internal CO2 levels) and triterpene content of select lines grown under field conditions were evaluated for three consecutive growing seasons. We observed that transgenic lines targeting enzymes to the chloroplasts accumulated 50‐150‐time more squalene than the lines targeting the enzymes to the cytoplasm, without compromising growth or photosynthesis. We also found that the transgenic lines directing botryococcene metabolism to the chloroplast accumulated 10‐33‐fold greater levels than the lines where the same enzymes were targeted to in the cytoplasm. However, growth of these high botryococcene accumulators was highly compromised, yet their photosynthesis rates remained unaffected. In addition, in the transgenic lines targeting a triterpene methyltransferase (TMT) to the chloroplasts of high squalene accumulators, 55‐65% of total squalene was methylated, whereas in the lines expressing a TMT in the cytoplasm only 6‐13% of squalene was methylated. The growth of these methylated triterpene accumulating lines was more compromised than that of non‐methylated squalene lines.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-25T11:25:30.092882-05:
      DOI: 10.1111/pbi.12855
  • Three AtCesA6‐like members enhance biomass production by distinctively
           promoting cell growth in Arabidopsis

    • Authors: Huizhen Hu; Ran Zhang, Shengqiu Feng, Youmei Wang, Yanting Wang, Chunfen Fan, Ying Li, Zengyu Liu, René Schneider, Tao Xia, Shi-You Ding, Staffan Persson, Liangcai Peng
      Abstract: Cellulose is an abundant biopolymer and a prominent constituent of plant cell walls. Cellulose is also a central component to plant morphogenesis and contributes the bulk of a plant's biomass. While cellulose synthase (CesA) genes were identified over two decades ago, genetic manipulation of this family to enhance cellulose production has remained difficult. In this study, we show that increasing the expression levels of the three primary cell wall AtCesA6‐like genes (AtCesA2, ‐5, ‐6), but not AtCesA3, AtCesA9 or secondary cell wall AtCesA7, can promote the expression of major primary wall CesA genes to accelerate primary wall CesA complex (CSCs) particle movement for acquiring long microfibrils and consequently increasing cellulose production in Arabidopsis transgenic lines, as compared with wild type. The over‐expression transgenic lines displayed changes in expression of genes related to cell growth and proliferation, perhaps explaining the enhanced growth of the transgenic seedlings. Notably, over‐expression of the three AtCesA6‐like genes also enhanced secondary cell wall deposition that led to improved mechanical strength and higher biomass production in transgenic mature plants. Hence, we propose that over‐expression of certain AtCesA genes can provide a biotechnological approach to increase cellulose synthesis and biomass accumulation in transgenic plants.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-23T23:36:33.191908-05:
      DOI: 10.1111/pbi.12842
  • Conversion of array‐based single nucleotide polymorphic markers for use
           in targeted genotyping by sequencing in hexaploid wheat (Triticum

    • Authors: Amanda J. Burridge; Paul A. Wilkinson, Mark O. Winfield, Gary L. A. Barker, Alexandra M. Allen, Jane A. Coghill, Christy Waterfall, Keith J. Edwards
      Abstract: Wheat breeders and academics alike use Single Nucleotide Polymorphisms (SNPs) as molecular markers to characterise regions of interest within the hexaploid wheat genome. A number of SNP‐based genotyping platforms are available and their utility depends upon factors such as the available technologies, number of data points required, budgets and the technical expertise required. Unfortunately, markers can rarely be exchanged between existing and newly developed platforms, meaning that previously generated data cannot be compared, or combined, with more recently generated datasets. We predict that genotyping by sequencing will become the predominant genotyping technology within the next five to ten years. With this in mind, to ensure that data generated from current genotyping platforms continues to be of use, we have designed and utilised SNP‐based capture probes from several thousand existing and publicly available probes from Axiom® and KASP™ genotyping platforms. We have validated our capture probes in a targeted genotyping by sequencing protocol using thirty one previously genotyped UK elite hexaploid wheat accessions. Data comparisons between targeted genotyping by sequencing, Axiom® array genotyping and KASP™ genotyping assays, identified a set of 3,256 probes which reliably bring together targeted genotyping by sequencing data with the previously available marker dataset. As such these probes are likely to be of considerable value to the wheat community. The probe details, full probe sequences and a custom built analysis pipeline may be freely downloaded from the CerealsDB.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-23T21:45:48.744544-05:
      DOI: 10.1111/pbi.12834
  • Host‐induced gene silencing of an important pathogenicity factor PsCPK1
           in Puccinia striiformis f. sp. tritici enhances resistance of wheat to
           stripe rust

    • Authors: Tuo Qi; Xiaoguo Zhu, Chenlong Tan, Peng Liu, Jia Guo, Zhensheng Kang, Jun Guo
      Abstract: Rust fungi are devastating plant pathogens and cause a large economic impact on wheat production worldwide. To overcome this rapidly loss of varieties resistance, we generated stable transgenic wheat plants expressing short interfering RNAs (siRNAs) targeting potentially vital genes of Puccinia striiformis f. sp. tritici (Pst). Protein kinase A (PKA) has been proved to play important roles in regulating the virulence of phytopathogenic fungi. PsCPK1, a PKA catalytic subunit gene from Pst, is highly induced at the early infection stage of Pst. The instantaneous silencing of PsCPK1 by barley stripe mosaic virus (BSMV)‐mediated host‐induced gene silencing (HIGS) results in a significant reduction in the length of infection hyphae and disease phenotype. These results indicate that PsCPK1 is an important pathogenicity factor by regulating Pst growth and development. Two transgenic lines expressing the RNA interference (RNAi) construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst throughout the T3 to T4 generations. The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting, and these RNAs were found to efficiently down‐regulate PsCPK1 expression in wheat. The present study addresses important aspects for the development of fungal‐derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-23T04:16:29.635304-05:
      DOI: 10.1111/pbi.12829
  • The transcriptome, extracellular proteome and active secretome of
           agroinfiltrated N. benthamiana uncover a large, diverse protease

    • Authors: Friederike Grosse-Holz; Steven Kelly, Svenja Blaskowski, Farnusch Kaschani, Markus Kaiser, Renier A.L. van der Hoorn
      Abstract: Infiltration of disarmed Agrobacterium tumefaciens into leaves of Nicotiana benthamiana (agroinfiltration) facilitates quick and safe production of antibodies, vaccines, enzymes and metabolites for industrial use (Molecular Farming). However, yield and purity of proteins produced by agroinfiltration are hampered by unintended proteolysis, restricting industrial viability of the agroinfiltration platform. Proteolysis may be linked to an immune response to agroinfiltration, but understanding of the response to agroinfiltration is limited. To identify the proteases, we studied the transcriptome, extracellular proteome and active secretome of agroinfiltrated leaves over a time course, with and without the P19 silencing inhibitor. Remarkably, P19 expression had little effect on the leaf transcriptome and no effect on the extracellular proteome. 25% of the detected transcripts changed in abundance upon agroinfiltration, associated with a gradual upregulation of immunity at the expense of photosynthesis. By contrast, 70% of the extracellular proteins increase in abundance, in many cases associated with increased efficiency of extracellular delivery. We detect a dynamic reprogramming of the proteolytic machinery upon agroinfiltration by detecting transcripts encoding for 975 different proteases and protease‐homologs. The extracellular proteome contains peptides derived from 196 proteases and protease homologs, and activity‐based proteomics displayed 17 active extracellular Ser and Cys proteases in agroinfiltrated leaves. We discuss unique features of the N. benthamiana protease repertoire and highlight abundant extracellular proteases in agroinfiltrated leaves, being targets for reverse genetics. This dataset increases our understanding of the plant response to agroinfiltration and indicates ways to improve a key expression platform for both plant science and Molecular Farming.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-21T00:31:01.561369-05:
      DOI: 10.1111/pbi.12852
  • Transcriptomic repertoires depict the initiation of lint and fuzz fibers
           in cotton (Gossypium hirsutum L.)

    • Authors: Haiyan Hu; Maojun Wang, Yuanhao Ding, Sitao Zhu, Guannan Zhao, Lili Tu, Xianlong Zhang
      Abstract: Cotton fiber is an important natural fiber for the textile industry. The number of fiber initials determines the lint percentage, which is an important factor for cotton fiber yield. Although fiber development has been described by transcriptomic analysis, the mechanism by which the long non‐coding RNA manipulate the initiation of lint and fuzz fibers remains unknown. In this study, three lines with different lint percentages were developed by crossing Xu142 with its fiberless mutant Xu142 fl. We collected the epidermal cells from the ovules with attached fibers at 0 and 5 days post‐anthesis (DPA) from Xu142, the fiberless mutant Xu142 fl and the three lint percent diversified lines for deep transcriptome sequencing. A total of 2,641 novel genes, 35,802 long non‐coding RNAs (lncRNAs) and 2,262 circular RNAs (circRNAs) were identified, of which 645 lncRNAs were preferentially expressed in the fiberless mutant Xu142 fl and 651 lncRNAs were preferentially expressed in the fiber‐attached lines. We demonstrated the functional roles of the three lncRNAs in fiber development via a virus‐induced gene silencing (VIGS) system. Our results showed that silencing XLOC_545639 and XLOC_039050 in Xu142 fl increased the number of fiber initials on the ovules, but silencing XLOC_079089 in Xu142 resulted in a short fiber phenotype. This study established the transcriptomic repertoires in cotton fiber initiation and provided evidence for the potential functions of lncRNAs in fiber development.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-18T02:05:43.410769-05:
      DOI: 10.1111/pbi.12844
  • Improved fruit α‐tocopherol, carotenoid, squalene and phytosterol
           content through manipulation of Brassica juncea
           3‐HYDROXY‐3‐METHYLGLUTARYL‐COA SYNTHASE1 in transgenic tomato

    • Authors: Pan Liao; Xinjian Chen, Mingfu Wang, Thomas J. Bach, Mee-Len Chye
      Abstract: 3‐Hydroxy‐3‐methylglutaryl‐coenzyme A synthase (HMGS) in the mevalonate (MVA) pathway generates isoprenoids including phytosterols. Dietary phytosterols are important because they can lower blood cholesterol levels. Previously, the overexpression of Brassica juncea wild‐type (wt) and mutant (S359A) BjHMGS1 in Arabidopsis up‐regulated several genes in sterol biosynthesis and increased sterol content. Recombinant S359A had earlier displayed a 10‐fold higher in vitro enzyme activity. Furthermore, tobacco HMGS‐overexpressors (OEs) exhibited improved sterol content, plant growth and seed yield. Increased growth and seed yield in tobacco OE‐S359A over OE‐wtBjHMGS1 coincided with elevations in NtSQS expression and sterol content. Herein, the overexpression of wt and mutant (S359A) BjHMGS1 in a crop plant, tomato (Solanum lycopersicum), caused an accumulation of MVA‐derived squalene and phytosterols, as well as methylerythritol phosphate (MEP)‐derived α‐tocopherol (vitamin E) and carotenoids, which are important to human health as antioxidants. In tomato HMGS‐OE seedlings, genes associated with the biosyntheses of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, brassinosteroids, dolichols, methylerythritol phosphate, carotenoid and vitamin E were up‐regulated. In OE‐S359A tomato fruits, increased squalene and phytosterol content over OE‐wtBjHMGS1 was attributed to heightened SlHMGR2, SlFPS1, SlSQS and SlCYP710A11 expression. In both tomato OE‐wtBjHMGS1 and OE‐S359A fruits, the up‐regulation of SlGPS and SlGGPPS1 in the MEP pathway that led to α‐tocopherol and carotenoid accumulation, indicated cross‐talk between the MVA and MEP pathways. Taken together, the manipulation of BjHMGS1 represents a promising strategy to simultaneously elevate health‐promoting squalene, phytosterols, α‐tocopherol and carotenoids in tomato, an edible fruit.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-17T03:40:18.748741-05:
      DOI: 10.1111/pbi.12828
  • Metabolite and transcript markers for the prediction of potato drought

    • Authors: Heike Sprenger; Alexander Erban, Sylvia Seddig, Katharina Rudack, Anja Thalhammer, Mai Q. Le, Dirk Walther, Ellen Zuther, Karin I. Köhl, Joachim Kopka, Dirk K. Hincha
      Abstract: Potato (Solanum tuberosum L.) is one of the most important food crops worldwide. Current potato varieties are highly susceptible to drought stress. In view of global climate change, selection of cultivars with improved drought tolerance and high yield potential is of paramount importance. Drought tolerance breeding of potato is currently based on direct selection according to yield and phenotypic traits and requires multiple trials under drought conditions. Marker‐assisted selection (MAS) is cheaper, faster and reduces classification errors caused by non‐controlled environmental effects. We analysed 31 potato cultivars grown under optimal and reduced water supply in six independent field trials. Drought tolerance was determined as tuber starch yield. Leaf samples from young plants were screened for pre‐selected transcript and non‐targeted metabolite abundances using qRT‐PCR and GC‐MS profiling, respectively. Transcript marker candidates were selected from a published RNA‐Seq data set. A Random Forest machine learning approach extracted metabolite and transcript markers for drought tolerance prediction with low error rates of 6% and 9%, respectively. Moreover, by combining transcript and metabolite markers, the prediction error was reduced to 4.3%. Feature selection from Random Forest models allowed model minimization, yielding a minimal combination of only 20 metabolite and transcript markers that were successfully tested for their reproducibility in 16 independent agronomic field trials. We demonstrate that a minimum combination of transcript and metabolite markers sampled at early cultivation stages predicts potato yield stability under drought largely independent of seasonal and regional agronomic conditions.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-17T02:50:32.614145-05:
      DOI: 10.1111/pbi.12840
  • Peanuts that keep aflatoxin at bay: A threshold that matters

    • Authors: Kiran K. Sharma; Arunima Pothana, Kalyani Prasad, Dilip Shah, Jagdeep Kaur, Deepak Bhatnagar, Zhi-Yuan Chen, Yenjit Raruang, Jeffrey W. Cary, Kanniah Rajasekaran, Hari Kishan Sudini, Pooja Bhatnagar-Mathur
      Abstract: Aflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub‐Saharan Africa and South Asia. Developing peanut varieties to combat pre‐harvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut‐Aspergilli pathosystem. Our study reports achieving high level of resistance in peanut by over expressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host‐induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/ lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE‐Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species‐scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near‐immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi‐arid regions.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-17T02:05:54.12962-05:0
      DOI: 10.1111/pbi.12846
  • From model to crop: functional characterization of SPL8 in M. truncatula
           led to genetic improvement of biomass yield and abiotic stress tolerance
           in alfalfa

    • Authors: Jiqing Gou; Smriti Debnath, Liang Sun, Amy Flanagan, Yuhong Tang, Qingzhen Jiang, Jiangqi Wen, Zeng-Yu Wang
      Abstract: Biomass yield, salt tolerance and drought tolerance are important targets for alfalfa (Medicago sativa L.) improvement. Medicago truncatula has been developed into a model plant for alfalfa and other legumes. By screening a Tnt1 retrotransposon–tagged M. truncatula mutant population, we identified three mutants with enhanced branching. Branch development determines shoot architecture which affects important plant functions like light acquisition, resource use and ultimately impacts biomass production. Molecular analyses revealed that the mutations were caused by Tnt1 insertions in the SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE 8 (SPL8) gene. The M. truncatula spl8 mutants had increased biomass yield, while overexpression of SPL8 in M. truncatula suppressed branching and reduced biomass yield. Scanning electron microscopy (SEM) analysis showed that SPL8 inhibited branching by directly suppressing axillary bud formation. Based on the M. truncatula SPL8 sequence, alfalfa SPL8 (MsSPL8) was cloned and transgenic alfalfa plants were produced. MsSPL8 downregulated or upregulated alfalfa plants exhibited similar phenotypes to the M. truncatula mutants or overexpression lines, respectively. Specifically, the MsSPL8 downregulated alfalfa plants showed up to 43% increase in biomass yield in the first harvest. The impact was even more prominent in the second harvest, with up to 86% increase in biomass production compared to the control. Furthermore, downregulation of MsSPL8 led to enhanced salt and drought tolerance in transgenic alfalfa. Results from this research offer a valuable approach to simultaneously improve biomass production and abiotic stress tolerance in legumes.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-17T01:41:44.979508-05:
      DOI: 10.1111/pbi.12841
  • Construction of Agropyron Gaertn. genetic linkage maps using a wheat 660K
           SNP array reveals a homoeologous relationship with the wheat genome

    • Authors: Shenghui Zhou; Jinpeng Zhang, Yonghe Che, Weihua Liu, Yuqing Lu, Xinming Yang, Xiuquan Li, Jizeng Jia, Xu Liu, Lihui Li
      Abstract: Agropyron Gaertn. (P genome) is a wild relative of wheat that harbours many genetic variations that could be used to increase the genetic diversity of wheat. To agronomically transfer important genes from the P genome to a wheat chromosome by induced homoeologous pairing and recombination, it is necessary to determine the chromosomal relationships between Agropyron and wheat. Here, we report using the wheat 660K single nucleotide polymorphism (SNP) array to genotype a segregating Agropyron F1 population derived from an interspecific cross between two cross‐pollinated diploid collections ‘Z1842′ [A. cristatum (L.) Beauv.] (male parent) and ‘Z2098′ [A. mongolicum Keng] (female parent) and 35 wheat‐A. cristatum addition/substitution lines. Genetic linkage maps were constructed using 913 SNP markers distributed among seven linkage groups spanning 839.7 cM. The average distance between adjacent markers was 1.8  cM. The maps identified the homoeologous relationship between the P genome and wheat and revealed that the P and wheat genomes are collinear and relatively conserved. In addition, obvious rearrangements and introgression spread were observed throughout the P genome compared with the wheat genome. Combined with genotyping data, the complete set of wheat‐A. cristatum addition/substitution lines was characterized according to their homoeologous relationships. In this study, the homoeologous relationship between the P genome and wheat was identified using genetic linkage maps, and the detection mean for wheat‐A. cristatum introgressions might significantly accelerate the introgression of genetic variation from Agropyron into wheat for exploitation in wheat improvement programmes.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-16T00:22:08.844592-05:
      DOI: 10.1111/pbi.12831
  • Genotyping‐by‐sequencing through transcriptomics: Implementation in a
           range of crop species with varying reproductive habits and ploidy levels

    • Authors: M. Michelle Malmberg; Luke W. Pembleton, Rebecca C. Baillie, Michelle C. Drayton, Shimna Sudheesh, Sukhjiwan Kaur, Hiroshi Shinozuka, Preeti Verma, German C. Spangenberg, Hans D. Daetwyler, John W. Forster, Noel O.I. Cogan
      Abstract: The application of genomics in crops has the ability to significantly improve genetic gain for agriculture. Many marker‐dense tools have been developed but few have seen broad adoption in plant genomics due to issues of significant variations of genome size, levels of ploidy, single nucleotide polymorphism (SNP) frequency and reproductive habit. When combined with limited breeding activities, small research communities and scant sequence resources, the suitability of popular systems is often sub‐optimal and routinely fails to effectively balance cost‐effectiveness and sample throughput.Genotyping‐by‐sequencing (GBS) encompasses a range of protocols including re‐sequencing of the transcriptome. The present study describes a skim GBS‐transcriptomics (GBS‐t) approach developed to be broadly applicable, cost‐effective and high‐throughput while still assaying a significant number of SNP loci. A range of crop species with differing levels of ploidy and degree of inbreeding/outbreeding were chosen, including; perennial ryegrass, a diploid outbreeding forage grass; phalaris, a putative segmental allotetraploid outbreeding forage grass; lentil, a diploid inbreeding grain legume; and canola, an allotetraploid partially outbreeding oilseed. GBS‐t was validated as a simple and largely automated, cost‐effective method which generates sufficient SNPs (from 89,738 to 231,977) with acceptable levels of missing data and even genome coverage from c. 3 million sequence reads per sample. GBS‐t is therefore a broadly applicable system suitable for many crops, offering advantages over other systems. The correct choice of subsequent sequence analysis software is important, and the bioinformatics process should be iterative and tailored to the specific challenges posed by ploidy variation and extent of heterozygosity.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-13T01:00:34.277056-05:
      DOI: 10.1111/pbi.12835
  • The potassium channel FaTPK1 plays a critical role in fruit quality
           formation in strawberry (Fragaria × ananassa)

    • Authors: Shufang Wang; Miaoyu Song, Jiaxuan Guo, Yun Huang, Fangfang Zhang, Cheng Xu, Yinghui Xiao, Lusheng Zhang
      Abstract: Potassium (K+), an abundant cation in plant cells, is important in fruit development and plant resistance. However, how cellular K+ is directed by potassium channels in fruit development and quality formation of strawberry (Fragaria × ananassa) is not yet fully clear. Here, a two-pore K+ (TPK) channel gene in strawberry, FaTPK1, was cloned using reverse transcription–PCR. A green fluorescent protein subcellular localization analysis showed that FaTPK1 localized in the vacuole membrane. A transcription analysis indicated that the mRNA expression level of FaTPK1 increased rapidly and was maintained at a high level in ripened fruit, which was coupled with the fruit's red colour development, suggesting that FaTPK1 is related to fruit quality formation. The down- and upregulation of the FaTPK1 mRNA expression levels using RNA interference and overexpression, respectively, inhibited and promoted fruit ripening, respectively, as demonstrated by consistent changes in firmness and the contents of soluble sugars, anthocyanin and abscisic acid, as well as the transcript levels of ripening-regulated genes PG1 (polygalacturonase), GAL6 (beta-galactosidase), XYL2 (D-xylulose reductase), SUT1 (sucrose transporter), CHS (chalcone synthase) and CHI (chalcone flavanone isomerase). Additionally, the regulatory changes influenced fruit resistance to Botrytis cinerea. An isothermal calorimetry analysis showed that the Escherichia coli-expressed FaTPK1 recombinant protein could bind K+ with a binding constant of 2.1×10–3 M−1 and a dissociation constant of 476 μM. Thus, the strawberry TPK1 is a ubiquitously expressed, tonoplast-localized two-pore potassium channel that plays important roles in fruit ripening and quality formation.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-12T21:16:21.984948-05:
      DOI: 10.1111/pbi.12824
  • OsASR2 regulates the expression of a defense‐related gene, Os2H16, by
           targeting the GT‐1 cis‐element

    • Authors: Ning Li; Shutong Wei, Jing Chen, Fangfang Yang, Lingguang Kong, Cuixia Chen, Xinhua Ding, Zhaohui Chu
      Abstract: The GT‐1 cis‐element widely exists in many plant gene promoters. However, the molecular mechanism that underlies the response of the GT‐1 cis‐element to abiotic and biotic stresses remains elusive in rice. We previously isolated a rice short‐chain peptide encoding gene, Os2H16, and demonstrated that it plays important roles in both disease resistance and drought tolerance. Here, we conducted a promoter assay of Os2H16 and identified GT‐1 as an important cis‐element that mediates Os2H16 expression in response to pathogen attack and osmotic stress. By using the repeated GT‐1 as bait, we characterized an abscisic acid, stress and ripening 2 (ASR2) protein from yeast‐one hybridization screening. Sequence alignments showed that the carboxy‐terminal domain of OsASR2 containing residues 80‐138 was the DNA‐binding domain. Furthermore, we identified that OsASR2 was specifically bound to GT‐1 and activated the expression of the target gene Os2H16, as well as GFP driven by the chimeric promoter of 2 × GT‐1‐35S mini construct. Additionally, the expression of OsASR2 was elevated by pathogens and osmotic stress challenges. Overexpression of OsASR2 enhanced the resistance against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, and tolerance to drought in rice. These results suggest that the interaction between OsASR2 and GT‐1 plays an important role in the crosstalk of the response of rice to biotic and abiotic stresses.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-10T00:30:53.994754-05:
      DOI: 10.1111/pbi.12827
  • Assembly and analysis of a qingke reference genome demonstrates its close
           genetic relation to modern cultivated barley

    • Authors: Fei Dai; Xiaolei Wang, Xiao-Qi Zhang, Zhonghua Chen, Eviatar Nevo, Gulei Jin, Dezhi Wu, Chengdao Li, Guoping Zhang
      Abstract: Qingke, the local name of hulless barley in the Tibetan Plateau, is a staple food for Tibetans. The availability of its reference genome sequences could be useful for studies on breeding and molecular evolution. Taking advantage of the third‐generation sequencer (PacBio), we de novo assembled a 4.84 Gb genome sequence of qingke, cv. Zangqing320 and anchored a 4.59 Gb sequence to seven chromosomes. Of the 46,787 annotated ‘high‐confidence’ genes, 31,564 were validated by RNA‐sequencing data of 39 wild and cultivated barley genotypes with wide genetic diversity, and the results were also confirmed by non‐redundant protein database from NCBI. As some gaps in the reference genome of Morex were covered in the reference genome of Zangqing320 by PacBio reads, we believe that the Zangqing320 genome provides the useful supplements for the Morex genome. Using the qingke genome as a reference, we conducted a genome comparison, revealing a close genetic relationship between a hulled barley (cv. Morex) and a hulless barley (cv. Zangqing320), which is strongly supported by the low diversity regions in the two genomes. Considering the origin of Morex from its breeding pedigree, we then demonstrated a close genomic relationship between modern cultivated barley and qingke. Given this genomic relationship and the large genetic diversity between qingke and modern cultivated barley, we propose that qingke could provide elite genes for barley improvement.This article is protected by copyright. All rights reserved.
      PubDate: 2017-10-05T21:51:04.877275-05:
      DOI: 10.1111/pbi.12826
  • Host‐induced silencing of essential genes in Puccinia triticina through
           transgenic expression of RNAi sequences reduces severity of leaf rust
           infection in wheat

    • Authors: Vinay Panwar; Mark Jordan, Brent McCallum, Guus Bakkeren
      Abstract: Leaf rust, caused by the pathogenic fungus Puccinia triticina (Pt), is one of the most serious biotic threats to sustainable wheat production worldwide. This obligate biotrophic pathogen is prevalent worldwide and is known for rapid adaptive evolution to overcome resistant wheat varieties. Novel disease control approaches are therefore required to minimize the yield losses caused by Pt. Having shown previously the potential of host‐delivered RNA interference (HD‐RNAi) in functional screening of Pt genes involved in pathogenesis, we here evaluated the use of this technology in transgenic wheat plants as a method to achieve protection against wheat leaf rust (WLR) infection. Stable expression of hairpin RNAi constructs with sequence homology to Pt MAP Kinase (PtMAPK1) or a cyclophilin (PtCYC1) encoding gene in susceptible wheat plants showed efficient silencing of the corresponding genes in the interacting fungus resulting in disease resistance throughout the T2 generation. Inhibition of Pt proliferation in transgenic lines by in planta‐induced RNAi was associated with significant reduction in target fungal transcript abundance and reduced fungal biomass accumulation in highly resistant plants. Disease protection was correlated with the presence of siRNA molecules specific to targeted fungal genes in the transgenic lines harbouring the complementary HD‐RNAi construct. This work demonstrates that generating transgenic wheat plants expressing RNAi‐inducing transgenes to silence essential genes in rust fungi can provide effective disease resistance, thus opening an alternative way for developing rust‐resistant crops.This article is protected by copyright. All rights reserved.
      PubDate: 2017-09-23T08:56:01.76493-05:0
      DOI: 10.1111/pbi.12845
  • A Synthetic Biology approach for consistent production of plant-made
           recombinant polyclonal antibodies against snake venom toxins

    • Authors: Jose Manuel Julve Parreño; Estefanía Huet, Asun Fernández-del-Carmen, Alvaro Segura, Micol Venturi, Antoni Gandía, Wei-song Pan, Irene Albaladejo, Javier Forment, Davinia Pla, Andrés Wigdorovitz, Juan J. Calvete, Carlos Gutiérrez, José María Gutiérrez, Antonio Granell, Diego Orzáez
      Abstract: Antivenoms developed from the plasma of hyper-immunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost-effective antivenom production based on plant-made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin-binding genetic information captured from peripheral blood lymphocytes of hyper-immunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant-made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins, and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice.This article is protected by copyright. All rights reserved.
      PubDate: 2017-09-22T02:01:16.548578-05:
      DOI: 10.1111/pbi.12823
  • Global Profiling of Alternative Splicing Landscape Responsive to Drought,
           Heat and Their Combination in Wheat (Triticum asetivum L.)

    • Authors: Zhenshan Liu; Jinxia Qin, Xuejun Tian, Shengbao Xu, Yu Wang, Hongxia Li, Xiaoming Wang, Huiru Peng, Yingyin Yao, Zhaorong Hu, Zhongfu Ni, Mingming Xin, Qixin Sun
      Abstract: Plant can acquire tolerance to environmental stresses via transcriptome reprogramming at transcriptional and alternative splicing (AS) levels. However, how AS coordinates with transcriptional regulation to contribute to abiotic stresses responses is still ambiguous. In the present study, we performed genome-wide analyses of AS responses to drought stress (DS), heat stress (HS) and their combination (HD) in wheat seedlings, and further compared them with transcriptional responses. In total, we found 200, 3,576, and 4,056 genes exhibiting significant AS pattern changes in response to DS, HS and HD, respectively, and combined drought and heat stress can induce specific AS compared with individual one. In addition, wheat homeologous genes exhibited differential AS responses under stress conditions that more AS events occurred on B subgenome than on A and D genomes. Comparison of genes regulated at AS and transcriptional levels showed that only 12% of DS induced AS genes were subjected to transcriptional regulation, whereas the proportion increased to ~40% under HS and HD. Functional enrichment analysis revealed that abiotic stress responsive pathways tended to be highly overrepresented among these overlapped genes under HS and HD. Thus, we proposed that transcriptional regulation may play a major role in response to DS, which coordinates with AS regulation to contribute to HS and HD tolerance in wheat.This article is protected by copyright. All rights reserved.
      PubDate: 2017-09-20T20:46:00.492959-05:
      DOI: 10.1111/pbi.12822
  • Identification of bottlenecks in the accumulation of cyclic fatty acids in
           camelina seed oil

    • Authors: Xiao-Hong Yu; Rebecca E. Cahoon, Patrick J. Horn, Hai Shi, Richa Rawat Prakash, Yuanheng Cai, Maegan Hearney, Kent D. Chapman, Edgar B. Cahoon, Jorg Schwender, John Shanklin
      Abstract: Modified fatty acids (mFA) have diverse uses, e.g., cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics, and cosmetics. The expression of mFA‐producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural‐occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co‐expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA‐accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation, and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon coexpression of SfLPAT with EcCPS, di‐CPA‐PC increased by ~50% relative to lines expressing EcCPS alone with the di‐CPA‐PC primarily observed in the embryonic axis and mono‐CPA‐PC primarily in cotyledon tissue. EcCPS‐SfLPAT lines revealed a redistribution of CPA from the sn‐1 to sn‐2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.This article is protected by copyright. All rights reserved.
      PubDate: 2017-09-20T02:21:24.109716-05:
      DOI: 10.1111/pbi.12839
  • Three unrelated protease inhibitors enhance accumulation of pharmaceutical
           recombinant proteins in N. benthamiana

    • Abstract: Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α‐Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose‐dependent. Activity‐based Protein Profiling (ABPP) revealed that the activities of Papain‐like Cys proteases (PLCPs), Ser hydrolases (SHs) or Vacuolar Processing Enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.This article is protected by copyright. All rights reserved.
  • Use of a draft genome of coffee (Coffea arabica) to identify SNPs
           associated with caffeine content

    • Abstract: Arabica coffee (Coffea arabica) has a small gene pool limiting genetic improvement. Selection for caffeine content within this gene pool would be assisted by identification of the genes controlling this important trait. Sequencing of DNA bulks from 18 genotypes with extreme high or low caffeine content from a population of 232 genotypes was used to identify linked polymorphisms. To obtain a reference genome a whole genome assembly of arabica coffee (variety K7) was achieved by sequencing using short read (Illumina) and long read (PacBio) technology. Assembly was performed using a range of assembly tools resulting in 76,409 scaffolds with a scaffold N50 of 54,544 bp and a total scaffold length of 1,448 Mb. Validation of the genome assembly using different tools showed high completeness of the genome. More than 99% of transcriptome sequences mapped to the C. arabica draft genome and 89% of BUSCOs were present. The assembled genome annotated using AUGUSTUS yielded 99,829 gene models. Using the draft arabica genome as reference in mapping and variant calling allowed the detection of 1,444 non‐synonymous SNPs associating with caffeine content. Based on KEGG pathway‐based analysis, 65 caffeine‐associated SNPs were discovered, among which 11 SNPs were associated with genes encoding enzymes involved in the conversion of substrates which participate in the caffeine biosynthesis pathways. This analysis demonstrated the complex genetic control of this key trait in coffee.This article is protected by copyright. All rights reserved.
  • Transcriptome reprogramming due to the introduction of a barley telosome
           into bread wheat affects more barley genes than wheat

    • Abstract: Despite a long history, the production of useful alien introgression lines in wheat remains difficult mainly due to linkage drag and incomplete genetic compensation. In addition, little is known about the molecular mechanisms underlying the impact of foreign chromatin on plant phenotype. Here, a comparison of the transcriptomes of barley, wheat and a wheat barley 7HL addition line allowed the transcriptional impact both on 7HL genes of a non‐native genetic background, and on the wheat gene complement as a result of the presence of 7HL to be assessed. Some 42% (389/923) of the 7HL genes assayed were differentially transcribed, which was the case for only 3% (960/35,301) of the wheat gene complement. The absence of any transcript in the addition line of a suite of chromosome 7A genes implied the presence of a 36 Mbp deletion at the distal end of the 7AL arm; this deletion was found to be in common across the full set of Chinese Spring/Betzes barley addition lines. The remaining differentially transcribed wheat genes were distributed across the whole genome. The up‐regulated barley genes were mostly located in the proximal part of the 7HL arm, while the down‐regulated ones were concentrated in the distal part; as a result, genes encoding basal cellular functions tended to be transcribed, while those encoding specific functions were suppressed. An insight has been gained into gene transcription in an alien introgression line, thereby providing a basis for understanding the interactions between wheat and exotic genes in introgression materials.This article is protected by copyright. All rights reserved.
  • The Rice Terpene Synthase Gene OsTPS19 Functions as an (S)‐Limonene
           Synthase in planta and its Overexpression Leads to Enhanced Resistance to
           the Blast Fungus Magnaporthe oryzae

    • Abstract: Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defense mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defense gene. Here, we report the functional characterization of OsTPS19, which is upregulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)‐limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)‐limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)‐limonene synthase in rice and plays a role in defense against M. oryzae, at least partly, by inhibiting spore germination.This article is protected by copyright. All rights reserved.
  • Seed‐specific RNAi in safflower generates a superhigh oleic oil with
           extended oxidative stability

    • Abstract: Vegetable oils extracted from oilseeds are an important component of foods, but are also used in a range of high value oleochemical applications. Despite being biodegradable, non‐toxic and renewable current plant oils suffer from the presence of residual polyunsaturated fatty acids that are prone to free radical formation that limit their oxidative stability, and consequently shelf‐life and functionality. Many decades of plant breeding have been successful in raising the oleic content to ~90%, but has come at the expense of overall field performance, including poor yields. Here we engineer superhigh oleic (SHO) safflower producing a seed oil with 93% oleic generated from seed produced in multi‐site field trials spanning five generations. SHO safflower oil is the result of seed‐specific hairpin‐based RNA interference of two safflower lipid biosynthetic genes, FAD2.2 and FATB, producing seed oil containing less than 1.5% polyunsaturates and only 4% saturates but with no impact on lipid profiles of leaves and roots. Transgenic SHO events were compared to non‐GM safflower in multi‐site trial plots with a wide range of growing season conditions, which showed no evidence of impact on seed yield. The oxidative stability of the field grown SHO oil produced from various sites was 50 hours at 110°C compared to 13 hours for conventional ~80% oleic safflower oils. SHO safflower produces a uniquely stable vegetable oil across different field conditions that can provide the scale of production that is required for meeting the global demands for high stability oils in food and the oleochemical industry.This article is protected by copyright. All rights reserved.
  • Bt rice could provide ecological resistance against non‐target

    • Abstract: Genetically engineered (GE) rice lines expressing Lepidoptera‐active insecticidal cry genes from the bacterium Bacillus thuringiensis (Bt) have been developed in China. Field surveys indicated that Bt rice harbours fewer rice planthoppers than non‐Bt rice although planthoppers are not sensitive to the produced Bt Cry proteins. The mechanisms underlying this phenomenon remain unknown. Here we show that the low numbers of planthoppers on Bt rice is associated with reduced caterpillar damage. In laboratory and field‐cage experiments, the rice planthopper Nilapavata lugens had no feeding preference for undamaged Bt or non‐Bt plants but exhibited a strong preference for caterpillar‐damaged plants whether Bt or non‐Bt. Under open field conditions, rice planthoppers were more abundant on caterpillar‐damaged non‐Bt rice than on neighbouring healthy Bt rice. GC‐MS analyses showed that caterpillar‐damage induced the release of rice plant volatiles known to be attractive to planthoppers, and metabolome analyses revealed increased amino acid contents and reduced sterol contents known to benefit planthopper development. That Lepidoptera‐resistant Bt rice is less attractive to this important non‐target pest in the field is therefore a first example of ecological resistance of Bt plants to non‐target pests. Our findings suggest that non‐Bt rice refuges established for delaying the development of Bt resistance may also act as a trap crop for N. lugens and possibly other planthoppers.This article is protected by copyright. All rights reserved.
  • Characterization of the hypersensitive response‐like cell death
           phenomenon induced by targeting antiviral lectin Griffithsin to the
           secretory pathway

    • Abstract: Griffithsin (GRFT) is an antiviral lectin, originally derived from a red alga, which is currently being investigated as a topical microbicide to prevent transmission of human immunodeficiency virus (HIV). Targeting GRFT to the apoplast for production in Nicotiana benthamiana resulted in necrotic symptoms associated with a hypersensitive response (HR)‐like cell death, accompanied by H2O2 generation and increased PR1 expression. Mannose‐binding lectins Surfactant protein D (SP‐D), cyanovirin‐N (CV‐N) and human mannose‐binding lectin (hMBL) also induce salicylic acid (SA) dependent HR‐like cell death in N. benthamiana, and this effect is mediated by the lectin's glycan binding activity. We found that secreted GRFT interacts with an endogenous glycoprotein, α‐xylosidase (XYL1), which is involved in cell wall organization. The necrotic effect could be mitigated by overexpression of Arabidopsis XYL1, and by co‐expression of SA‐degrading enzyme NahG, providing strategies for enhancing expression of oligomannose‐binding lectins in plants.This article is protected by copyright. All rights reserved.
  • Upregulation of GhTT2‐3A in Cotton Fibers during Secondary Wall
           Thickening Results in Brown Fibers with Improved Quality

    • Abstract: Brown cotton fibers are the most widely used naturally colored raw materials for the eco‐friendly textile industry. Previous studies have indicated that brown fiber pigments belong to proanthocyanidins (PAs) or their derivatives, and fiber coloration is negatively associated with cotton productivity and fiber quality. To date, the molecular basis controlling the biosynthesis and accumulation of brown pigments in cotton fibers is largely unknown. In the present study, based on expressional and transgenic analyses of cotton homologs of Arabidopsis PA regulator TRANSPARENT TESTA 2 (TT2) and fine mapping of the cotton dark brown fiber gene (Lc1), we show that a TT2 homolog, GhTT2‐3A, controls PA biosynthesis and brown pigmentation in cotton fibers. We observed that GhTT2‐3A activated GhbHLH130D, a homolog of Arabidopsis TT8, which in turn synergistically acted with GhTT2‐3A to activate downstream PA structural genes and PA synthesis and accumulation in cotton fibers. Furthermore, the upregulation of GhTT2‐3A in fibers at the secondary wall‐thickening stage resulted in brown mature fibers, and fiber quality and lint percentage were comparable to that of the white‐fiber control. The findings of the present study reveal the regulatory mechanism controlling brown pigmentation in cotton fibers and demonstrate a promising biotechnological strategy to break the negative linkage between coloration and fiber quality and/or productivity.This article is protected by copyright. All rights reserved.
  • Combining mutations at genes encoding key enzymes involved in starch
           synthesis affects the amylose content, carbohydrate allocation and
           hardness in the wheat grain

    • Abstract: Modifications to the composition of starch, the major component of wheat flour, can have a profound effect on the nutritional and technological characteristics of the flour's end‐products. The starch synthesized in the grain of conventional wheats (Triticum aestivum) is a 3:1 mixture of the two polysaccharides amylopectin and amylose. Altering the activity of certain key starch synthesis enzymes (GBSSI, SSIIa and SBEIIa) has succeeded in generating starches containing a different polysaccharide ratio. Here, mutagenesis, followed by a conventional marker‐assisted breeding exercise, has been used to generate three mutant lines which produce starch with an amylose contents of 0%, 46% and 79%. The direct and pleiotropic effects of the multiple mutation lines were identified at both the biochemical and molecular levels. Both the structure and composition of the starch were materially altered, changes which affected the functionality of the starch. An analysis of sugar and non‐starch polysaccharide content in the endosperm suggested an impact of the mutations on the carbon allocation process, suggesting the existence of cross‐talk between the starch and carbohydrate synthesis pathways.This article is protected by copyright. All rights reserved.
  • Trans‐species synthetic gene design allows resistance pyramiding and
           broad‐spectrum engineering of virus resistance in plants

    • Abstract: To infect plants, viruses rely heavily on their host's machinery. Plant genetic resistances based on host factor modifications can be found among existing natural variability and are widely used for some but not all crops. While biotechnology can supply for the lack of natural resistance alleles, new strategies need to be developed to increase resistance spectra and durability without impairing plant development. Here, we assess how the targeted allele modification of the Arabidopsis thaliana translation initiation factor eIF4E1 can lead to broad and efficient resistance to the major group of potyviruses. A synthetic Arabidopsis thaliana eIF4E1 allele was designed by introducing multiple amino acid changes associated with resistance to potyvirus in naturally occurring Pisum sativum alleles. This new allele encodes a functional protein while maintaining plant resistance to a potyvirus isolate that usually hijacks eIF4E1. Due to its biological functionality, this synthetic allele allows, at no developmental cost, the pyramiding of resistances to potyviruses that selectively use the two major translation initiation factors, eIF4E1 or its isoform eIFiso4E. Moreover, this combination extends the resistance spectrum to potyvirus isolates for which no efficient resistance has so far been found, including resistance‐breaking isolates and an unrelated virus belonging to the Luteoviridae family. This study is a proof‐of‐concept for the efficiency of gene engineering combined with knowledge of natural variation to generate trans‐species virus resistance at no developmental cost to the plant. This has implications for breeding of crops with broad‐spectrum and high durability resistance using recent genome editing techniques.
  • An oligosaccharyltransferase from Leishmania major increases the
           N‐glycan occupancy on recombinant glycoproteins produced in Nicotiana

    • Abstract: N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central protein‐complex facilitating the N‐glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N‐glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single‐subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well‐established production platform for recombinant proteins. A fluorescent protein‐tagged LmSTT3D variant was predominately found in the ER and co‐located with plant oligosaccharyltransferase subunits. Co‐expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N‐glycosylation site occupancy on all N‐glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N‐glycosylation efficiency in plants.This article is protected by copyright. All rights reserved.
  • Blocking Amino acid transporter OsAAP3 improves grain yield by promoting
           outgrowth buds and increasing tiller number in rice

    • Abstract: Amino acid transporters (AATs) play indispensable roles in nutrient allocation during plant development. In this study, we demonstrated that inhibiting expression of the rice amino acid transporter OsAAP3 increased grain yield due to a formation of larger numbers of tillers as a result of increased bud outgrowth. Elevated expression of OsAAP3 in transgenic plants resulted in significantly higher amino acid concentrations of Lys, Arg, His, Asp, Ala, Gln, Gly, Thr and Tyr, and inhibited bud outgrowth and rice tillering. However, RNAi of OsAAP3 decreased significantly Arg, Lys, Asp, and Thr concentrations to a small extent, and thus promoted bud outgrowth, increased significantly tiller numbers and effective panicle numbers per plant, and further enhanced significantly grain yield and nitrogen use efficiency (NUE). The promoter sequences of OsAAP3 showed some divergence between Japonica and Indica rice, and expression of the gene was higher in Japonica, which produced fewer tillers than Indica. We generated knockout‐lines of OsAAP3 on Japonica ZH11 and KY131 using CRISPR technology and found that grain yield could be increased significantly. These results suggest that manipulation of OsAAP3 expression could be used to increase grain yield in rice.This article is protected by copyright. All rights reserved.
  • Synthetic biology approach for plant protection using dsRNA

    • Abstract: Pathogens induce severe damages on cultivated plants and represent a serious threat to global food security. Emerging strategies for crop protection involve the external treatment of plants with double‐stranded (ds)RNA to trigger RNA interference. However, applying this technology in greenhouses and fields depends on dsRNA quality, stability, and efficient large‐scale production. Using components of the bacteriophage phi6, we engineered a stable and accurate in vivo dsRNA production system in Pseudomonas syringae bacteria. Unlike other in vitro or in vivo dsRNA‐production systems that rely on DNA transcription and post‐synthetic alignment of single‐stranded RNA molecules, the phi6 system is based on the replication of dsRNA by an RNA‐dependent RNA polymerase, thus allowing production of high‐quality, long dsRNA molecules. The phi6 replication complex was reprogrammed to multiply dsRNA sequences homologous to tobacco mosaic virus (TMV) by replacing the coding regions within two of the three phi6 genome segments with TMV sequences and introduction of these constructs into P. syringae together with the third phi6 segment, which encodes the components of the phi6 replication complex. The stable production of TMV dsRNA was achieved by combining all the three phi6 genome segments and by maintaining the natural dsRNA sizes and sequence elements required for efficient replication and packaging of the segments. The produced TMV‐derived dsRNAs inhibited TMV propagation when applied to infected Nicotiana benthamiana plants. The established dsRNA production system enables the broad application of dsRNA molecules as an efficient, highly flexible, non‐transgenic and environmentally friendly approach for protecting crops against viruses and other pathogens.This article is protected by copyright. All rights reserved.
  • Engineering rice with lower grain arsenic

    • Abstract: Arsenic (As) is a poisonous element that causes severe skin lesions and cancer in humans. Rice (Oryza sativa L.) is a major dietary source of As in humans who consume this cereal as a staple food. We hypothesized that increasing As vacuolar sequestration would inhibit its translocation into the grain and reduce the amount of As entering the food chain. We developed transgenic rice plants expressing two different vacuolar As sequestration genes, ScYCF1 and OsABCC1, under the control of the RCc3 promoter in the root cortical and internode phloem cells, along with a bacterial γ‐glutamylcysteine synthetase driven by the maize UBI promoter. The transgenic rice plants exhibited reduced root‐to‐shoot and internode‐to‐grain As translocation, resulting in a 70% reduction in As accumulation in the brown rice without jeopardizing agronomic traits. This technology could be used to reduce As intake, particularly in populations of South East Asia suffering from As toxicity, and thereby improve human health.This article is protected by copyright. All rights reserved.
  • Alternative splicing of OsLG3b controls grain length and yield in
           japonica rice

    • Abstract: Grain size, one of the important components determining grain yield in rice, is controlled by the multiple quantitative trait loci (QTLs). Intensive artificial selection for grain size during domestication is evidenced in modern cultivars compared to their wild relatives. Here, we report the molecular cloning and characterization of OsLG3b, a QTL for grain length in tropical japonica rice that encodes MADS‐box transcription factor 1 (OsMADS1). Six SNPs in the OsLG3b region led to alternative splicing, which were associated with grain length in an association analysis of candidate region. Quantitative PCR analysis indicated that OsLG3b expression was higher during the panicle and seed development stages. Analysis of haplotypes and introgression regions revealed that the long‐grain allele of OsLG3b might have arisen after domestication of tropical japonica and spread to subspecies indica or temperate japonica by natural crossing and artificial selection. OsLG3b is therefore a target of human selection for adaptation to tropical regions during domestication and/or improvement of rice. Phylogenetic analysis and pedigree records showed that OsLG3b had been employed by breeders, but the gene still has much breeding potential for increasing grain length in indica. These findings will not only aid efforts to elucidate the molecular basis of grain development and domestication, but also facilitate the genetic improvement of rice yield.This article is protected by copyright. All rights reserved.
  • Linkage and association mapping reveals the genetic basis of brown fibre
           (Gossypium hirsutum)

    • Abstract: Brown fibre cotton is an environmental‐friendly resource that plays a key role in the textile industry. However, the fibre quality and yield of natural brown cotton are poor, and fundamental research on brown cotton is relatively scarce. To understand the genetic basis of brown fibre cotton, we constructed linkage and association populations to systematically examine brown fibre accessions. We fine‐mapped the brown fibre region, Lc1, and dissected it into two loci, qBF‐A07‐1 and qBF‐A07‐2. The qBF‐A07‐1 locus mediates the initiation of brown fibre production, whereas the shade of the brown fibre is affected by the interaction between qBF‐A07‐1 and qBF‐A07‐2. Gh_A07G2341 and Gh_A07G0100 were identified as candidate genes for qBF‐A07‐1 and qBF‐A07‐2, respectively. Haploid analysis of the signals significantly associated with these two loci showed that most tetraploid modern brown cotton accessions exhibit the introgression signature of Gossypium barbadense. We identified 10 quantitative trait loci (QTLs) for fibre yield and 19 QTLs for fibre quality through a genome‐wide association study (GWAS) and found that qBF‐A07‐2 negatively affects fibre yield and quality through an epistatic interaction with qBF‐A07‐1. This study sheds light on the genetics of fibre colour and lint‐related traits in brown fibre cotton, which will guide the elite cultivars breeding of brown fibre cotton.This article is protected by copyright. All rights reserved.
  • Induction and recovery of copy number variation in banana through gamma
           irradiation and low coverage whole genome sequencing

    • Abstract: Traditional breeding methods are hindered in bananas due to the fact that major cultivars are sterile, parthenocarpic, triploid, and thus clonally propagated. This has resulted in a narrow genetic base and limited resilience to biotic and abiotic stresses. Mutagenesis of in vitro propagated bananas is one method to introduce novel alleles and broaden genetic diversity. We previously established a method for the induction and recovery of single nucleotide mutations generated with the chemical mutagen EMS. However, officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic insertions and deletions (indels). Such dosage mutations may be important for generating observable phenotypes in polyploids. In this study we establish a low coverage whole genome sequencing approach in triploid bananas to recover large genomic indels caused by treatment with gamma irradiation. We first evaluated the commercially released mutant cultivar ‘Novaria’ and found that it harbors multiple predicted deletions, ranging from 0.3 to 3.8 million base pairs (Mbp). In total, predicted deleted regions span 189 coding regions. To evaluate the feasibility of generating and maintaining new mutations, we developed a pipeline for mutagenesis and screening for copy number variation in Cavendish bananas using the cultivar ‘Williams’. Putative mutations were recovered in 70% of lines treated with 20 Gy and 60% of the lines treated with 40 Gy. While deletion events predominate, insertions were identified in 20 Gy treated material. Based on these results, we believe this approach can be scaled up to support large breeding projects.This article is protected by copyright. All rights reserved.
  • Issue Information

  • Deciphering drought‐induced metabolic responses and regulation in
           developing maize kernels

    • Abstract: Drought stress conditions decrease maize growth and yield, and aggravate pre‐harvest aflatoxin contamination. While several studies have been performed on mature kernels responding to drought stress, the metabolic profiles of developing kernels are not as well characterized, particularly in germplasm with contrasting resistance to both drought and mycotoxin contamination. Here, following screening for drought tolerance, a drought‐sensitive line, B73, and a drought‐tolerant line, Lo964, were selected and stressed beginning at 14 days after pollination. Developing kernels were sampled 7 and 14 days after drought induction (DAI) from both stressed and irrigated plants. Comparative biochemical and metabolomic analyses profiled 409 differentially accumulated metabolites. Multivariate statistics and pathway analyses showed that drought stress induced an accumulation of simple sugars and polyunsaturated fatty acids and a decrease in amines, polyamines, and dipeptides in B73. Conversely, sphingolipid, sterol, phenylpropanoid, and dipeptide metabolites accumulated in Lo964 under drought stress. Drought stress also resulted in the greater accumulation of reactive oxygen species (ROS) and aflatoxin in kernels of B73 in comparison to Lo964 implying a correlation in their production. Overall, field drought treatments disordered a cascade of normal metabolic programming during development of maize kernels and subsequently caused oxidative stress. The glutathione and urea cycles along with the metabolism of carbohydrates and lipids for osmoprotection, membrane maintenance, and antioxidant protection were central among the drought stress responses observed in developing kernels. These results also provide novel targets to enhance host drought tolerance and disease resistance through the use of biotechnologies such as transgenics and genome editing.This article is protected by copyright. All rights reserved.
  • Host‐induced gene silencing of a regulator of G protein signaling gene
           (VdRGS1) confers resistance to Verticillium wilt in cotton

    • Abstract: Verticillium wilt (VW), caused by soil‐borne fungi of the genus Verticillium, is a serious disease affecting a wide range of plants and leading to a constant and major challenge to agriculture worldwide. Cotton (Gossypium hirsutum) is the world's most important natural textile fiber and oil crop. VW of cotton is a highly devastating vascular disease, however, few resistant germplasms have been reported in cotton. An increasing number of studies have shown that RNA interference (RNAi) based host‐induced gene silencing (HIGS) is an effective strategy for improving plant resistance to pathogens by silencing genes essential for the pathogenicity of these pathogens. Here, we have identified and characterized multifunctional regulators of G protein signaling (RGS) in the Verticillium dahliae virulence strain, Vd8. Of eight VdRGS genes, VdRGS1 showed the most significant increase in expression in V. dahliae after treating with the roots of cotton seedlings. Based on the phenotype detection of VdRGS1 deletion and complementation mutants, we found that VdRGS1 played crucial roles in spore production, hyphal development, microsclerotia formation, and pathogenicity. Tobacco rattle virus (TRV)‐mediated HIGS in cotton plants silenced VdRGS1 transcripts in invaded V. dahliae strains, and enhanced broad spectrum resistance to cotton Verticillium wilt. Our data demonstrate that VdRGS1 is a conserved and essential gene for V. dahliae virulence. HIGS of VdRGS1 provide effective control against V. dahliae infection and could obtain the durable disease resistance in cotton, and in other Verticillium wilt‐susceptible host crops by developing the stable transformants.This article is protected by copyright. All rights reserved.
  • Transcriptome‐wide comparison of selenium hyperaccumulator and
           non‐accumulator Stanleya species provides new insight into key processes
           mediating the hyperaccumulation syndrome

    • Abstract: To obtain better insight into the mechanisms of selenium hyperaccumulation in Stanleya pinnata, transcriptome‐wide differences in root and shoot gene expression levels were investigated in S. pinnata and related nonaccumulator Stanleya elata grown with or without 20 μM selenate. Genes predicted to be involved in sulfate/selenate transport and assimilation or in oxidative stress resistance (glutathione‐related genes and peroxidases) were among the most differentially expressed between species; many showed constitutively elevated expression in S. pinnata. A number of defense‐related genes predicted to mediate synthesis and signaling of defense hormones jasmonic acid (JA, reported to induce sulfur assimilatory and glutathione biosynthesis genes), salicylic acid (SA) and ethylene were also more expressed in S. pinnata than S. elata. Several upstream signaling genes that upregulate defense hormone synthesis showed higher expression in S. pinnata than S. elata and might trigger these selenium‐mediated defense responses. Thus, selenium hyperaccumulation and hypertolerance in S. pinnata may be mediated by constitutive, upregulated JA, SA and ethylene‐mediated defense systems, associated with elevated expression of genes involved in sulfate/selenate uptake and assimilation or in antioxidant activity. Genes pinpointed in this study may be targets of genetic engineering of plants that may be employed in biofortification or phytoremediation.This article is protected by copyright. All rights reserved.
  • QTL mapping and GWAS reveal candidate genes controlling capsaicinoid
           content in Capsicum

    • Abstract: Capsaicinoids are unique compounds produced only in peppers (Capsicum spp.). Several studies using classical quantitative trait loci (QTL) mapping and genome‐wide association studies (GWAS) have identified QTLs controlling capsaicinoid content in peppers; however, neither the QTLs common to each population nor the candidate genes underlying them have been identified due to the limitations of each approach used. Here, we performed QTL mapping and GWAS for capsaicinoid content in peppers using two recombinant inbred line (RIL) populations and one GWAS population. Whole‐genome resequencing and genotyping‐by‐sequencing (GBS) were used to construct high‐density single‐nucleotide polymorphism (SNP) maps. Five QTL regions on chromosomes 1, 2, 3, 4, and 10 were commonly identified in both RIL populations over multiple locations and years. Furthermore, a total of 109,610 SNPs derived from two GBS libraries were used to analyze the GWAS population consisting of 208 C. annuum‐clade accessions. A total of 69 QTL regions were identified from the GWAS, 10 of which were co‐located with the QTLs identified from the two biparental populations. Within these regions, we were able to identify five candidate genes known to be involved in capsaicinoid biosynthesis. Our results demonstrate that QTL mapping and GBS‐GWAS represent a powerful combined approach for the identification of loci controlling complex traits.This article is protected by copyright. All rights reserved.
  • Allelic variation for broad‐spectrum resistance and susceptibility to
           bacterial pathogens identified in a rice MAGIC population

    • Abstract: Quantitative trait loci (QTL) that confer broad‐spectrum resistance (BSR), or resistance that is effective against multiple and diverse plant pathogens, have been elusive targets of crop breeding programs. Multi‐parent Advanced Generation Inter‐Cross (MAGIC) populations, with their diverse genetic composition and high levels of recombination, are potential resources for identification of QTL for BSR. In this study, a rice MAGIC population was used to map QTL conferring BSR to two major rice diseases, bacterial leaf streak (BLS) and bacterial blight (BB), caused by Xanthomonas oryzae pathovars (pv.) oryzicola (Xoc) and oryzae (Xoo), respectively. Controlling these diseases is particularly important in Sub‐Saharan Africa, where no sources of BSR are currently available in deployed varieties. The MAGIC founders and lines were genotyped by sequencing and phenotyped in the greenhouse and field by inoculation with multiple strains of Xoc and Xoo. A combination of genome‐wide association studies (GWAS) and interval mapping analyses revealed 11 BSR QTL, effective against both diseases, and three pathovar‐specific QTL. The most promising BSR QTL (qXO‐2‐1, qXO‐4‐1 and qXO‐11‐2) conferred resistance to more than nine Xoc and Xoo strains. GWAS detected 369 significant SNP markers with distinguishable phenotypic effects, allowing the identification of alleles conferring disease resistance and susceptibility. The BSR and susceptibility QTL will improve our understanding of the mechanisms of both resistance and susceptibility in the long term, and will be immediately useful resources for rice breeding programs.This article is protected by copyright. All rights reserved.
  • PeCHYR1, a ubiquitin E3 ligase from Populus euphratica, enhances drought

    • Abstract: Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba×Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H2O2 production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild‐type) controls. Moreover, up‐regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA‐induced stomatal closure caused by hydrogen peroxide (H2O2) production in transgenic poplar plants.This article is protected by copyright. All rights reserved.
  • WRKY1 acts as a key component improving resistance against Alternaria
           solani in wild tomato, Solanum arcanum Peralta

    • Abstract: Early blight (EB), caused by Alternaria solani, is a major threat to global tomato production. In comparison to cultivated tomato (Solanum lycopersicum), a wild relative, S. arcanum exhibits strong resistance against EB. However, molecular cascades operating during EB resistance in wild or cultivated tomato plants are largely obscure. Here, we provide novel insight on spatio‐temporal molecular events in S. arcanum against A. solani. Transcriptome and co‐expression analysis presented 33‐WRKYs as promising candidates of which 12 SaWRKYs displayed differential expression patterns in resistant and susceptible accessions during EB disease progression. Among these, SaWRKY1 exhibited induced expression with significant modulation in xyloglucan endotrans hydrolase 5 (XTH5) and MYB2 expressions that correlated with disease phenotypes. Electro‐mobility shift assay confirmed physical interaction of recombinant SaWRKY1 to SaXTH5 and SaMYB2 promoters. Comparative WRKY1 promoter analysis between resistant and susceptible plants revealed presence of crucial motifs for defence mechanism exclusively in resistant accession. Additionally, many defence related genes displayed significant expression variations in both the accessions. Further, WRKY1 overexpressing transgenic plants exhibited higher levels of EB resistance while RNAi silencing lines had increased susceptibility to A. solani with altered expression of XTH5 and MYB2. Overall, these findings demonstrate the positive influence of WRKY1 in improving EB resistance in wild tomato and this could be further utilized as a potential target through genetic engineering to augment protection against A. solani in crop plants.This article is protected by copyright. All rights reserved.
  • Grapevine VpPR10.1 functions in resistance to Plasmopara viticola through
           triggering a cell death‐like defense response by interacting with

    • Abstract: As one of the most serious diseases in grape, downy mildew caused by Plasmopara viticola is a worldwide grape disease. Much effort has been focused on improving susceptible grapevine’ resistance and wild resistant grapevine species are important for germplasm improvement of commercial cultivars. Using yeast two‐hybrid screen followed by a series of immunoprecipitation experiments, we identified Voltage‐dependent Anion Channel 3 (VDAC3) protein from Vitis piasezkii ‘Liuba‐8′ as an interacting partner of VpPR10.1 cloned from Vitis pseudoreticulata ‘Baihe‐35‐1′, which is an important germplasm for its resistance to a range of pathogens. Co‐expression of VpPR10.1/VpVDAC3 induced cell death in Nicotiana benthamiana which accompanied with ROS accumulation. VpPR10.1 transgenic grapevine line showed resistance to Plasmopara viticola. We conclude that the VpPR10.1/VpVDAC3 complex is responsible for cell death‐mediated defense response to P. viticola in grapevine.This article is protected by copyright. All rights reserved.
  • Rice copine genes OsBON1 and OsBON3 function as suppressors of
           broad‐spectrum disease resistance

    • Abstract: Breeding for disease resistance is the most effective strategy to control diseases, particularly with broad‐spectrum disease resistance in many crops. However, knowledge on genes and mechanism of broad‐spectrum resistance and tradeoff between defense and growth in crops is limited. Here, we show that the rice copine genes OsBON1 and OsBON3 are critical suppressors of immunity. Both OsBON1 and OsBON3 changed their protein subcellular localization upon pathogen challenge. Knockdown of OsBON1 and dominant negative mutant of OsBON3 enhanced resistance to rice bacterial and fungal pathogens with either hemibiotrophic or necrotrophic life styles. The defense activation in OsBON1 knockdown mutants was associated with reduced growth, both of which were largely suppressed under high temperature. In contrast, overexpression of OsBON1 or OsBON3 decreased disease resistance and promoted plant growth. However, neither OsBON1 nor OsBON3 could rescue the dwarf phenotype of the Arabidopsis BON1 knockout mutant, suggesting an evolutionary divergence of the rice and Arabidopsis copine genes. Our study therefore shows that the rice copine genes play a negative role in regulating disease resistance and their expression level and protein location likely have a large impact on the balance between immunity and agronomic traits.This article is protected by copyright. All rights reserved.
  • Beyond pathways: genetic dissection of tocopherol content in maize kernels
           by combining linkage and association analyses

    • Abstract: Although tocopherols play an important role in plants and animals, the genetic architecture of tocopherol content in maize kernels has remained largely unknown. In this study, linkage and association analyses were conducted to examine the genetic architecture of tocopherol content in maize kernels. Forty‐one unique quantitative trait loci (QTLs) were identified by linkage mapping in six populations of recombinant inbred lines (RILs). In addition, 32 significant loci were detected via genome‐wide association study (GWAS), 18 of which co‐localized with the QTLs identified by linkage mapping. Fine‐mapping of a major QTL validated the accuracy of GWAS and QTL mapping results and suggested a role for non‐tocopherol pathway genes in the modulation of natural tocopherol variation. We provided genome‐wide evidence that genes involved in fatty acid metabolism, chlorophyll metabolism and chloroplast function may affect natural variation in tocopherols. These findings were confirmed through mutant analysis of a particular gene from the fatty acid pathway. In addition, the favourable alleles for many of the significant SNPs/QTLs represented rare alleles in natural populations. Together, our results revealed many novel genes that are potentially involved in the variation of tocopherol content in maize kernels. Pyramiding of the favourable alleles of the newly elucidated genes and well‐known tocopherol pathway genes would greatly improve tocopherol content in maize.This article is protected by copyright. All rights reserved.
  • Haplotype based genotyping‐by‐sequencing in oat genome

    • Abstract: In a de‐novo genotyping‐by‐sequencing (GBS) analysis of short, 64‐base tag‐level haplotypes in 4657 accessions of cultivated oat, we discovered 164741 tag‐level (TL) genetic variants containing 241224 SNPs. From this, the marker‐density of an oat consensus map was increased by the addition of more than 70000 loci. The mapped TL genotypes of a 635‐line diversity panel were used to infer chromosome‐level (CL) haplotype maps. These maps revealed differences in the number and size of haplotype blocks, as well as differences in haplotype diversity between chromosomes and subsets of the diversity panel. We then explored potential benefits of SNP vs. TL vs. CL GBS variants for mapping, high resolution genome analysis, and genomic selection in oats. A combined genome‐wide association study (GWAS) of heading date from multiple locations using both TL haplotypes and individual SNP markers identified 184 significant associations. A comparative GWAS using TL haplotypes, CL haplotype blocks, and their combinations demonstrated the superiority of using TL haplotype markers. Using a principal‐component‐based genome‐wide scan, genomic regions containing signatures of selection were identified. These regions may contain genes that are responsible for the local adaptation of oats to Northern American conditions. Genomic selection for heading date using TL haplotypes or SNP markers gave comparable and promising prediction accuracies of up to r=0.74. Genomic selection carried out in an independent calibration and test population for heading date gave promising prediction accuracies that ranged between r=0.42 and 0.67. In conclusion, TL haplotype GBS‐derived markers facilitate genome analysis and genomic selection in oat.This article is protected by copyright. All rights reserved.
  • Modulates Brassinosteroids‐mediated Growth Regulation and Influences
           Architecture and Grain Shape

    • Abstract: Rice (Oryza sativa) is the most important crop and feeds more than half of the world population. The architecture of rice, including height, leaf inclination, and tiller number, is important for rice planting and yield. Plant hormone, brassinosteroids (BR), plays crucial roles in modulating plant architecture and seed yield. But BR cannot be applied in agriculture production directly because BR regulated multiple processes and could not be transcriptional regulated in different tissues.This article is protected by copyright. All rights reserved.
  • Engineering of “Purple Embryo Maize” with a multigene expression
           system derived from a bidirectional promoter and self‐cleaving 2A

    • Abstract: Anthocyanins are polyhydroxy and polymethoxy 2‐phenylbenzopyrylium glucosides that belong to a large group of plant secondary metabolites termed flavonoids. The most common anthocyanidins (anthocyanin aglycones) in higher plants are cyanidin, pelargonidin, peonidin, delphinidin, petunidin and malvidin. Flavonoid biosynthesis has been suggested to occur on the cytoplasmic face of the endoplasmic reticulum (ER), where sequential enzymes of the pathway loosely form a metabolon (Winkel‐Shirley, 1999). Known structural genes along the anthocyanin biosynthesis pathway in maize include chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3‐hydroxylase (F3H), flavonoid 3′‐hydroxylase (F3′H), dihydroflavonol 4‐reductase (DFR), anthocyanidin synthase (ANS), flavonoid 3‐O‐glucosyltransferase (3UFGT) and glutathione S‐transferase (GST).This article is protected by copyright. All rights reserved.
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