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  • Guard cells integrate light and temperature signals to control stomatal aperture
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-16
    Kalliopi-Ioanna Kostaki, Aude Coupel-Ledru, Verity C Bonnell, Mathilda Gustavsson, Peng Sun, Fiona J Mclaughlin, Donald P Fraser, Deirdre H McLachlan, Alistair M. Hetherington, Antony N Dodd, Keara A. Franklin

    High temperature promotes guard cell expansion, which opens stomatal pores to facilitate leaf cooling. How the high temperature signal is perceived and transmitted to regulate stomatal aperture is, however, unknown. Here, we used a reverse-genetics approach to understand high temperature-mediated stomatal opening in Arabidopsis thaliana. Our findings reveal that high temperature-induced guard cell movement requires components involved in blue light-mediated stomatal opening, suggesting crosstalk between light and temperature signalling pathways. The molecular players involved include phototropin photoreceptors, plasma membrane H+-ATPases, and multiple members of the 14-3-3 protein family. We further show that phototropin-deficient mutants display impaired rosette evapotranspiration and leaf cooling at high temperatures. Blocking the interaction of 14-3-3 proteins with their client proteins severely impairs high temperature-induced stomatal opening, but has no effect on the induction of heat-sensitive guard cell transcripts, supporting the existence of an additional intracellular high-temperature response pathway in plants.

    更新日期:2020-01-17
  • Target of Rapamycin Signaling in Plant Stress Responses
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-16
    Liwen Fu, Pengcheng Wang, Yan Xiong

    Target of Rapamycin (TOR) is an atypical serine/threonine protein kinase that is evolutionally conserved among yeasts, plants, and mammals. In plants, TOR signaling functions as a central hub to integrate different kinds of nutrient, energy, hormone, and environmental signals. TOR thereby orchestrates every stage of plant life, from embryogenesis, meristem activation, root and leaf growth to flowering, senescence, and life span determination. Besides its essential role in the control of plant growth and development, recent research has also shed light on its multifaceted roles in plant environmental stress responses. Here, we review recent findings on the involvement of TOR signaling in plant adaptation to nutrient deficiency and various abiotic stresses. We also discuss the mechanisms underlying how plants cope with such unfavorable conditions via TOR-ABA crosstalk and TOR-mediated autophagy, both of which play crucial roles in plant stress responses. Until now, little was known about the upstream regulators and downstream effectors of TOR in plant stress responses. We propose that the SnRKs-TOR axis plays a role in sensing various stress signals, and predict the key downstream effectors based on recent high-throughput proteomic analyses.

    更新日期:2020-01-17
  • MicroRNA397b-LACCASE2 module regulates root lignification under water- and phosphate deficiency
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-16
    Hitaishi Khandal, Amar Pal Singh, Debasis Chattopadhyay

    Deficiency of water and phosphate induce lignin deposition in roots. LACCASEs, a family of cell wall-localized multi-copper oxidases, are involved in lignin biosynthesis. We demonstrate here that LACCASE2 (LAC2) acts as a negative regulator of lignin deposition in root vascular tissues during water deficit. An Arabidopsis T-DNA insertion mutant of LAC2 displayed a short primary root and high lignin deposition in root vascular tissues. However, restoration of LAC2 expression rescued these phenotypes. LAC2 expression was significantly downregulated under water deficit and post-transcriptionally regulated by microRNA397b (miR397b) in roots under normal and water deficit conditions. Downregulation of miR397b activity increased LAC2 expression and root length, and decreased lignin content in root vasculature. Similarly, phosphate (Pi) deficiency inversely affected miR397b and LAC2 expression. Lignin deposition in the root elongation zone under Pi-limited conditions was dependent on LAC2 expression. Localized iron accumulation and callose deposition in the root elongation zone under Pi-deficiency increased with LAC2-dependent lignification, suggesting a direct relationship between these processes. Our study reveals a regulatory role for the miR397b-LAC2 module in root lignification during water- and phosphate deficiency.

    更新日期:2020-01-17
  • CER16 inhibits post-transcriptional gene silencing of CER3 to regulate alkane biosynthesis
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-15
    Xianpeng Yang, Tao Feng, Shipeng LI, Huayan Zhao, Shuangshuang Zhao, Changle Ma, Matthew A Jenks, Shiyou Lu

    The aerial surfaces of land plants have a protective layer of cuticular wax. Alkanes are common components of these waxes, and their abundance is affected by a range of stresses. The CER16 protein has been implicated in alkane biosynthesis in the cuticular wax of Arabidopsis. Here, we identified two new mutant alleles of CER16 in Arabidopsis resulting in production of less wax with dramatically fewer alkanes than the wild type. Map-based cloning with genetic analysis revealed that the cer16 phenotype was caused by complete loss of AT5G44150, encoding a protein with no known domains or motifs. Comparative transcriptomic analysis revealed that transcripts of CER3, previously shown to play a principal role in alkane production, were markedly reduced in the cer16 mutants. To define the relationship between CER3 and CER16, we transformed the full CER3 gene into a cer16 mutant. Transgenic CER3 expression was silenced, and levels of small interfering RNAs targeting CER3 were significantly increased. Mutating two major components of the RNA-silencing machinery in a cer16 genetic background restored CER3 transcript levels to wild-type levels, with the stems restored to wild-type glaucousness. We suggest that CER16 deficiency induces post-transcriptional gene silencing of both endogenous and exogenous expression of CER3.

    更新日期:2020-01-16
  • Autophagy Increases Zinc Bioavailability to Avoid Light-Mediated ROS Production under Zn Deficiency
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-15
    Daiki Shinozaki, Ekaterina A Merkulova, Loreto Naya, Tetsuro Horie, Yuri Kanno, Mitsunori Seo, Yoshinori Ohsumi, Céline Masclaux Daubresse, Kohki Yoshimoto

    Zinc (Zn) is an essential micronutrient for plant growth. Accordingly, Zn deficiency in agricultural fields is a serious problem, especially in developing regions. Autophagy, a major intracellular degradation system in eukaryotes, plays important roles in nutrient recycling under nitrogen and carbon starvation. However, the relationship between autophagy and deficiencies of other essential elements remains poorly understood, especially in plants. In this study, we focused on Zn due to the property that within cells most Zn is tightly bound to proteins, which can be targets of autophagy. We found that autophagy plays a critical role during Zn deficiency in Arabidopsis thaliana. Autophagy-defective plants (atg mutants) failed to grow and developed accelerated chlorosis under Zn starvation. As expected, a shortage of Zn induced autophagy in wild-type plants, whereas in atg mutants, various organelle proteins accumulated to high levels. Additionally, the amount of free Zn2+ was lower in atg mutants than in control plants. Interestingly, Zn-deficiency symptoms in atg mutants recovered under low-light, iron-limited conditions. The levels of hydroxyl radicals in chloroplasts were elevated, and the levels of superoxide were reduced in Zn-deficient atg mutants. These results imply that the photosynthesis-mediated Fenton-like reaction, which is responsible for the chlorotic symptom of Zn deficiency, is accelerated in atg mutants. Together, our data indicate that autophagic degradation plays important functions in maintaining Zn pools to increase Zn bioavailability and maintain ROS homeostasis under Zn starvation in plants.

    更新日期:2020-01-15
  • The Arabidopsis Protein CGL20 is Required for Plastid 50S Ribosome Biogenesis
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-14
    Bennet Reiter, Evgenia Vamvaka, Giada Marino, Tatjana Kleine, Peter Jahns, Cordelia Bolle, Dario Leister, Thilo Ruehle

    Biogenesis of plastid ribosomes is facilitated by auxiliary factors that process and modify ribosomal RNAs (rRNA) or are involved in ribosome assembly. In comparison to their bacterial and mitochondrial counterparts, the biogenesis of plastid ribosomes is less well understood and few auxiliary factors have been described so far. In this study, we report the functional characterization of CONSERVED ONLY IN THE GREEN LINEAGE20 (CGL20) in Arabidopsis thaliana (AtCGL20), which is a proline-rich, ~10 kD protein that is targeted to mitochondria and chloroplasts. In Arabidopsis, CGL20 is encoded by segmentally duplicated genes of high sequence similarity (AtCGL20A and AtCGL20B). Inactivation of these genes in the atcgl20ab mutant led to a visible virescent phenotype and growth arrest at low temperature. The chloroplast proteome, pigment composition, and photosynthetic performance were significantly affected in atcgl20ab mutants. Loss of AtCGL20 impaired plastid translation, perturbing the formation of a hidden break in the 23S rRNA and causing abnormal accumulation of 50S ribosomal subunits in the high-molecular-mass fraction of chloroplast stromal extracts. Moreover, AtCGL20A-eGFP fusion proteins co-migrated with 50S ribosomal subunits in sucrose density gradients, even after RNase treatment of stromal extracts. Therefore, we propose that AtCGL20 participates in the late stages of the biogenesis of 50S ribosomal subunits in plastids - a role which presumably evolved in the green lineage as a consequence of structural divergence of plastid ribosomes.

    更新日期:2020-01-15
  • TaANR1-TaBG1 and TaWabi5-TaNRT2s/NARs link ABA metabolism and nitrate acquisition in wheat roots
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-14
    Meng Wang, Pengli Zhang, Qian Liu, Guangjie Li, Dongwei Di, Guangmin Xia, Herbert Kronzucker, Shuang Fang, Jinfang Chu, Weiming Shi

    Nitrate is the preferred form of nitrogen for most plants, acting both as a nutrient and a signaling molecule. However, the components and regulatory factors governing nitrate uptake in bread wheat (Triticum aestivum), one of the world's most important crop species, have remained unclear, largely due to the complexity of its hexaploid genome. Here, based on recently released whole-genome information for bread wheat, the high-affinity nitrate transporter 2 (NRT2) and the nitrate-assimilation-related (NAR) gene family are characterized. We show that ABA-GE deconjugation is stimulated in bread wheat roots by nitrate resupply following nitrate withdrawal, leading to enhanced root-tissue ABA accumulation, and that this enhancement, in turn, affects the expression of root-type NRT2/NAR genes. TaANR1 is shown to regulate nitrate-mediated ABA accumulation by directly activating TaBG1, while TaWabi5 is involved in ABA-mediated NO3- induction of NRT2/NAR genes. Building on previous evidence establishing ABA involvement in the developmental response to high-nitrate stress, our study suggests that ABA also contributes to the optimisation of nitrate uptake by regulating the expression of NRT2/NAR genes under limited nitrate supply, offering a new target for improvement of nitrate absorption in crops.

    更新日期:2020-01-15
  • Putative cis-regulatory elements predict iron deficiency responses in Arabidopsis roots
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-14
    Birte Schwarz, Christina B Azodi, Shin-Han Shiu, Petra Bauer

    Plant iron deficiency ( Fe) activates a complex regulatory network which coordinates root Fe uptake and distribution to sink tissues. In Arabidopsis (Arabidopsis thaliana), FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), a basic helix-loop-helix (bHLH) transcription factor (TF), regulates root Fe acquisition genes. Many other Fe-induced genes are FIT-independent, and instead regulated by other bHLH TFs and by yet unknown TFs. The cis-regulatory code, i.e. the cis-regulatory elements (CREs) and their combinations that regulate plant -Fe-responses, remains largely elusive. Using Arabidopsis root transcriptome data and co-expression clustering, we identified over 100 putative CREs (pCREs) that predicted -Fe-induced gene expression in computational models. To assess pCRE properties and possible functions, we used large-scale in vitro TF binding data, positional bias, and evolutionary conservation. As one example, our approach uncovered pCREs resembling IDE1 (iron deficiency-responsive element 1), a known grass Fe response CRE. Arabidopsis IDE1-likes were associated with FIT-dependent gene expression, more specifically with biosynthesis of Fe-chelating compounds. Thus, IDE1 seems to be conserved in grass and non-grass species. Our pCREs matched amongst others in vitro binding sites of B3, NAC, bZIP, and TCP TFs, which might be regulators of Fe responses. Altogether, our findings provide a comprehensive source of cis-regulatory information for -Fe-responsive genes, that advances our mechanistic understanding and informs future efforts in engineering plants with more efficient Fe uptake or transport systems.

    更新日期:2020-01-15
  • Identification of chloroplast envelope proteins with critical importance for cold acclimation
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-13
    Oliver Trentmann, Timo Mühlhaus, David Zimmer, Frederik K Sommer, Michael Schroda, Ilka Haferkamp, Isabel Keller, Benjamin Pommerrenig, H. Ekkehard Neuhaus

    The ability of plants to withstand cold temperatures relies on their photosynthetic activity. Thus, the chloroplast is of utmost importance for cold acclimation and acquisition of freezing tolerance. During cold acclimation, the properties of the chloroplast change markedly. To provide the most comprehensive view of the protein repertoire of the chloroplast envelope, we analysed this membrane system in Arabidopsis (Arabidopsis thaliana) using mass spectrometry-based proteomics. Profiling chloroplast envelope membranes was achieved by a cross comparison of protein intensities across the plastid and the enriched membrane fraction both under normal and cold conditions. We used multivariable logistic regression to model the probabilities for the classification of an envelope localization. In total, we identified 38 envelope membrane intrinsic or associated proteins exhibiting altered abundance after cold acclimation. These proteins comprise several solute carriers, such as the ATP/ADP antiporter NTT2 (nucleotide transporter 2, substantially increased abundance) or the maltose exporter MEX1 (maltose exporter 1, substantially decreased abundance). Remarkably, analysis of the frost recovery of ntt loss-of-function and mex1 overexpressor mutants confirmed that the comparative proteome is well suited to identify key factors involved in cold acclimation and acquisition of freezing tolerance. Moreover, for proteins with known physiological function, we propose scenarios explaining their possible roles in cold acclimation. Furthermore, spatial proteomics introduces an additional layer of complexity and enables the identification of proteins differentially localized at the envelope membrane under the changing environmental regime.

    更新日期:2020-01-14
  • ZmEHD1 is required for kernel development and vegetative growth through regulating auxin homeostasis
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-10
    Yafei Wang, Wenwen Liu, Hongqiu Wang, Qingguo Du, Zhiyuan Fu, Wen-Xue Li, Jihua Tang

    The roles of C-terminal Eps15 homology domain (EHD) proteins in clathrin-mediated endocytosis (CME) in plants are poorly understood. Here, we isolated a maize (Zea mays) mutant, designated as ehd1, which showed defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that ehd1 encodes an EHD protein. Internalization of the endocytic tracer FM4-64 was substantially reduced in the ehd1 mutant and ZmEHD1 knock-out mutants. We further demonstrated that ZmEHD1 and ZmAP2 subunit physically interact at the plasma membrane. Auxin distribution and ZmPIN1a-YFP localization were altered in the ehd1 mutant. Kernel IAA levels were substantially lower in the ehd1 mutant than in wild-type maize. Exogenous application of 1-NAA, but not GA3 or 2-NAA, rescued the seed germination and seedling emergency phenotypic defects of ehd1 mutants. Taken together, these results indicate that ZmEHD1 regulates auxin homeostasis by mediating CME through its interaction with the ZmAP2 subunit, which is crucial for kernel development and vegetative growth of maize.

    更新日期:2020-01-13
  • Structural Aspects of Plant Hormone Signal Perception and Regulation by Ubiquitin Ligases
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-09
    Lior Tal, M Ximena Anleu Gil, Angelica Miriam Guercio, Nitzan Shabek

    Hormonal cues regulate many aspects of plant growth and development, facilitating the plant's ability to systemically respond to environmental changes. Elucidating the molecular mechanisms governing these signaling pathways is crucial to understanding how plants function. Structural and functional biology methods have been essential in decoding plant genetic findings and revealing precise molecular actions at the protein level. Past studies of plant hormone signaling have uncovered mechanisms that involves highly coordinated protein turnover to elicit immediate cellular responses. Many phytohormone signaling pathways rely on the ubiquitin (Ub) proteasome system, specifically E3 Ub ligases, for perception and initiation of signaling transduction. In this review, we highlight structural aspects of plant hormone-sensing mechanisms by Ub ligases and discuss our current understanding of the emerging field of strigolactone signaling.

    更新日期:2020-01-10
  • Rapid affinity purification of tagged plant mitochondria (Mito-AP) for metabolome and proteome analyses
    Plant Physiol. (IF 6.305) Pub Date : 2020-01-07
    Markus Niehaus, Henryk Straube, Patrick Künzler, Nils Rugen, Jan Hegermann, Patrick Giavalisco, Holger Eubel, Claus-Peter Witte, Marco Herde

    The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep-tag in Arabidopsis exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30-60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of over 90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shot-gun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods - among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment for typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinons in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool to reach higher spatial and temporal resolution for the analysis of the metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle sub-populations.

    更新日期:2020-01-08
  • Analysis of Soybean Long Non-coding RNAs Reveals a Subset of Small Peptide-Coding Transcripts
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-27
    Xiao Lin, Wengui Lin, Yee-Shan Ku, Fuk-Ling Wong, Man-Wah Li, Hon-Ming Lam, Sai-Ming Ngai, Ting-Fung Chan

    Long non-coding RNAs (lncRNAs) are defined as non-protein-coding transcripts that are at least 200 nucleotides long. They are known to play pivotal roles in regulating gene expression, especially during stress responses in plants. We used a large collection of in-house transcriptome data from various soybean (Glycine max and Glycine soja) tissues treated under different conditions to perform a comprehensive identification of soybean lncRNAs. We also retrieved publicly available soybean transcriptome data that were of sufficient quality and sequencing depth to enrich our analysis. In total, RNA-seq data of 332 samples were used for this analysis. An integrated reference-based, de novo transcript assembly was developed that identified ~69,000 lncRNA gene loci. We showed that lncRNAs are distinct from both protein-coding transcripts and genomic background noise in terms of length, number of exons, transposable element composition, and sequence conservation level across legume species. The tissue-specific and time-specific transcriptional responses of the lncRNA genes under some stress conditions may suggest their biological relevance. The transcription start sites of lncRNA gene loci tend to be close to their nearest protein-coding genes, and they may be transcriptionally related to the protein-coding genes, particularly for antisense and intronic lncRNAs. A previously unreported subset of small peptide-coding transcripts was identified from these lncRNA loci via tandem mass spectrometry, which paved the way for investigating their functional roles. Our results also highlight the current inadequacy of the bioinformatic definition of lncRNA, which excludes those lncRNA gene loci with small open reading frames (ORFs) from being regarded as protein-coding.

    更新日期:2019-12-30
  • A SAC phosphoinositide phosphatase controls rice development via hydrolyzing PI4P and PI(4,5)P2
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-27
    Tao Guo, Hua-Chang Chen, Zi-Qi Lu, Min Diao, Ke Chen, Nai-Qian Dong, Jun-Xiang Shan, Wang-Wei Ye, Shanjin Huang, Hong-Xuan Lin

    Phosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PI-dependent modulation remain largely elusive, the effects of disturbed PI metabolism could be employed to identify regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice (Oryza sativa). Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related proteins 2 and 3 (Arp2/3) complex-nucleated actin branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effects in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulting from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering cell development. These findings imply that the Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing insights into the relationship between PI homeostasis and plant growth and development.

    更新日期:2019-12-30
  • Failure to Maintain Acetate Homeostasis by Acetate-Activating Enzymes Impacts Plant Development
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-24
    Xinyu Fu, Hannah Yang, Febriana Pangestu, Basil J. Nikolau

    The metabolic intermediate acetyl-coenzyme A (acetyl-CoA) links anabolic and catabolic processes and coordinates metabolism with cellular signaling by influencing protein acetylation. In this study we demonstrate that in Arabidopsis thaliana, two distinctly localized acetate-activating enzymes, ACETYL-COA SYNTHETASE (ACS) in plastids and ACETATE NON-UTILIZING 1 (ACN1) in peroxisomes, function redundantly to prevent the accumulation of excess acetate. In contrast to the near wild-type morphological and metabolic phenotypes of acs or acn1 mutants, the acs acn1 double mutant is delayed in growth and sterile, which is associated with hyperaccumulation of cellular acetate and decreased accumulation of acetyl-CoA-derived intermediates of central metabolism. Using multiple mutant stocks and stable isotope-assisted metabolic analyses, we demonstrate the twin metabolic origins of acetate from the oxidation of ethanol and the non-oxidative decarboxylation of pyruvate, with acetaldehyde being the common intermediate precursor of acetate. Conversion from pyruvate to acetate is activated under hypoxic conditions, and ACS recovers carbon that would otherwise be lost from the plant as ethanol. Plastid-localized ACS metabolizes cellular acetate and contributes to the de novo biosynthesis of fatty acids and leucine; peroxisome-localized ACN1 enables the incorporation of acetate into organic acids and amino acids. Thus, the activation of acetate in distinct subcellular compartments provides plants with the metabolic flexibility to maintain physiological levels of acetate and a metabolic mechanism for the recovery of carbon that would otherwise be lost as ethanol, for example following hypoxia.

    更新日期:2019-12-25
  • Arabidopsis thaliana Ureide Permease 5 (AtUPS5) connects cell compartments involved in Ureide metabolism
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-23
    Ignacio Lescano, María Florencia Bogino, Carolina Martini, Tomás María Tessi, Claudio Alejandro González, Karin Schumacher, Marcelo Desimone

    Allantoin is a purine oxidative product that serves for long distance transport of organic nitrogen in nodulating legumes and was recently related with stress tolerance in other plants. The subcellular localization of enzymes that lead to allantoin synthesis and degradation indicates that allantoin is produced in peroxisomes and degraded in the endoplasmic reticulum (ER). Although it has been determined that allantoin is mostly synthesized in roots and transported to shoots either for organic nitrogen translocation in legumes or for plant protection during stress in Arabidopsis, the mechanism and molecular components for allantoin export from root cells is still unknown. AtUPS5 (Arabidopsis thaliana Ureide Permease 5) is a transmembrane protein that transports allantoin with high affinity when expressed in yeast. The subcellular fate of splicing variants AtUPS5L (Long) and AtUPS5S (Short) was studied by tagging them with fluorescent proteins in their cytosolic loops. The capability of these fusion proteins to complement the function of the native proteins was demonstrated by nutritional and salt stress experiments. Both variants were localized in the ER, but the AtUPS5L variant was also detected in the trans-Golgi network/early endosome (TGN/EE) and at the plasma membrane. AtUPS5L and AtUPS5S localization indicates that they could have different roles in allantoin distribution between subcellular compartments. Our data predict that under non-stress conditions UPS5L and UPS5S may function in allantoin degradation pathway for nutrient recycling, whereas under stress, both genes would be involved in cell export via vesicles of the secretory pathway allowing allantoin translocation from roots to shoots.

    更新日期:2019-12-25
  • MYB20, MYB42, MYB43 and MYB85 Regulate Phenylalanine and Lignin Biosynthesis during Secondary Cell Wall Formation
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-23
    Pan Geng, Su Zhang, Jinyue Liu, Cuihuan Zhao, Jie Wu, Yingping Cao, Chunxiang Fu, Xue Han, Hang He, Qiao Zhao

    Lignin is a phenylpropanoid-derived polymer that functions as a major component of cell walls in plant vascular tissues. Biosynthesis of the aromatic amino acid phenylalanine provides precursors for many secondary metabolites including lignins and flavonoids. Here, we discovered that MYB transcription factors MYB20, MYB42, MYB43, and MYB85 are transcriptional regulators that directly activate lignin biosynthesis genes and phenylalanine biosynthesis genes during secondary wall formation in Arabidopsis (Arabidopsis thaliana). Disruption of MYB20, MYB42, MYB43, and MYB85 resulted in growth development defects and substantial reductions in lignin biosynthesis. In addition, our data showed that these MYB proteins directly activated transcriptional repressors that specifically inhibit flavonoid biosynthesis, which competes with lignin biosynthesis for phenylalanine precursors. Together, our results provide important insights into the molecular framework for the lignin biosynthesis pathway.

    更新日期:2019-12-25
  • Promotion of BR biosynthesis by miR444 is required for ammonium-triggered inhibition of root growth
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-23
    Xiaoming Jiao, Huacai Wang, Jijun Yan, Xiaoyu Kong, Yawen Liu, Jinfang Chu, Xiaoying Chen, Rongxiang Fang, Yongsheng Yan

    Rice (Oryza sativa), the staple food for almost half of the world's population, prefers ammonium (NH4+) as the major nitrogen resource, and while NH4+ has profound effects on rice growth and yields, the underlying regulatory mechanisms remain largely unknown. Brassinosteroids (BRs) are a class of steroidal hormones playing key roles in plant growth and development. In this study, we show that NH4+ promotes BR biosynthesis through miR444 to regulate rice root growth. miR444 targeted five homologous MADS-box transcription repressors potentially forming homologous or heterogeneous complexes in rice. miR444 positively regulated BR biosynthesis through its MADS-box targets, which directly repress the transcription of BR-deficient dwarf1 (OsBRD1), a key BR biosynthetic gene. NH4+ induced the miR444-OsBRD1 signaling cascade in roots, thereby increasing the amount of BRs, whose biosynthesis and signaling were required for NH4+-dependent root elongation inhibition. Consistently, miR444-overexpressing rice roots were hypersensitive to NH4+ depending on BR biosynthesis, and overexpression of miR444's target, OsMADS57, resulted in rice hyposensitivity to NH4+ in root elongation, which was associated with a reduction of BR content. In summary, our findings reveal a crosstalk mechanism between NH4+ and BR in which NH4+ activates miR444-OsBRD1, an undescribed BR biosynthesis-promoting signaling cascade, to increase BR content, inhibiting root elongation in rice.

    更新日期:2019-12-25
  • AtMOB1 genes regulate jasmonate accumulation and plant development
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-20
    Zhiai Guo, Xiaozhen Yue, Xiaona Cui, Lizhen Song, Youfa Cheng

    The MOB1 proteins are highly conserved in yeasts, animals, and plants. Previously, we showed that the Arabidopsis MOB1A gene (AtMOB1A/NCP1) plays critical roles in auxin-mediated plant development. Here, we report that AtMOB1A and AtMOB1B redundantly and negatively regulate jasmonate (JA) accumulation and function in Arabidopsis development. The two MOB1 genes exhibited similar expression patterns and the MOB1 proteins displayed similar subcellular localizations and physically interacted in vivo. Furthermore, the atmob1a atmob1b (mob1a/1b) double mutant displayed severe developmental defects, which were much stronger than those of either single mutant. Interestingly, many jasmonate-related genes were up-regulated in mob1a/1b, suggesting that AtMOB1A and AtMOB1B negatively regulate the JA pathways. mob1a/1b plants accumulated more JA and were hypersensitive to exogenous JA treatments. Disruption of MYC2, a key gene in JA signaling, in the mob1a/1b background partially alleviated the root defects and JA hypersensitivity observed in mob1a/1b. Moreover, the expression levels of the MYC2-repressed genes PLT1 and PLT2 were significantly decreased in the mob1a/1b double mutant. Our results showed that MOB1A/1B genetically interact with SIK1 and antagonistically modulate JA-related gene expression. Taken together, our findings indicate that AtMOB1A and AtMOB1B play important roles in regulating JA accumulation and Arabidopsis development.

    更新日期:2019-12-21
  • Subdivision of light signalling networks contributes to partitioning of C4 photosynthesis.
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-20
    Ross-William Hendron, Steven Kelly

    Plants coordinate the expression of photosynthesis-related genes in response to growth and environmental changes. In species that conduct two-cell C4 photosynthesis, expression of photosynthesis genes is partitioned such that leaf mesophyll and bundle sheath cells accumulate different components of the photosynthetic pathway. The identities of the regulatory networks that facilitate this partitioning are unknown. Here we show that differences in light perception between mesophyll and bundle sheath cells facilitate differential regulation and accumulation of photosynthesis gene transcripts in the C4 crop maize (Zea mays). Key components of the photosynthesis gene regulatory network differentially accumulated between mesophyll and bundle sheath cells, indicative of differential network activity across cell types. We further show that blue light (but not red) is necessary and sufficient to activate Photosystem II assembly in mesophyll cells in etiolated maize. Finally, we demonstrate that 61% of all light-induced mesophyll and bundle sheath genes were induced only by blue light or only by red light but not both. These findings provide evidence that subdivision of light signalling networks is a component of cellular partitioning of C4 photosynthesis in maize.

    更新日期:2019-12-21
  • Full-length transcript-based proteogenomics of rice improves its genome and proteome annotation
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-20
    Mo-Xian Chen, Fu-Yuan Zhu, Bei Gao, Kai-Long Ma, Youjun Zhang, Alisdair R. Fernie, Xi Chen, Lei Dai, Neng-Hui Ye, Xue Zhang, Yuan Tian, Di Zhang, Shi Xiao, Jianhua Zhang, Ying-Gao Liu

    Rice (Oryza sativa) molecular breeding has gained considerable attention in recent years but inaccurate genome annotation hampers its progress and functional studies of the rice genome. In this study, we applied single-molecule long-read RNA sequencing (lrRNA_seq)-based proteogenomics to reveal the complexity of the rice transcriptome and its coding abilities. Surprisingly, approximately 60% of loci identified by lrRNA_seq are associated with natural antisense transcripts (NATs). The high-density genomic arrangement of NAT genes suggests their potential roles in the multifaceted control of gene expression. In addition, a large number of fusion and intergenic transcripts have been observed. Furthermore, a total of 906,456 transcript isoforms were identified, and 72.9% of the genes can generate splicing isoforms. 706,075 post-transcriptional events were subsequently categorized into ten subtypes, demonstrating the interdependence of post-transcriptional mechanisms that contribute to transcriptome diversity. Parallel short-read RNA sequencing indicated that lrRNA_seq has a superior capacity for the identification of longer transcripts. In addition, over 190,000 unique peptides belonging to 9,706 proteoforms/protein groups were identified, expanding the diversity of the rice proteome. Our findings indicate that the genome organization, transcriptome diversity, and coding potential of the rice transcriptome are far more complex than previously anticipated.

    更新日期:2019-12-21
  • The nanoscale organization of the plasma membrane and its importance in signaling - a proteolipid perspective
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-19
    Yvon Jaillais, Thomas Ott

    Plasma membranes provide a highly selective environment for a large number of transmembrane and membrane-associated proteins. Whereas the lateral movement of proteins in this lipid bilayer is possible, it is rather limited in turgid and cell wall-shielded plant cells. However, membrane-resident signaling processes occur on sub-second scales that cannot be explained by simple diffusion models. Accordingly, several receptors and other membrane-associated proteins are organized and functional in membrane nanodomains. Although the general presence of membrane nanodomains has become a widely-accepted fact, fundamental functional aspects, the roles of individual lipid species and their interplay with proteins, and aspects of nanodomain maintenance and persistence remain poorly understood. Here, we review the current knowledge of nanodomain organization and function, with a particular focus on signaling processes involving proteins, lipids, and their interactions. Furthermore, we propose new and hypothetical aspects of plant membrane biology that we consider important for future research.

    更新日期:2019-12-20
  • Low-phosphate chromatin dynamics predict a cell wall remodeling network in rice shoots
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-19
    Maryam Foroozani, Sara Zahraeifard, Dong-Ha Oh, Guannan Wang, Maheshi Dassanayake, Aaron P Smith

    Phosphorus (P) is an essential plant macronutrient vital to fundamental metabolic processes. Plant-available P is low in most soils, making it a frequent limiter of growth. Declining P reserves for fertilizer production exacerbates this agricultural challenge. Plants modulate complex responses to fluctuating P levels via global transcriptional regulatory networks. Although chromatin structure plays a substantial role in controlling gene expression, the chromatin dynamics involved in regulating P homeostasis have not been determined. Here we define distinct chromatin states across the rice (Oryza sativa) genome by integrating multiple chromatin marks, including the H2A.Z histone variant, H3K4me3 modification, and nucleosome positioning. In response to P starvation, 40% of all protein-coding genes exhibit a transition from one chromatin state to another at their transcription start site. Several of these transitions are enriched in subsets of genes differentially expressed under P deficiency. The most prominent subset supports the presence of a coordinated signaling network that targets cell wall structure and is regulated in part via a decrease of H3K4me3 at transcription start sites. The P starvation-induced chromatin dynamics and correlated genes identified here will aid in enhancing P use efficiency in crop plants, benefitting global agriculture.

    更新日期:2019-12-20
  • FLOWERING LOCUS T Improved Cucumber Adaptation to Higher Latitudes
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-16
    Shenhao Wang, Hongbo Li, Yangyang Li, Zheng Li, Jianjian Qi, Tao Lin, Xueyong Yang, Zhonghua Zhang, Sanwen Huang

    Flowering time plays a crucial role in the geographical adaptation of most crops during domestication. Cucumber (Cucumis sativus L.) is a major vegetable crop worldwide. From its tropical origin on the Southern Asian continent, cucumber has spread over a wide latitudinal cline, but the molecular mechanisms underlying this latitudinal adaptation and the expansion of domesticated cucumber are largely unclear. Here, we report the cloning of two flowering-time loci from two distinct cucumber populations, and show that two large deletions upstream from FLOWERING LOCUS T (FT) are associated with higher expression of FT and earlier flowering. We determined that the two large deletions are pervasive and occurred independently in Eurasian and East-Asian populations. Nucleotide diversity analysis further revealed that the FT locus region of the cucumber genome contains a signature for a selective sweep during domestication. Our results suggest that large genetic structural variations upstream from FT were selected for and have been important in the geographic spread of cucumber from its tropical origin to higher latitudes.

    更新日期:2019-12-17
  • Treatment analogous to seasonal change demonstrates the integration of cold responses in Brachypodium distachyon
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-16
    Boris F. Mayer, Annick Bertrand, Jean-Benoit Charron

    Anthropogenic climate change precipitates the need to understand plant adaptation. Crucial in temperate climates, adaptation to winter is characterized by cold acclimation and vernalization, which respectively lead to freezing tolerance and flowering competence. However, the progression of these responses during fall and their interaction with plant development are not completely understood. By identifying key seasonal cues found in the native range of the cereal model Brachypodium distachyon, we designed a diurnal-freezing treatment (DF) that emulates summer-to-winter change. DF induced unique cold acclimation and vernalization responses characterized by low VERNALIZATION1 (VRN1) expression. Flowering under DF is characterized by an upregulation of FLOWERING LOCUS T (FT) post-vernalization independent of VRN1 expression. DF, while conferring flowering competence, favors a high tolerance to freezing and the development of a winter-hardy plant structure. The findings of this study highlight the contribution of phenotypic plasticity to freezing tolerance and demonstrate the integration of key morphological, physiological, and molecular responses in cold-adaptation. The results suggest a fundamental role for VRN1 in regulating cold acclimation, vernalization, and morphological development in B. distachyon. This study also establishes the usefulness of reproducing natural cues in laboratory settings.

    更新日期:2019-12-17
  • An online database for exploring over 2,000 Arabidopsis small RNA libraries
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-16
    Li Feng, Fei Zhang, Hong Zhang, Yan Zhao, Blake C. Meyers, Jixian Zhai

    Small RNAs (sRNAs) play a wide range of important roles in plants, from maintaining genome stability and enhancing disease resistance to regulating developmental processes. Over the past decade, next-generation sequencing (NGS) technologies have allowed us to explore the sRNA populations with unprecedented depth and accuracy. The community has accumulated a tremendous amount of sRNA sequencing (sRNA-seq) data from various genotypes, tissues, and treatments. However, it has become increasingly challenging to access these "big data" and extract useful information, particularly for researchers lacking sophisticated bioinformatics tools and expensive computational resources. Here, we constructed an online website, Arabidopsis Small RNA Database (ASRD, http://ipf.sustech.edu.cn/pub/asrd), that allows users to easily explore the information from publicly available Arabidopsis (Arabidopsis thaliana) sRNA libraries. Our database contains ~2.3 billion sRNA reads, representing ~250 million unique sequences from 2,024 sRNA-seq libraries. We downloaded the raw data for all libraries and re-processed them with a unified pipeline so that the normalized abundance of any particular sRNA or the sum of abundances of sRNAs from a genic or transposable element (TE) region can be compared across all libraries. We also integrated an online Integrative Genomics Viewer (IGV) browser into our website for convenient visualization. ASRD is a free, web-accessible, and user-friendly database that supports the direct query of over 2,000 Arabidopsis sRNA-seq libraries. We believe this resource will help plant researchers take advantage of the vast NGS datasets available in the public domain.

    更新日期:2019-12-17
  • Identification of low-abundant lipid droplet proteins in seeds and seedlings
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-11
    Franziska K. Kretzschmar, Nathan Doner, Hannah E Krawczyk, Patricia Scholz, Kerstin Schmitt, Oliver Valerius, Gerhard Braus, Robert T Mullen, Till Ischebeck

    The developmental program of seed formation, germination, and early seedling growth requires not only the tight regulation of cell division and metabolism, but also the concerted control of the structure and function of organelles, which relies on specific changes in their protein composition. Of particular interest is the switch from heterotrophic to photoautotrophic seedling growth, for which cytoplasmic lipid droplets (LDs) play a critical role as depots for energy-rich storage lipids. Here, we present the results of a bottom-up proteomics study analyzing the total protein fractions and LD-enriched fractions in eight different developmental phases during silique (seed) development, seed germination, and seedling establishment in Arabidopsis (Arabidopsis thaliana). The quantitative analysis of the LD proteome using LD-enrichment factors led to the identification of six previously unidentified and comparably low-abundant LD proteins, each of which was confirmed by intracellular localization studies with fluorescent protein fusions. In addition to these advances in LD protein discovery and the potential insights provided to as-of-yet unexplored aspects in plant LD functions, our dataset allowed for a comparative analysis of the LD protein composition throughout the various developmental phases examined. Among the most notable of the alterations in the LD proteome were those during seedling establishment, indicating a switch in the physiological function(s) of LDs after greening of the cotyledons. This work highlights LDs as dynamic organelles with functions beyond lipid storage.

    更新日期:2019-12-13
  • Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-09
    Diann Achor, Stacy Welker, Sulley K Ben-Mahmoud, Chunxia Wang, Svetlana Yuryevna Folimonova, Manjul Dutt, Siddarame Gowda, Amit Levy

    Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and p-protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated non-sieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE 7 and PHLOEM PROTEIN 2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

    更新日期:2019-12-11
  • Switching the direction of stem gravitropism by altering two amino acids in AtLAZY1
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-09
    Takeshi Yoshihara, Edgar P. Spalding

    From germination to flowering, gravity influences plant growth and development. A rice (Oryza sativa) mutant with a distinctly prostrate growth habit led to the discovery of a gene category that participates in the shaping of plant form by gravity. Each so-called LAZY gene includes five short regions of conserved sequence. The importance of each of these regions in the LAZY1 gene of Arabidopsis thaliana (AtLAZY1) was tested by mutating each region and measuring how well transgenic expression of the resulting protein variant rescued the large inflorescence branch angle of an atlazy1 mutant. The effect of each alteration on subcellular localization was also determined. Region I was required for AtLAZY1 to reside at the plasma membrane, which is necessary for its function. Mutating region V severely disrupted function without affecting subcellular localization. Regions III and IV could be mutated without large impacts on function or localization. Altering region II with two conservative amino acid substitutions (L92A/I94A) had the profound effect of switching shoot gravity responses from negative (upward bending) to positive (downward bending), resulting in a 'weeping' inflorescence phenotype. Mechanical weakness of the stem was ruled out as an explanation for the downward bending. Instead, experiments demonstrated that the L92A/I94A change to AtLAZY1 reversed the auxin gradient normally established across stems by the gravity-sensing mechanism. This discovery opens up new avenues for studying how auxin gradients form across organs and new approaches for engineering plant architecture for agronomic and other practical purposes.

    更新日期:2019-12-11
  • MADS78 and MADS79 are essential regulators of early seed development in rice
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-09
    Puneet Paul, Balpreet K Dhatt, Michael Miller, Jing J Folsom, Zhen Wang, Inga Krassovskaya, Kan Liu, Jaspreet Sandhu, Huihui Yu, Chi Zhang, Toshihiro Obata, Paul Staswick, Harkamal Walia

    MADS-box transcription factors (TFs) are subdivided into type-I and II based on phylogenetic analysis. The type II's regulate floral organ identity and flowering time, but type I's are relatively less characterized. Here, we report functional characterization of two type-I MADS-box TFs in rice (Oryza sativa), MADS78 and MADS79. Transcript abundance of both these genes in developing seed peaked at 48 hours after fertilization (HAF) and was suppressed by 96 HAF, corresponding to syncytial and cellularized stages of endosperm development, respectively. Seeds overexpressing (OE) MADS78 and 79 exhibited delayed endosperm cellularization, while CRISPR-Cas9 mediated single knock-out mutants showed precocious endosperm cellularization. MADS78 and 79 were indispensable for seed development, as a double knock-out mutant failed to make viable seeds. Both MADS78 and 79 interacted with MADS89, another type-I MADS-box, which enhances nuclear localization. The expression analysis of Fie1, a rice FERTILIZATION-INDEPENDENT SEED-polycomb repressor complex 2 (FIS-PRC2) component, in MADS78 and 79 mutants and vice versa established an antithetical relation, suggesting Fie1 could be involved in negative regulation of MADS78 and 79. Mis-regulation of MADS78 and 79 perturbed auxin homeostasis and carbon metabolism, as evident by mis-regulation of genes involved in auxin transport and signaling as well as starch biosynthesis genes causing structural abnormalities in starch granules at maturity. Collectively, we show MADS78 and 79 are essential regulators of early seed developmental transition and impact both seed size and quality in rice.

    更新日期:2019-12-11
  • ErbB-3 BINDING PROTEIN 1 Regulates Translation and Counteracts RETINOBLASTOMA RELATED to Maintain the Root Meristem
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-09
    Ansul Lokdarshi, Csaba Papdi, Aladar Pettko-Szandtner, Stefan Dorokhov, Ben Scheres, Zoltan Magyar, Albrecht G. von Arnim, Laszlo Bogre, Beatrix Horváth

    The ErbB-3 BINDING PROTEIN 1 (EBP1) drives growth, but the mechanism of how it acts in plants is little understood. Here, we show that EBP1 expression and protein abundance in Arabidopsis (Arabidopsis thaliana) are predominantly confined to meristematic cells and are induced by sucrose and partially dependent on TARGET OF RAPAMYCIN (TOR) kinase activity. Consistent with being downstream of TOR, silencing of EBP1 restrains, while overexpression promotes, root growth, mostly under sucrose-limiting conditions. Inducible overexpression of RETINOBLASTOMA RELATED (RBR), a sugar-dependent transcriptional repressor of cell proliferation, depletes meristematic activity and causes precocious differentiation, which is attenuated by EBP1. To understand the molecular mechanism, we searched for EBP1- and RBR-interacting proteins by affinity purification and mass spectrometry. In line with the double-stranded RNA-binding activity of EBP1 in human (Homo sapiens) cells, the overwhelming majority of EBP1 interactors are part of ribonucleoprotein complexes regulating many aspects of protein synthesis, including ribosome biogenesis and mRNA translation. We confirmed that EBP1 associates with ribosomes and that EBP1 silencing hinders rRNA processing. Unexpectedly, we revealed that RBR also interacts with a set of EBP1-associated nucleolar proteins as well as factors that function in protein translation. This suggests EBP1 and RBR act antagonistically on common processes that determine the capacity for translation to tune meristematic activity in relation to available resources.

    更新日期:2019-12-11
  • Rapid single-step affinity purification of HA-tagged plant mitochondria
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-09
    Franziska Kuhnert, Anja Stefanski, Nina Overbeck, Leonie Drews, Andreas S Reichert, Kai Stühler, Andreas P.M. Weber

    Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria from plant cells for physiological and biochemical analyses is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis thaliana with a triple haemagglutinin-tag for rapid purification via a single affinity purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant deficient in the mitochondrial glutamate transporter À BOUT DE SOUFFLE (BOU) and identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 controls. Our work sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.

    更新日期:2019-12-11
  • CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) mediates plant resistance against Pseudomonas syringae
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-05
    Baoda Han, Yunhe Jiang, Guoxin Cui, Jianing Mi, Rob Roelfsema, Grégory Mouille, Julien Sechet, Salim Al-Babili, Manuel Aranda, Heribert Hirt

    Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na+:K+:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as Pathogenesis-Related (PR) proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively downregulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.

    更新日期:2019-12-06
  • Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-05
    Edward J Whittle, Yuanheng Cai, Jantana Keereetaweep, Jin Chai, Peter H Buist, John Shanklin

    In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in the present work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9, 10-dihydroxystearate, that we identified via its methyl ester through gas chromatography/mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9, 10-dihydroxystearate, which presents a likely explanation for its accumulation to approximately 0.7% in castor oil, of which the biosynthetic origin had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of diiron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.

    更新日期:2019-12-06
  • CLE40 signalling regulates the fate of root stem cells in Arabidopsis
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-05
    Barbara Berckmans, Gwendolyn Kirschner, Nadja Gerlitz, Ruth Stadler, Rüdiger Simon

    The quiescent center (QC) of the Arabidopsis root meristem acts as an organiser that promotes stem cell fate in adjacent cells and patterns the stem cell niche. This stem cell niche is covered by cells of the columella, which are continuously replaced during root growth by a layer of columella stem cells (CSCs) that are maintained in an undifferentiated state by the QC-expressed transcription factor WOX5. The differentiated columella cells provide a feedback signal via secretion of the peptide CLE40, which acts through the plasmodesmata-localised receptor kinases ACR4 and CLV1 to control WOX5 expression. Previously, WOX5 protein movement from the QC into CSCs was proposed to be required for CSC maintenance, and the CLE40/CLV1/ACR4 signalling module was suggested to restrict WOX5 mobility or function. Here, these assumptions were tested, and the function of CLE40/CLV1/ACR4 in CSC maintenance was investigated. However, no role for CLE40/CLV1/ACR4 in constricting mobility of WOX5 or other fluorescent test proteins was found. Furthermore, in contrast to previous observations, WOX5 mobility was not required to inhibit CSC differentiation. Instead, we propose that WOX5 acts mainly in the QC, where other short-range signals are generated that not only inhibit differentiation but also promote stem cell division in adjacent cells. Therefore, the main function of columella-derived CLE40 signals is to position the QC at a defined distance from the root tip by repressing QC-specific gene expression via the ACR4 and CLV1 receptors in the distal domain and by promoting WOX5 expression via the CLV2 receptor in the proximal meristem.

    更新日期:2019-12-06
  • Non-catalytic subunits facilitate quaternary organization of plastidic acetyl-CoA carboxylase
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-02
    Kiran-Kumar Shivaiah, Geng Ding, Bryon Upton, Basil J. Nikolau

    Arabidopsis (Arabidopsis thaliana), like most dicotyledonous plants, expresses a multicomponent, heteromeric acetyl-CoA carboxylase (htACCase), which catalyzes the generation of the malonyl-CoA precursor of de novo fatty acid biosynthesis. This enzyme consists of four catalytic subunits: biotin carboxylase (BC), carboxyltransferase (CT)-α, CT-β, and biotin carboxyl carrier protein (BCCP1 or BCCP2). By co-expressing combinations of components in a bacterial expression system, we demonstrate non-catalytic BADCs facilitate the assembly and activation of BCCP-BADC-BC subcomplexes catalyzing the bicarbonate-dependent hydrolysis of ATP, which is the first half-reaction catalyzed by the htACCase enzyme. Although BADC proteins do not directly impact formation of the CT-αβ subcomplex, the BADC-facilitated BCCP-BADC-BC subcomplex can more readily interact with the CT-αβ subcomplex to facilitate the generation of malonyl-CoA. The Arabidopsis genome encodes three BADC isoforms (BADC1, BADC2, BADC3), and BADC2 and BADC3 (rather than BADC1), in combination with BCCP1, best support this quaternary-structural organizational and catalytic activation of the htACCase enzyme. Physiological genetic studies validate these attributes as Arabidopsis double mutants singularly expressing BADC2, BADC3 or BADC1 present increasingly greater deleterious impacts on morphological and biochemical phenotypes. Specifically, plants expressing only BADC2 develop normally, plants only expressing BADC3 suffer a stunted root-growth phenotype, and plants expressing only BADC1 are embryo-lethal. The latter phenotype may also be associated with the distinct sub-organelle localization of BADC1 in plastids as compared to the localization of the other two BADC homologs. These finding can inspire novel strategies to improve the biological sources of fats and oils for dietary and industrial applications.

    更新日期:2019-12-03
  • Dark-induced senescence causes localized changes in DNA methylation
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-02
    Minerva S. Trejo-Arellano, Saher Mehdi, Jennifer de Jonge, Eva Dvorák Tomastíková, Claudia Köhler, Lars Hennig

    Senescence occurs in a programmed manner to dismantle the vegetative tissues and redirect nutrients towards metabolic pathways supporting reproductive success. External factors can trigger the senescence program as an adaptive strategy, indicating that this terminal program is controlled at different levels. It has been proposed that epigenetic factors accompany the reprogramming of the senescent genome; however, the mechanism and extent of this reprogramming remain unknown. Using bisulphite conversion followed by sequencing, we assessed changes in the methylome of senescent Arabidopsis (Arabidopsis thaliana) leaves induced by darkness, and monitored their effect on gene and transposable element (TE) expression with transcriptome sequencing. Upon dark-induced senescence, genes controlling chromatin silencing were collectively downregulated. As a consequence, the silencing of TEs was impaired, causing in particular young TEs to become preferentially reactivated. In parallel, heterochromatin at chromocenters was decondensed. Despite the disruption of the chromatin maintenance network, the global DNA methylation landscape remained highly stable, with localized changes mainly restricted to CHH methylation. Together, our data show that the terminal stage of plant life is accompanied by global changes in chromatin structure but only localized changes in DNA methylation, adding another example of the dynamics of DNA methylation during plant development.

    更新日期:2019-12-03
  • VENOSA4, a human dNTPase SAMHD1 homolog, contributes to chloroplast development and abiotic stress tolerance
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-02
    Duorong Xu, Dario Leister, Tatjana Kleine

    Chloroplast biogenesis depends on an extensive interplay between the nucleus, cytosol and chloroplasts, involving regulatory nucleus-encoded chloroplast proteins, as well as nucleocytosolic photoreceptors such as phytochromes (phys) and other extrachloroplastic factors. However, this whole process is only partially understood. Here, we describe the role of VENOSA4 (VEN4) in chloroplast development and acclimation to adverse growth conditions. A 35S:VEN4-eGFP fusion protein localizes to the nucleus in Arabidopsis protoplasts, and VEN4 homologues are present in a wide range of eukaryotes including humans, where the corresponding homolog (SAMHD1) cleaves dNTPs. Defective photosynthesis in ven4 seedlings results from reduced accumulation of photosynthetic proteins and appears to be caused by a reduction in the translational capacity of chloroplasts. The negative effect of the ven4 mutation on photosynthesis can be phenotypically suppressed by germinating seeds in the presence of excess dCTP or a pool of dNTPs, implying that VEN4, like human SAMHD1, is involved in dNTP catabolism. Moreover, VEN4 activity is also required for optimal responses to cold and salt stresses. In conclusion, our work emphasizes the importance of the nucleocytosolic compartment and the fine-tuning of dNTP levels for chloroplast translation and development.

    更新日期:2019-12-02
  • Reorganization of acyl flux through the lipid metabolic network in oil-accumulating tobacco leaves
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-02
    Xue-Rong Zhou, Sajina Bhandari, Brandon S. Johnson, Hari Kiran Kotapati, Douglas K. Allen, Thomas Vanhercke, Philip David Bates

    The triacylglycerols (TAGs; i.e., oils) that accumulate in plants represent the most energy dense form of biological carbon storage, and are used for food, fuels, and chemicals. The increasing human population and decreasing amount of arable land have amplified the need to produce plant oil more efficiently. Engineering plants to accumulate oils in vegetative tissues is a novel strategy, because most plants only accumulate large amounts of lipids in the seeds. Recently, tobacco (Nicotiana tabacum) leaves were engineered to accumulate oil at 15% of dry weight due to a push (increased fatty acid synthesis) and pull (increased final step of TAG biosynthesis) engineering strategy. However, to accumulate both TAG and essential membrane lipids, fatty acid flux through non-engineered reactions of the endogenous metabolic network must also adapt, which is not evident from total oil analysis. To increase our understanding of endogenous leaf lipid metabolism and its ability to adapt to metabolic engineering, we utilized a series of in vitro and in vivo experiments to characterize the path of acyl flux in wild-type and transgenic oil-accumulating tobacco leaves. Acyl flux around the phosphatidylcholine acyl editing cycle was the largest acyl flux reaction in wild-type and engineered tobacco leaves. In oil-accumulating leaves, acyl flux into the eukaryotic pathway of glycerolipid assembly was enhanced at the expense of the prokaryotic pathway. However, a direct Kennedy pathway of TAG biosynthesis was not detected as acyl flux through phosphatidylcholine preceded the incorporation into TAG. These results provide insight into the plasticity and control of acyl lipid metabolism in leaves.

    更新日期:2019-12-02
  • Brassinosteroids Antagonize Jasmonates-Activated Plant Defense Responses through BRI1-EMS-SUPPRESSOR1 (BES1)
    Plant Physiol. (IF 6.305) Pub Date : 2019-11-27
    Ke Liao, Yu-Jun Peng, Li-Bing Yuan, Yang-Shuo Dai, Qin-Fang Chen, Lu-Jun Yu, Ming-Yi Bai, Wen-Qing Zhang, Li-Juan Xie, Shi Xiao

    Brassinosteroids (BRs) and jasmonates (JAs) regulate plant growth, development, and defense responses, but how these phytohormones mediate the growth-defense trade-off is unclear. Here, we identified the Arabidopsis thaliana dwarf at early stages1 (dwe1) mutant, which exhibits enhanced expression of defensin genes PLANT DEFENSIN1.2a (PDF1.2a) and PDF1.2b. The dwe1 mutant showed increased resistance to herbivory by beet armyworms (Spodoptera exigua) and infection by botrytis (Botrytis cinerea). DWE1 encodes ROTUNDIFOLIA3, a cytochrome P-450 protein essential for BR biosynthesis. The JA-inducible transcription of PDF1.2a and PDF1.2b was significantly reduced in the BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) gain-of-function mutant bes1-D, which was highly susceptible to S. exigua and B. cinerea. BES1 directly targeted the terminator regions of PDF1.2a/PDF1.2b and suppressed their expression. PDF1.2a overexpression diminished the enhanced susceptibility of bes1-D to B. cinerea, but did not improve resistance of bes1-D to S. exigua. In response to S. exigua herbivory, BES1 inhibited biosynthesis of the JA-induced insect defense-related metabolite indolic glucosinolate by interacting with transcription factors MYB DOMAIN PROTEIN34 (MYB34), MYB51, and MYB122, and suppressing expression of genes encoding CYTOCHROME P450 FAMILY79 SUBFAMILY B POLYPEPTIDE3 (CYP79B3) and UDP-GLUCOSYL TRANSFERASE 74B1 (UGT74B1). Thus, BR contributes to the growth-defense trade-off by suppressing expression of defensin and glucosinolate biosynthesis genes.

    更新日期:2019-11-28
  • Two Chloroplast Proteins Negatively Regulate Plant Drought Resistance Through Separate Pathways
    Plant Physiol. (IF 6.305) Pub Date : 2019-11-27
    Yechun Hong, Zhen Wang, Xue Liu, Juanjuan Yao, Xiangfeng Kong, Huazhong Shi, Jian-Kang Zhu

    Drought is one of the most deleterious environmental conditions affecting crop growth and productivity. Here we report the important roles of a nuclear-encoded chloroplast protein, PsbP Domain Protein 5 (PPD5), in drought resistance in Arabidopsis. From a forward genetic screen, a drought-resistant mutant named ppd5-2 was identified, which has a knockout mutation in PPD5. The ppd5 mutants showed increased H2O2 accumulation in guard cells and enhanced stomatal closure in response to drought stress. Further analysis revealed that the chloroplast-localized PPD5 protein interacts with and is phosphorylated by OST1, and phosphorylation of PPD5 increases its protein stability. Double mutant ppd5-2ost1-3 exhibited phenotypes resembling the ost1-3 single mutant with decreased stomatal closure, increased water loss, reduced H2O2 accumulation in guard cells and hypersensitivity to drought stress. These results indicate that the chloroplast protein PPD5 negatively regulates drought resistance by modulating guard cell H2O2 accumulation via an OST1-dependent pathway. Interestingly, the thf1-1 mutant defective in the chloroplast protein THF1 displayed drought-resistance and H2O2 accumulation similar to the ppd5 mutants, but the thf1-1ost1-3 double mutant resembled the phenotypes of the thf1-1 single mutant. These results indicate that both OST1-dependent and OST1-independent pathways exist in the regulation of H2O2 production in chloroplasts of guard cells under drought stress conditions. Additionally, our findings suggest a strategy to improve plant drought resistance through manipulation of chloroplast proteins.

    更新日期:2019-11-28
  • Apple ALMT9 requires a conserved C-terminal domain for malate transport underlying fruit acidity
    Plant Physiol. (IF 6.305) Pub Date : 2019-11-26
    Chun-Long Li, Laura Dougherty, Alison E Coluccio, Dong Meng, Islam El-Sharkawy, Ewa Borejsza-Wysocka, Dong Liang, Miguel A Pineros, Kenong Xu, Lailiang Cheng

    Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal. Here, we report both the full length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating ma1 has significantly lower malate transport activity than Ma1. RNAi suppression of Ma1 expression in 'McIntosh' apple leaves, 'Empire' apple fruit, and 'Orin' apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate Ma1 plays a key role in determining fruit acidity and the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lowers Ma1 transport activity. We conclude the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.

    更新日期:2019-11-27
  • PERSISTENT TAPETAL CELL 2 is required for normal tapetal programmed cell death and pollen wall patterning
    Plant Physiol. (IF 6.305) Pub Date : 2019-11-26
    Muhammad Uzair, Dawei Xu, Lukas Schreiber, Jianxin Shi, Wanqi Liang, Ki-Hong Jung, Mingjiao Chen, Zhijing Luo, Yueya Zhang, Jing Yu, Dabing Zhang

    The timely programmed cell death (PCD) of the tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development, including the deposition and patterning of the pollen wall. Although several genes involved in tapetal PCD and pollen wall development have been characterized, the underlying regulatory mechanism remains elusive. Here we report that PERSISTENT TAPETAL CELL2 (PTC2), which encodes an AT-hook nuclear localized (AHL) protein in rice (Oryza sativa), is required for normal tapetal PCD and pollen wall development. ptc2 showed persistent tapetal cells and abnormal pollen wall patterning including absent nexine, collapsed bacula and disordered tectum. The defective tapetal PCD phenotype of ptc2 was similar to that of a PCD delayed mutant, ptc1, in rice, while the abnormal pollen wall patterning resembled that of a pollen wall defective mutant, tek, in Arabidopsis thaliana. Levels of anther cutin monomers in ptc2 anthers were significantly reduced, as was expression of a series of lipid biosynthetic genes. PTC2 transcript and protein were shown to be present in the anther after meiosis, consistent with the observed phenotype. Based on these data, we propose a model explaining how PTC2 affects anther and pollen development. The characterization of PTC2 in tapetal PCD and pollen wall patterning expands our understanding of the regulatory network of male reproductive development in rice and will aid future breeding approaches. -

    更新日期:2019-11-27
  • A role for plant KASH proteins in regulating stomatal dynamics
    Plant Physiol. (IF 6.305) Pub Date : 2019-11-25
    Alecia M Biel, Morgan Moser, Iris Meier

    Stomatal movement, which regulates gas exchange in plants, is controlled by a variety of environmental factors, including biotic and abiotic stresses. The stress hormone abscisic acid (ABA) initiates a signaling cascade, which leads to increased H2O2 and Ca2+ levels and F-actin reorganization, but the mechanism of, and connection between, these events is unclear. SINE1, an outer nuclear envelope component of a plant Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, associates with F-actin and is, along with its putative paralog SINE2, expressed in guard cells. Here, we have determined that Arabidopsis (Arabidopsis thaliana) SINE1 and SINE2 play an important role in stomatal opening and closing. Loss of SINE1 or SINE2 results in ABA hyposensitivity and impaired stomatal dynamics but does not affect stomatal closure induced by the bacterial elicitor flg22. The ABA-induced stomatal closure phenotype is, in part, attributed to impairments in Ca2+ and F-actin regulation. Together, the data suggest that SINE1 and SINE2 act downstream of ABA but upstream of Ca2+ and F-actin. While there is a large degree of functional overlap between the two proteins, there are also critical differences. Our study makes an unanticipated connection between stomatal regulation and nuclear envelope-associated proteins, and adds two new players to the increasingly complex system of guard cell regulation.

    更新日期:2019-11-26
  • PlantAPAdb: a comprehensive database for alternative polyadenylation sites in plants
    Plant Physiol. (IF 6.305) Pub Date : 2019-11-25
    Sheng Zhu, Wenbin Ye, Lishan Ye, Hongjuan Fu, Congting Ye, Xuesong Xiao, Yuanhaowei Ji, Weixu Lin, Guoli Ji, Xiaohui Wu

    Alternative cleavage and polyadenylation (APA) is increasingly recognized as an important regulatory mechanism in eukaryotic gene expression and is dynamically modulated in a developmental, tissue-specific, or environmentally responsive manner. Given the functional importance of APA and the rapid accumulation of APA sites in plants, a comprehensive and easily accessible APA site database is necessary for improved understanding of APA-mediated gene expression regulation. We present a database called PlantAPAdb that catalogues the most comprehensive APA site data derived from sequences from diverse 3' sequencing protocols and biological samples in plants. Currently, PlantAPAdb contains APA sites in six species, including Oryza sativa L. (japonica and indica), Arabidopsis thaliana, Medicago truncatula, Trifolium pratense, Phyllostachys edulis, and Chlamydomonas reinhardtii. APA sites in PlantAPAdb are available for bulk download and can be queried in a Google-like manner. PlantAPAdb provides rich information of the whole genome APA sites, including genomic locations, heterogeneous cleavage sites, expression levels, and sample information. It also provides comprehensive poly(A) signals for APA sites in different genomic regions according to distinct profiles of cis elements in plants. In addition, PlantAPAdb contains events of 3' UTR shortening/lengthening resulting from APA, which helps to understand the mechanisms underlying systematic changes in 3' UTR lengths. Additional information about conservation of APA sites in plants is also available, providing insights into the evolutionary polyadenylation configuration across species. As a user-friendly database, PlantAPAdb is a large and extendable resource for elucidating APA mechanisms, APA conservation, and gene expression regulation.

    更新日期:2019-11-26
  • Exogenous Auxin Induces Transverse Microtubule Arrays Through TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX Receptors
    Plant Physiol. (IF 6.305) Pub Date : 2019-11-25
    Jillian H. True, Sidney L. Shaw

    Auxin plays a central role in controlling plant cell growth and morphogenesis. Application of auxin to light-grown seedlings elicits both axial growth and transverse patterning of the cortical microtubule cytoskeleton in hypocotyl cells. Microtubules respond to exogenous auxin within five minutes, though repatterning of the array does not initiate until 30 minutes after application and is complete by 2 hours. To examine the requirements for auxin-induced microtubule array patterning, we used an Arabidopsis thaliana double auxin f-box (afb) receptor mutant, afb4-8 afb5-5, that responds to conventional auxin (IAA) but has a strongly diminished response to the auxin analog, picloram. We show that 5 µM picloram induces immediate changes to microtubule density and later transverse microtubule patterning in wild-type plants, but does not cause microtubule array reorganization in the afb4-8 afb5-5 mutant. Additionally, a dominant mutant (axr2-1) for the auxin co-receptor AUXIN RESPONSIVE2 (AXR2) was strongly suppressed for auxin-induced microtubule array reorganization, providing additional evidence that auxin functions through a transcriptional pathway for transverse patterning. We observed that brassinosteroid application mimicked the auxin response, showing both early and late microtubule array effects, and induced transverse patterning in the axr2-1 mutant. Application of auxin to the brassinosteroid synthesis mutant, diminuto1, induced transverse array patterning but did not produce significant axial growth. Thus, exogenous auxin induces transverse microtubule patterning through the TRANSPORT INHIBITOR 1/AUXIN F-BOX (TIR1/AFB) transcriptional pathway and can act independently of brassinosteroids.

    更新日期:2019-11-26
  • The FATTY ACID DESATURASE2 family in tomato contributes to primary metabolism and stress responses
    Plant Physiol. (IF 6.305) Pub Date : 2019-11-25
    Min Woo Lee, Carmen S. Padilla, Chirag Gupta, Aravind Galla, Andy Pereira, Jiamei Li, Fiona L. Goggin

    The conversion of oleic acid (C18:1) to linoleic acid (C18:2) in the endoplasmic reticulum is critical to the accumulation of polyunsaturated fatty acids in seeds and other tissues, and this reaction is catalyzed by a -12 desaturase, FATTY ACID DESATURASE2 (FAD2). Here, we report that the tomato (Solanum lycopersicum) genome harbors two genes, SlFAD2-1 and SlFAD2-2, which encode proteins with in vitro -12 desaturase activity. In addition, tomato has seven divergent FAD2 members that lack -12 desaturase activity and differ from canonical FAD2 enzymes at multiple amino acid positions important to enzyme function. Whereas SlFAD2-1 and SlFAD2-2 are downregulated by biotic stress, the majority of divergent FAD2 genes in tomato are upregulated by one or more stresses. In particular, SlFAD2-7 is induced by the potato aphid (Macrosiphum euphorbiae) and has elevated constitutive expression levels in suppressor of prosystemin-mediated responses2 (spr2), a tomato mutant with enhanced aphid resistance and altered fatty acid profiles. Virus induced gene silencing of SlFAD2-7 in spr2 results in significant increases in aphid population growth, indicating that a divergent FAD2 gene contributes to aphid resistance in this genotype. Thus, the FAD2 gene family in tomato is important both to primary fatty acid metabolism and to responses to biotic stress.

    更新日期:2019-11-26
  • A Sequence-Indexed Mutator Insertional Library for Maize Functional Genomics Study
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Lei Liang, Ling Zhou, Yuanping Tang, Niankui Li, Teng Song, Wen Shao, Ziru Zhang, Peng Cai, Fan Feng, Yafei Ma, Dongsheng Yao, Yang Feng, Zeyang Ma, Han Zhao, Rentao Song

    Sequence-indexed insertional libraries are important resources for functional gene study in model plants. However, the maize (Zea mays) UniformMu library covers only 36% of the annotated maize genes. Here, we generated a new sequence-indexed maize Mutator insertional library named ChinaMu through high-throughput sequencing of enriched Mu-tagged sequences. A total of 2,581 Mu F2 lines were analyzed, and 311,924 nonredundant Mu insertion sites were obtained. Based on experimental validation, ChinaMu contains about 97,000 germinal Mu insertions, about twice as many as UniformMu. About two-thirds (66,565) of the insertions are high-quality germinal insertions (positive rate > 90%), 89.6% of which are located in genic regions. Furthermore, 45.7% (20,244) of the 44,300 annotated maize genes are effectively tagged and about two-thirds (13,425) of these genes harbor multiple insertions. We tested the utility of ChinaMu using pentatricopeptide repeat (PPR) genes. For published PPR genes with defective kernel phenotypes, 17 out of 20 were tagged, 11 of which had the previously reported mutant phenotype. For 16 unstudied PPR genes with both Mu insertions and defective kernel phenotypes, 6 contained insertions that cosegregated with the mutant phenotype. Our sequence-indexed Mu insertional library provides an important resource for functional genomics study in maize.

    更新日期:2019-11-26
  • A Deep Learning-Based Approach for High-Throughput Hypocotyl Phenotyping
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Orsolya Dobos, Peter Horvath, Ferenc Nagy, Tivadar Danka, András Viczián

    Hypocotyl length determination is a widely used method to phenotype young seedlings. The measurement itself has advanced from using rulers and millimeter papers to assessing digitized images but remains a labor-intensive, monotonous, and time-consuming procedure. To make high-throughput plant phenotyping possible, we developed a deep-learning–based approach to simplify and accelerate this method. Our pipeline does not require a specialized imaging system but works well with low-quality images produced with a simple flatbed scanner or a smartphone camera. Moreover, it is easily adaptable for a diverse range of datasets not restricted to Arabidopsis (Arabidopsis thaliana). Furthermore, we show that the accuracy of the method reaches human performance. We not only provide the full code at https://github.com/biomag-lab/hypocotyl-UNet, but also give detailed instructions on how the algorithm can be trained with custom data, tailoring it for the requirements and imaging setup of the user.

    更新日期:2019-11-26
  • Machine Learning Approaches to Improve Three Basic Plant Phenotyping Tasks Using Three-Dimensional Point Clouds
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Illia Ziamtsov, Saket Navlakha

    Developing automated methods to efficiently process large volumes of point cloud data remains a challenge for three-dimensional (3D) plant phenotyping applications. Here, we describe the development of machine learning methods to tackle three primary challenges in plant phenotyping: lamina/stem classification, lamina counting, and stem skeletonization. For classification, we assessed and validated the accuracy of our methods on a dataset of 54 3D shoot architectures, representing multiple growth conditions and developmental time points for two Solanaceous species, tomato (Solanum lycopersicum cv 75 m82D) and Nicotiana benthamiana. Using deep learning, we classified lamina versus stems with 97.8% accuracy. Critically, we also demonstrated the robustness of our method to growth conditions and species that have not been trained on, which is important in practical applications but is often untested. For lamina counting, we developed an enhanced region-growing algorithm to reduce oversegmentation; this method achieved 86.6% accuracy, outperforming prior methods developed for this problem. Finally, for stem skeletonization, we developed an enhanced tip detection technique, which ran an order of magnitude faster and generated more precise skeleton architectures than prior methods. Overall, our improvements enable higher throughput and accurate extraction of phenotypic properties from 3D point cloud data.

    更新日期:2019-11-26
  • A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Qiang Zhang, Yu Zhang, Min-Hui Lu, Yi-Ping Chai, Yuan-Yuan Jiang, Yun Zhou, Xue-Chen Wang, Qi-Jun Chen

    The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens. The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.

    更新日期:2019-11-26
  • Identification of the Arabidopsis Calmodulin-Dependent NAD+ Kinase That Sustains the Elicitor-Induced Oxidative Burst
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Elisa Dell’Aglio, Cécile Giustini, Alexandra Kraut, Yohann Couté, Alex Costa, Guillaume Decros, Yves Gibon, Christian Mazars, Michel Matringe, Giovanni Finazzi, Gilles Curien

    NADP(H) is an essential cofactor of multiple metabolic processes in all living organisms, and in plants, NADP(H) is required as the substrate of Ca2+-dependent NADPH oxidases, which catalyze a reactive oxygen species burst in response to various stimuli. While NADP+ production in plants has long been known to involve a calmodulin (CaM)/Ca2+-dependent NAD+ kinase, the nature of the enzyme catalyzing this activity has remained enigmatic, as has its role in plant physiology. Here, we used proteomic, biochemical, molecular, and in vivo analyses to identify an Arabidopsis (Arabidopsis thaliana) protein that catalyzes NADP+ production exclusively in the presence of CaM/Ca2+. This enzyme, which we named NAD kinase-CaM dependent (NADKc), has a CaM-binding peptide located in its N-terminal region and displays peculiar biochemical properties as well as different domain organization compared with known plant NAD+ kinases. In response to a pathogen elicitor, the activity of NADKc, which is associated with the mitochondrial periphery, contributes to an increase in the cellular NADP+ concentration and to the amplification of the elicitor-induced oxidative burst. Based on a phylogenetic analysis and enzymatic assays, we propose that the CaM/Ca2+-dependent NAD+ kinase activity found in photosynthetic organisms is carried out by NADKc-related proteins. Thus, NADKc represents the missing link between Ca2+ signaling, metabolism, and the oxidative burst.

    更新日期:2019-11-26
  • Plant Cell-Cell Transport via Plasmodesmata Is Regulated by Light and the Circadian Clock
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Jacob O. Brunkard, Patricia Zambryski

    Plasmodesmata (PD) are essential for plant development, but little is known about their regulation. Several studies have linked PD transport to chloroplast-centered signaling networks, but the physiological significance of this connection remains unclear. Here, we show that PD transport is strongly regulated by light and the circadian clock. Light promotes PD transport during the day, but light is not sufficient to increase rates of PD transport at night, suggesting a circadian gating mechanism. Silencing expression of the core circadian clock gene, LHY/CCA1, allows light to strongly promote PD transport during subjective night, confirming that the canonical plant circadian clock controls the PD transport light response. We conclude that PD transport is dynamically regulated during the day/night cycle. Due to the many roles of PD in plant biology, this discovery has strong implications for plant development, physiology, and pathogenesis.

    更新日期:2019-11-26
  • Isoforms of Acyl-CoA:Diacylglycerol Acyltransferase2 Differ Substantially in Their Specificities toward Erucic Acid
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Kamil Demski, Simon Jeppson, Ida Lager, Agnieszka Misztak, Katarzyna Jasieniecka-Gazarkiewicz, Małgorzata Waleron, Sten Stymne, Antoni Banaś

    In most oilseeds, two evolutionarily unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, are the main contributors to the acylation of diacylglycerols in the synthesis of triacylglycerol. DGAT1 and DGAT2 are both present in the important crop oilseed rape (Brassica napus), with each type having four isoforms. We studied the activities of DGAT isoforms during seed development in microsomal fractions from two oilseed rape cultivars: edible, low-erucic acid (22:1) MONOLIT and nonedible high-erucic acid MAPLUS. Whereas the specific activities of DGATs were similar with most of the tested acyl-CoA substrates in both cultivars, MAPLUS had 6- to 14-fold higher activity with 22:1-CoA than did MONOLIT. Thus, DGAT isoforms with different acyl-CoA specificities are differentially active in the two cultivars. We characterized the acyl-CoA specificities of all DGAT isoforms in oilseed rape in the microsomal fractions of yeast cells heterologously expressing these enzymes. All four DGAT1 isoforms showed similar and broad acyl-CoA specificities. However, DGAT2 isoforms had much narrower acyl-CoA specificities: two DGAT2 isoforms were highly active with 22:1-CoA, while the ability of the other two isoforms to use this substrate was impaired. These findings elucidate the importance, which a DGAT isoform with suitable acyl-CoA specificity may have, when aiming for high content of a particular fatty acid in plant triacylglycerol reservoirs.

    更新日期:2019-11-26
  • The Chlamydomonas deg1c Mutant Accumulates Proteins Involved in High Light Acclimation
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Jasmine Theis, Julia Lang, Benjamin Spaniol, Suzanne Ferté, Justus Niemeyer, Frederik Sommer, David Zimmer, Benedikt Venn, Shima Farazandeh Mehr, Timo Mühlhaus, Francis-André Wollman, Michael Schroda

    Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Sec-mediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knock-out mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.

    更新日期:2019-11-26
  • Natural Variation Reveals a Key Role for Rhamnogalacturonan I in Seed Outer Mucilage and Underlying Genes
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Isabelle Fabrissin, Gwendal Cueff, Adeline Berger, Fabienne Granier, Christine Sallé, Damien Poulain, Marie-Christine Ralet, Helen M. North

    On imbibition, Arabidopsis (Arabidopsis thaliana) seeds release polysaccharides from their epidermal cells that form a two-layered hydrogel, termed mucilage. Analysis of a publicly available data set of outer seed mucilage traits of over 300 accessions showed little natural variation in composition. This mucilage is almost exclusively made up of rhamnogalacturonan I (RGI), highlighting the importance of this pectin for outer mucilage function. In a genome-wide association study, observed variations in polymer amount and macromolecular characteristics were linked to several genome polymorphisms, indicating the complexity of their genetic regulation. Natural variants with high molar mass were associated with a gene encoding a putative glycosyltransferase called MUCILAGE-RELATED70 (MUCI70). muci70 insertion mutants produced many short RGI polymers that were highly substituted with xylan, confirming that polymorphism in this gene can affect RGI polymer size. A second gene encoding a putative copper amine oxidase of clade 1a (CuAOα1) was associated with natural variation in the amount of RGI present in the outer mucilage layer; cuaoα1 mutants validated its role in pectin production. As the mutant phenotype is unique, with RGI production only impaired for outer mucilage, this indicates that CuAOα1 contributes to a further mechanism controlling mucilage synthesis.

    更新日期:2019-11-26
  • Aldo-keto Reductase Metabolizes Glyphosate and Confers Glyphosate Resistance in Echinochloa colona
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Lang Pan, Qin Yu, Heping Han, Lingfeng Mao, Alex Nyporko, LongJiang Fan, Lianyang Bai, Stephen Powles

    Glyphosate, the most commonly used herbicide in the world, controls a wide range of plant species, mainly because plants have little capacity to metabolize (detoxify) glyphosate. Massive glyphosate use has led to world-wide evolution of glyphosate-resistant (GR) weed species, including the economically damaging grass weed Echinochloa colona. An Australian population of E. colona has evolved resistance to glyphosate with unknown mechanisms that do not involve the glyphosate target enzyme 5-enolpyruvylshikimate-3-P synthase. GR and glyphosate-susceptible (S) lines were isolated from this population and used for resistance gene discovery. RNA sequencing analysis and phenotype/genotype validation experiments revealed that one aldo-keto reductase (AKR) contig had higher expression and higher resultant AKR activity in GR than S plants. Two full-length AKR (EcAKR4-1 and EcAKR4-2) complementary DNA transcripts were cloned with identical sequences between the GR and S plants but were upregulated in the GR plants. Rice (Oryza sativa) calli and seedlings overexpressing EcAKR4-1 and displaying increased AKR activity were resistant to glyphosate. EcAKR4-1 expressed in Escherichia coli can metabolize glyphosate to produce aminomethylphosphonic acid and glyoxylate. Consistent with these results, GR E. colona plants exhibited enhanced capacity for detoxifying glyphosate into aminomethylphosphonic acid and glyoxylate. Structural modeling predicted that glyphosate binds to EcAKR4-1 for oxidation, and metabolomics analysis of EcAKR4-1 transgenic rice seedlings revealed possible redox pathways involved in glyphosate metabolism. Our study provides direct experimental evidence of the evolution of a plant AKR that metabolizes glyphosate and thereby confers glyphosate resistance.

    更新日期:2019-11-26
  • Basic Proline-Rich Protein-Mediated Microtubules Are Essential for Lobe Growth and Flattened Cell Geometry
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Jeh Haur Wong, Takehide Kato, Samuel A. Belteton, Rie Shimizu, Nene Kinoshita, Takumi Higaki, Yuichi Sakumura, Daniel B. Szymanski, Takashi Hashimoto

    Complex cell shapes are generated first by breaking symmetry, and subsequent polar growth. Localized bending of anticlinal walls initiates lobe formation in the epidermal pavement cells of cotyledons and leaves, but how the microtubule cytoskeleton mediates local cell growth, and how plant pavement cells benefit from adopting jigsaw puzzle-like shapes, are poorly understood. In Arabidopsis (Arabidopsis thaliana), the basic Pro-rich protein (BPP) microtubule-associated protein family comprises seven members. We analyzed lobe morphogenesis in cotyledon pavement cells of a BPP1;BPP2;BPP5 triple knockout mutant. New image analysis methods (MtCurv and BQuant) showed that anticlinal microtubule bundles were significantly reduced and cortical microtubules that fan out radially across the periclinal wall did not enrich at the convex side of developing lobes. Despite these microtubule defects, new lobes were initiated at the same frequency as in wild-type cells, but they did not expand into well-defined protrusions. Eventually, mutant cells formed nearly polygonal shapes and adopted concentric microtubule patterns. The mutant periclinal cell wall bulged outward. The radius of the calculated inscribed circle of the pavement cells, a proposed proxy for maximal stress in the cell wall, was consistently larger in the mutant cells during cotyledon development, and correlated with an increase in cell height. These bpp mutant phenotypes provide genetic and cell biological evidence that initiation and growth of lobes are distinct morphogenetic processes, and that interdigitated cell geometry effectively suppresses large outward bulging of pavement cells.

    更新日期:2019-11-26
  • Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Olga Blokhina, Teresa Laitinen, Yuto Hatakeyama, Nicolas Delhomme, Tanja Paasela, Lei Zhao, Nathaniel R. Street, Hiroshi Wada, Anna Kärkönen, Kurt Fagerstedt

    A comparative transcriptomic study and a single-cell metabolome analysis were combined to determine whether parenchymal ray cells contribute to the biosynthesis of monolignols in the lignifying xylem of Norway spruce (Picea abies). Ray parenchymal cells may function in the lignification of upright tracheids by supplying monolignols. To test this hypothesis, parenchymal ray cells and upright tracheids were dissected with laser-capture microdissection from tangential cryosections of developing xylem of spruce trees. The transcriptome analysis revealed that among the genes involved in processes typical for vascular tissues, genes encoding cell wall biogenesis-related enzymes were highly expressed in both developing tracheids and ray cells. Interestingly, most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Nonetheless, 1,073 differentially expressed genes were detected between developing ray cells and tracheids, among which a set of genes expressed only in ray cells was identified. In situ single cell metabolomics of semi-intact plants by picoliter pressure probe-electrospray ionization-mass spectrometry detected monolignols and their glycoconjugates in both cell types, indicating that the biosynthetic route for monolignols is active in both upright tracheids and parenchymal ray cells. The data strongly support the hypothesis that in developing xylem, ray cells produce monolignols that contribute to lignification of tracheid cell walls.

    更新日期:2019-11-26
  • Effect of Vapor Pressure Deficit on Gas Exchange in Wild-Type and Abscisic Acid-Insensitive Plants
    Plant Physiol. (IF 6.305) Pub Date : 2019-12-01
    Lucas A. Cernusak, Gregory R. Goldsmith, Matthias Arend, Rolf T. W. Siegwolf

    Stomata control the gas exchange of terrestrial plant leaves, and are therefore essential to plant growth and survival. We investigated gas exchange responses to vapor pressure deficit (VPD) in two gray poplar (Populus x canescens) lines: wild type and abscisic acid–insensitive (abi1) with functionally impaired stomata. Transpiration rate in abi1 increased linearly with VPD, up to about 2 kPa. Above this, sharply declining transpiration was followed by leaf death. In contrast, wild type showed a steady or slightly declining transpiration rate up to VPD of nearly 7 kPa, and fully recovered photosynthetic function afterward. There were marked differences in discrimination against 13CO2 (13C) and C18OO (18O) between abi1 and wild-type plants. The 13C indicated that intercellular CO2 concentrations decreased with VPD in wild-type plants, but not in abi1 plants. The 18O reflected progressive stomatal closure in wild type in response to increasing VPD; however, in abi1, stomata remained open and oxygen atoms of CO2 continued to exchange with 18O enriched leaf water. Coupled measurements of 18O and gas exchange were used to estimate intercellular vapor pressure, ei. In wild-type leaves, there was no evidence of unsaturation of ei, even at VPD above 6 kPa. In abi1 leaves, ei approached 0.6 times saturation vapor pressure before the precipitous decline in transpiration rate. For wild type, a sensitive stomatal response to increasing VPD was pivotal in preventing unsaturation of ei. In abi1, after taking unsaturation into account, stomatal conductance increased with increasing VPD, consistent with a disabled active response of guard cell osmotic pressure.

    更新日期:2019-11-26
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