Showing papers in "Plant and Cell Physiology in 2018"

ATTED-II in 2018: A Plant Coexpression Database Based on Investigation of the Statistical Property of the Mutual Rank Index

Abstract: ATTED-II (http://atted.jp) is a coexpression database for plant species to aid in the discovery of relationships of unknown genes within a species. As an advanced coexpression analysis method, multispecies comparisons have the potential to detect alterations in gene relationships within an evolutionary context. However, determining the validity of comparative coexpression studies is difficult without quantitative assessments of the quality of coexpression data. ATTED-II (version 9) provides 16 coexpression platforms for nine plant species, including seven species supported by both microarray- and RNA sequencing (RNAseq)-based coexpression data. Two independent sources of coexpression data enable the assessment of the reproducibility of coexpression. The latest coexpression data for Arabidopsis (Ath-m.c7-1 and Ath-r.c3-0) showed the highest reproducibility (Jaccard coefficient = 0.13) among previous coexpression data in ATTED-II. We also investigated the statistical basis of the mutual rank (MR) index as a coexpression measure by bootstrap sampling of experimental units. We found that the error distribution of the logit-transformed MR index showed normality with equal variances for each coexpression platform. Because the MR error was strongly correlated with the number of samples for the coexpression data, typical confidence intervals for the MR index can be estimated for any coexpression platform. These new, high-quality coexpression data can be analyzed with any tool in ATTED-II and combined with external resources to obtain insight into plant biology.

Salicylic Acid and Jasmonic Acid Pathways are Activated in Spatially Different Domains Around the Infection Site During Effector-Triggered Immunity in Arabidopsis thaliana

Abstract: The innate immune response is, in the first place, elicited at the site of infection. Thus, the host response can be different among the infected cells and the cells surrounding them. Effector-triggered immunity (ETI), a form of innate immunity in plants, is triggered by specific recognition between pathogen effectors and their corresponding plant cytosolic immune receptors, resulting in rapid localized cell death known as hypersensitive response (HR). HR cell death is usually limited to a few cells at the infection site, and is surrounded by a few layers of cells massively expressing defense genes such as Pathogenesis-Related Gene 1 (PR1). This virtually concentric pattern of the cellular responses in ETI is proposed to be regulated by a concentration gradient of salicylic acid (SA), a phytohormone accumulated around the infection site. Recent studies demonstrated that jasmonic acid (JA), another phytohormone known to be mutually antagonistic to SA in many cases, is also accumulated in and required for ETI, suggesting that ETI is a unique case. However, the molecular basis for this uniqueness remained largely to be solved. Here, we found that, using intravital time-lapse imaging, the JA signaling pathway is activated in the cells surrounding the central SA-active cells around the infection sites in Arabidopsis thaliana. This distinct spatial organization explains how these two phythormone pathways in a mutually antagonistic relationship can be activated simultaneously during ETI. Our results re-emphasize that the spatial consideration is a key strategy to gain mechanistic insights into the apparently complex signaling cross-talk in immunity.

Chloride: from Nutrient to Toxicant.

Abstract: In salinized soils in which chloride (Cl-) is the dominant salt anion, growth of plants that tolerate only low concentrations of salt (glycophytes) is disturbed by Cl- toxicity. Chlorotic discolorations precede necrotic lesions, causing yield reductions. Little is known about the effects of Cl- toxicity on these dysfunctions. A lack of understanding exists regarding (i) the molecular and physiological mechanisms that lead to Cl--induced damage and (ii) the adaptive aspects of induced tolerance to Cl- salinity. Here, mechanistic explanations for the Cl--induced stress responses are proposed and novel ideas and strategies by which glycophytic plants avoid the excessive accumulation of Cl- are reviewed. New experiments are suggested to test the proposed hypotheses. Cl- salinity constrains global food security and thus we urgently need more research into the causes and consequences of Cl- salinity.

TFL1-Like Proteins in Rice Antagonize Rice FT-Like Protein in Inflorescence Development by Competition for Complex Formation with 14-3-3 and FD.

Abstract: Hd3a, a rice homolog of FLOWERING LOCUS T (FT), is a florigen that induces flowering. Hd3a forms a ternary 'florigen activation complex' (FAC) with 14-3-3 protein and OsFD1 transcription factor, a rice homolog of FD that induces transcription of OsMADS15, a rice homolog of APETALA1 (AP1), which leads to flowering. TERMINAL FLOWER 1 (TFL1) represses flowering and controls inflorescence architecture. However, the molecular basis for floral repression by TFL1 remains poorly understood. Here we show that RICE CENTRORADIALIS (RCN), rice TFL1-like proteins, compete with Hd3a for 14-3-3 binding. All four RCN genes are predominantly expressed in the vasculature, and RCN proteins are transported to the shoot apex to antagonize florigen activity and regulate inflorescence development. The antagonistic function of RCN to Hd3a is dependent on its 14-3-3 binding activity. Our results suggest a molecular basis for regulation of the balance between florigen FT and anti-florigen TFL1.

Beyond Defense: Multiple Functions of Benzoxazinoids in Maize Metabolism.

Abstract: Benzoxazinoids are a class of indole-derived plant metabolites that function in defense against numerous pests and pathogens. Due to their abundance in maize (Zea mays) and other important cereal crops, benzoxazinoids have been the subject of extensive research for >50 years. Whereas benzoxazinoids can account for 1% or more of the dry weight in young seedlings constitutively, their accumulation in older plants is induced locally by pest and pathogen attack. Although the biosynthetic pathways for most maize benzoxazinoids have been identified, unanswered questions remain about the developmental and defense-induced regulation of benzoxazinoid metabolism. Recent research shows that, in addition to their central role in the maize chemical defense repertoire, benzoxazinoids may have important functions in regulating other defense responses, flowering time, auxin metabolism, iron uptake and perhaps aluminum tolerance. Investigation of natural variation in maize benzoxazinoid accumulation, which is greatly facilitated by recent genomics advances, will have a major impact in this research area by leading to the discovery of previously unknown genes and functions of benzoxazinoid metabolism.

BZR1 Transcription Factor Regulates Heat Stress Tolerance Through FERONIA Receptor-Like Kinase-Mediated Reactive Oxygen Species Signaling in Tomato

Abstract: BRASSINAZOLE RESISTANT 1 (BZR1), the critical regulator of brassinosteroid (BR) response, participates in various BR-mediated developmental processes. However, the roles of BZR1 in stress tolerance are less clear. Here, we found that BZR1-like protein in tomato controls BR response and is involved in thermotolerance by regulating the FERONIA (FER) homologs. The CRISPR-bzr1 mutant showed reduced growth and was not responsive to 24-epibrassinolide (EBR) with regard to the promotion of plant growth. Mutation in BZR1 impaired the induction of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1), production of H2O2 in the apoplast and heat tolerance. Exogenous H2O2 recovered the heat tolerance of the tomato bzr1 mutant. Overexpression of BZR1 enhanced the production of apoplastic H2O2 and heat stress responses. However, silencing of RBOH1 abolished the BZR1-mediated heat tolerance. Further analysis showed that BZR1 bound to the promoters of FERONIA2 (FER2) and FER3 and induced their expression. Silencing of FER2/3 suppressed BZR1-dependent BR signaling for the induction of RBOH1 transcripts, accumulation of apoplastic H2O2 and heat tolerance. These results indicate that BZR1 regulates heat stress responses in tomato through RBOH1-dependent reactive oxygen species (ROS) signaling, which is at least partially mediated by FER2 and FER3.

The Putative Peptide Gene FEP1 Regulates Iron Deficiency Response in Arabidopsis.

Abstract: Iron is an essential element for all organisms, and plants have developed sophisticated systems to acquire iron and maintain iron homeostasis. We found that an Arabidopsis thaliana ABA-hypersensitive mutant, aba hypersensitive germination2-1 (ahg2-1), that is known to be defective in mitochondrial mRNA regulation, had increased expression of iron deficiency response genes. The ahg2-1 mutant had lower heme levels than the wild type. Transcriptome data further revealed that novel genes encoding short polypeptides were highly expressed in this mutant. The expression of one of these genes, which we named FE-UPTAKE-INDUCING PEPTIDE 1 (FEP1), was induced under iron-deficient conditions and was observed in the vascular tissues of the leaves and roots, as well as in leaf mesophyll cells. Notably, deletion or insertion mutations of FEP1 exhibited impaired iron accumulation in shoots but normal iron levels in roots. Artificially induced expression of FEP1 was sufficient to induce iron deficiency response genes, such as basic HELIX-LOOP-HELIX 38 (bHLH38), bHLH39, IRON-REGULATED TRANSPORTER1 (IRT1) and FERRIC REDUCTION OXIDASE2 (FRO2), and led to iron accumulation in planta. Further analysis confirmed that the encoded peptide, but not the FEP1 RNA, was responsible for this activity. Remarkably, the activation of bHLH39 by FEP1 was independent of FER-LIKE IRON DEFICIENCY INDUCED (FIT), a key transcription factor in the iron deficiency response. Taken together, our results indicate that FEP1 functions in iron homeostasis through a previously undescribed regulatory mechanism for iron acquisition in Arabidopsis.

In Concert: Orchestrated Changes in Carbohydrate Homeostasis Are Critical for Plant Abiotic Stress Tolerance

Abstract: The sessile lifestyle of higher plants is accompanied by their remarkable ability to tolerate unfavorable environmental conditions. This is because, during evolution, plants developed a sophisticated repertoire of molecular and metabolic reactions to cope with changing biotic and abiotic challenges. In particular, the abiotic factors light intensity and ambient temperature are characterized by altering their amplitude within comparably short periods of time and are causative for onset of dynamic plant responses. These rapid responses in plants are also classified as 'acclimation reactions' which differ, due to their reversibility and duration, from non-reversible 'adaptation reactions'. In this review, we demonstrate the remarkable importance of stress-induced changes in carbohydrate homeostasis of plants exposed to high light or low temperatures. These changes represent a co-ordinated process comprising modifications of (i) the concentrations of selected sugars; (ii) starch turnover; (iii) intracellular sugar compartmentation; and (iv) corresponding gene expression patterns. The critical importance of these individual processes has been underlined in the recent past by the analyses of a large number of mutant plants. The outcome of these analyses raised our understanding of acclimation processes in plants per se but might even become instrumental to develop new concepts for directed breeding approaches with the aim to increase abiotic stress tolerance of crop species, which in most cases have high stress sensitivity. The latter direction of plant research is of special importance since abiotic stress stimuli strongly impact on crop productivity and are expected to become even more pronounced because of human activities which alter environmental conditions rapidly.

Combinatorial Regulation of Stilbene Synthase Genes by WRKY and MYB Transcription Factors in Grapevine (Vitis vinifera L.)

Abstract: Stilbene synthase (STS) is the key enzyme leading to the biosynthesis of resveratrol. Recently we reported two R2R3-MYB transcription factor (TF) genes that regulate the stilbene biosynthetic pathway in grapevine: VviMYB14 and VviMYB15. These genes are strongly co-expressed with STS genes under a range of stress and developmental conditions, in agreement with the specific activation of STS promoters by these TFs. Genome-wide gene co-expression analysis using two separate transcriptome compendia based on microarray and RNA sequencing data revealed that WRKY TFs were the top TF family correlated with STS genes. On the basis of correlation frequency, four WRKY genes, namely VviWRKY03, VviWRKY24, VviWRKY43 and VviWRKY53, were further shortlisted and functionally validated. Expression analyses under both unstressed and stressed conditions, together with promoter-luciferase reporter assays, suggested different hierarchies for these TFs in the regulation of the stilbene biosynthetic pathway. In particular, VviWRKY24 seems to act as a singular effector in the activation of the VviSTS29 promoter, while VviWRKY03 acts through a combinatorial effect with VviMYB14, suggesting that these two regulators may interact at the protein level as previously reported in other species.

Vacuolar Transporters for Cadmium and Arsenic in Plants and their Applications in Phytoremediation and Crop Development.

Abstract: Soil contamination by heavy metals and metalloids such as cadmium (Cd) and arsenic (As) poses a major threat to the environment and to human health. Vacuolar sequestration is one of the main mechanisms by which plants control toxic materials including Cd and As. Understanding the mechanisms of heavy metal tolerance and accumulation can be useful for both phytoremediation and safe crop development. In this review, we summarize recent advances in deciphering the molecular mechanisms underlying vacuolar sequestration of Cd and As, and discuss potential biotechnological applications of this knowledge and efforts towards attaining these goals.

Strigolactone Biosynthesis Genes of Rice are Required for the Punctual Entry of Arbuscular Mycorrhizal Fungi into the Roots.

Abstract: Arbuscular mycorrhiza (AM) is a mutualistic association between most plant species and the ancient fungal phylum Glomeromycota in roots, and it plays a key role in a plant's nutrient uptake from the soil. Roots synthesize strigolactones (SLs), derivatives of carotenoids, and exude them to induce energy metabolism and hyphal branching of AM fungi. Despite the well-documented roles of SLs in the pre-symbiotic phase, little is known about the role of SLs in the process of root colonization. Here we show that the expansion of root colonization is suppressed in the mutants of rice (Oryza sativa) SL biosynthesis genes, carotenoid cleavage dioxygenase D10 and more severely in D17. Interestingly, most of the colonization process is normal, i.e. AM fungal hyphae approach the roots and cling around them, and epidermal penetration, arbuscule size, arbuscule number per hyphopodium and metabolic activity of the intraradical mycelium are not affected in d10 and d17 mutants. In contrast, hyphopodium formation is severely attenuated. Our observations establish the requirement for SL biosynthesis genes for efficient hyphopodium formation, suggesting that SLs are required in this process. Efficient hyphopodium formation is required for the punctual internalization of hyphae into roots and maintaining the expansion of colonization.

The WOX11-LBD16 Pathway Promotes Pluripotency Acquisition in Callus Cells During De Novo Shoot Regeneration in Tissue Culture.

Abstract: De novo shoot regeneration in tissue culture undergoes at least two phases. Explants are first cultured on auxin-rich callus-inducing medium (CIM) to produce a group of pluripotent cells termed callus; the callus is then transferred to cytokinin rich shoot-inducing medium (SIM) to promote the formation of shoot progenitor cells, from which adventitious shoots may differentiate. Here, we show that the Arabidopsis thaliana transcription factor gene LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) is involved in pluripotency acquisition in callus cells. LBD16, which is activated by WUSCHEL RELATED HOMEOBOX11 (WOX11), is specifically expressed in the newly formed callus on CIM and its expression decreases quickly when callus is moved to SIM. Blocking the WOX11-LBD16 pathway results in the loss of pluripotency in callus cultured on CIM, leading to shooting defects on SIM. Further analysis showed that LBD16 may function in the establishment of the root primordium-like identity in the newly formed callus, indicating that the root primordium-like identity is the cellular nature of pluripotency in callus cells. Additionally, LBD16 promotes cell division during callus initiation. Our study clarified that the WOX11-LBD16 pathway promotes pluripotency acquisition in callus cells.

A Gene Regulatory Network for Cellular Reprogramming in Plant Regeneration

Abstract: Wounding triggers organ regeneration in many plant species, and application of plant hormones, such as auxin and cytokinin, enhances their regenerative capacities in tissue culture. Recent studies have identified several key players mediating wound- and/or plant hormone-induced cellular reprogramming, but the global architecture of gene regulatory relationships underlying plant cellular reprogramming is still far from clear. In this study, we uncovered a gene regulatory network (GRN) associated with plant cellular reprogramming by using an enhanced yeast one-hybrid (eY1H) screen systematically to identify regulatory relationships between 252 transcription factors (TFs) and 48 promoters. Our network analyses suggest that wound- and/or hormone-invoked signals exhibit extensive cross-talk and regulate many common reprogramming-associated genes via multilayered regulatory cascades. Our data suggest that PLETHORA 3 (PLT3), ENHANCER OF SHOOT REGENERATION 1 (ESR1) and HEAT SHOCK FACTOR B 1 (HSFB1) act as critical nodes that have many overlapping targets and potentially connect upstream stimuli to downstream developmental decisions. Interestingly, a set of wound-inducible APETALA 2/ETHYLENE RESPONSE FACTORs (AP2/ERFs) appear to regulate these key genes, which, in turn, form feed-forward cascades that control downstream targets associated with callus formation and organ regeneration. In addition, we found another regulatory pathway, mediated by LATERAL ORGAN BOUNDARY/ASYMMETRIC LEAVES 2 (LOB/AS2) TFs, which probably plays a distinct but partially overlapping role alongside the AP2/ERFs in the putative gene regulatory cascades. Taken together, our findings provide the first global picture of the GRN governing plant cell reprogramming, which will serve as a valuable resource for future studies.

Transcriptional Regulation of Arbuscular Mycorrhiza Development.

Abstract: Arbuscular mycorrhiza (AM) is an ancient symbiosis between land plants and fungi of the glomeromycotina that is widespread in the plant kingdom. AM improves plant nutrition, stress resistance and general plant performance, and thus represents a promising addition to sustainable agricultural practices. In return for delivering mineral nutrients, the obligate biotrophic AM fungi receive up to 20% of the photosynthetically fixed carbon from the plant. AM fungi colonize the inside of roots and form highly branched tree-shaped structures, called arbuscules, in cortex cells. The pair of the arbuscule and its host cell is considered the central functional unit of the symbiosis as it mediates the bidirectional nutrient exchange between the symbionts. The development and spread of AM fungi within the root is predominantly under the control of the host plant and depends on its developmental and physiological status. Intracellular accommodation of fungal structures is enabled by the remarkable plasticity of plant cells, which undergo drastic subcellular rearrangements. These are promoted and accompanied by cell-autonomous transcriptional reprogramming. AM development can be dissected into distinct stages using plant mutants. Progress in the application of laser dissection technology has allowed the assignment of transcriptional responses to specific stages and cell types. The first transcription factors controlling AM-specific gene expression and AM development have been discovered, and cis-elements required for AM-responsive promoter activity have been identified. An understanding of their connectivity and elucidation of transcriptional networks orchestrating AM development can be expected in the near future.

Unveiling the Molecular Mechanisms of Plant Autophagy-From Autophagosomes to Vacuoles in Plants.

Abstract: Autophagy is an evolutionarily conserved intracellular vacuolar process. Since Christian de Duve first coined the term 'autophagy' in 1963, it had not been well understood at the molecular level until much later, due to limitations in biochemical approaches and/or morphological approaches posed by electron microscopy. An important milestone was achieved with the isolation and identification of autophagy-related (ATG) genes by genetic screening using yeast Saccharomyces cerevisiae. ATG genes are well conserved in most eukaryotic organisms, which allowed the subsequent isolation of ATG gene-knockouts in plants. From the phenotypic analyses of the autophagy-defective plants, the physiological roles of autophagy have been predicted. However, in some cases, all the phenotypes cannot be simply explained by defects in autophagy. Therefore, in order to fully understand the physiological implications of plant autophagy, it is quite important to elucidate the molecular mechanisms involved in each process in macro-/micro-autophagy. Although, until recently, our understanding of the molecular mechanisms of plant autophagy was lagging compared to similar research in yeast and animals, current studies have made many great advances in the plant research field. In this review, we discuss current knowledge of the molecular mechanisms of plant autophagy, from autophagy-induction/autophagosome-formation to vacuolar degradation, comparing these to processes in yeast and mammals. We also review aspects of plant autophagy research that require further investigation in the future.

Sensing and Signaling of Phosphate Starvation: From Local to Long Distance.

Abstract: Phosphorus (P) is an essential nutrient, but low concentrations of phosphate (Pi), the predominant form in which it is acquired, in the soil often limits plant growth and reproduction. To adapt to low Pi availability, plants have developed intricate regulatory mechanisms that integrate the environmental stimuli with internal cues in order to exploit the use of P. These mechanisms include sensing external and internal Pi concentrations along with co-ordination between local and long-distance signaling pathways. The downstream actions governed by these signaling pathways include local responses for remodeling the root system architecture and systemic responses for modulating the activities of Pi uptake, remobilization and recycling. As an initially acquired molecule, Pi is considered to be a primary signal that directly regulates Pi starvation responses and sets in motion the generation of subsequent signals, such as hormones, sugars, P-containing metabolites, peptides and mobile RNAs. In this review, we summarize recent progress in understanding the regulatory pathways mediated by these signaling molecules that underlie both local and systemic responses to Pi deprivation, and discuss the potential cross-talk among these signaling pathways.

New Perspectives on Chloroplast Protein Import.

Abstract: Virtually all chloroplasts in extant photosynthetic eukaryotes derive from a single endosymbiotic event that probably occurred more than a billion years ago between a host eukaryotic cell and a cyanobacterium-like ancestor. Many endosymbiont genes were subsequently transferred to the host nuclear genome, concomitant with the establishment of a system for protein transport through the chloroplast double-membrane envelope. Presently, 2,000-3,000 different nucleus-encoded chloroplast proteins must be imported into the chloroplast following their synthesis in the cytosol. The TOC (translocon at the outer envelope membrane of chloroplasts) and TIC (translocon at the inner envelope membrane of chloroplasts) complexes are protein translocation machineries at the outer and inner envelope membranes, respectively, that facilitate this chloroplast protein import with the aid of a TIC-associated ATP-driven import motor. All the essential components of this protein import system seemed to have been identified through biochemical analyses and subsequent genetic studies that initiated in the late 1990s. However, in 2013, the Nakai group reported a novel inner envelope membrane TIC complex, for which a novel ATP-driven import motor associated with this TIC complex is likely to exist. In this mini review, I will summarize these recent discoveries together with new, or reanalyzed, data presented by other groups in recent years. Whereas the precise concurrent view of chloroplast protein import is still a matter of some debate, it is anticipated that the entire TOC/TIC/ATP motor system, including any novel components, will be conclusively established in the next decade. Such findings may lead to an extensively revised view of the evolution and molecular mechanisms of chloroplast protein import.

Changes in Membrane Lipid Composition and Function Accompanying Chilling Injury in Bell Peppers.

Abstract: Bell peppers are vulnerable to low temperature (<7°C) and subject to chilling injury (CI). To elucidate the relationship between cell membrane lipid composition and CI, a membrane lipidomic approach was taken. In addition, we performed microstructural analysis and low-field nuclear magnetic resonance to better understand CI. We also monitored primary physiological metabolism parameters to explain lipidomics. Our study indicated that cellular structure damage was more serious at 4°C, mostly represented by damage to the plasmalemma and plastid degradation. Membrane lipidomic data analysis reveals monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid as crucial biomarkers during CI. Furthermore, the significant increase in proline, electrolyte leakage and phospholipase D in chilled fruits also proved that membrane lipid metabolism is involved in the response to low temperature stress. To our knowledge, this study is the first attempt to describe the CI mechanisms in bell peppers based on membrane lipidomics.

Stress-Inducible Galactinol Synthase of Chickpea (CaGolS) is Implicated in Heat and Oxidative Stress Tolerance Through Reducing Stress-Induced Excessive Reactive Oxygen Species Accumulation.

Abstract: Raffinose family oligosaccharides (RFOs) participate in various aspects of plant physiology, and galactinol synthase (GolS; EC 2.4.1.123) catalyzes the key step of RFO biosynthesis. Stress-induced accumulation of RFOs, in particular galactinol and raffinose, has been reported in a few plants; however, their precise role and mechanistic insight in stress adaptation remain elusive. In the present study, we have shown that the GolS activity as well as galactinol and raffinose content are significantly increased in response to various abiotic stresses in chickpea. Transcriptional analysis indicated that the CaGolS1 and CaGolS2 genes are induced in response to different abiotic stresses. Interestingly, heat and oxidative stress preferentially induce CaGolS1 over CaGolS2. In silco analysis revealed several common yet distinct cis-acting regulatory elements in their 5'-upstream regulatory sequences. Further, in vitro biochemical analysis revealed that the CaGolS1 enzyme functions better in stressful conditions than the CaGolS2 enzyme. Finally, Arabidopsis transgenic plants constitutively overexpressing CaGolS1 or CaGolS2 exhibit not only significantly increased galactinol but also raffinose content, and display better growth responses than wild-type or vector control plants when exposed to heat and oxidative stress. Further, improved tolerance of transgenic lines is associated with reduced accumulation of reactive oxygen species (ROS) and consequent lipid peroxidation as compared with control plants. Collectively, our data imply that GolS enzyme activity and consequent galactinol and raffinose content are significantly increased in response to stresses to mitigate stress-induced growth inhibition by restricting excessive ROS accumulation and consequent lipid peroxidation in plants.

Characterization and Alternative Splicing Profiles of the Lipoxygenase Gene Family in Tea Plant (Camellia sinensis).

Abstract: Oxylipins, including jasmonic acid (JA) and volatiles, are important for signaling in plants, and these are formed by the lipoxygenase (LOX) enzyme family. There is a large gap in understanding of the underlying molecular basis of their roles in tea plants. Here, we identified 11 CsLOX genes from the tea plant (Camellia sinensis), and characterized their phylogeny, gene structure and protein features into three subclasses. We then examined their enzymatic activities, LOX expression and alternative splicing of transcripts during development and in response to abiotic or biotic stresses in tea plants. In vitro expressed protein assays showed that the CsLOX2, 3 and 9 enzymatically function to produce 9/13-HPOT, 13-HPOT and 9-HPOT, respectively. CsLOX2 and CsLOX9 green fluorescent protein (GFP) fusion proteins localized to chloroplasts and the cytoplasm, respectively. RNA sequencing, quantitative reverse transcription-PCR and Northern blot analysis suggested that CsLOX5, 6 and 9 were predominantly expressed in seeds, flowers and roots, respectively. CsLOX2, 3, 4, 6 and 7 were up-regulated after attack by the insect Ectropis oblique, while CsLOX1 was induced after infection with the pathogen Glomerella cingulata. CsLOX3, 7 and 10 were up-regulated by JA but not ABA or salicylic acid. Long-term cold stress down-regulated CsLOX expression while a short duration of cold induced the expression of CsLOX1, 6 and 7. Alternatively spliced transcripts of six CsLOX genes were dynamically regulated through time and varied in relative abundances under the investigated stresses; we propose a mechanism of competing or compensating regulation between isoforms. This study improves our understanding of evolution of LOXs and regulation of their diverse functions in plants.

BES1 and BZR1 Redundantly Promote Phloem and Xylem Differentiation.

Abstract: Vascular development is a good model for studying cell differentiation in plants. Two conductive tissues, the xylem and phloem, are derived from common stem cells known as procambial/cambial cells. Glycogen synthase kinase 3 proteins (GSK3s) play crucial roles in maintaining procambial/cambial cells by suppressing their differentiation into xylem or phloem cells. We previously designed an in vitro culture system for analyzing vascular cell differentiation named VISUAL (Vascular cell Induction culture System Using Arabidopsis Leaves). Using this system, we found that the transcription factor BRI1-EMS-SUPPRESSOR 1 (BES1) functions as a downstream target of GSK3s during xylem differentiation. However, the function of BES1 in vascular development remains largely unknown. Here, we found that, in addition to xylem differentiation, BES1 positively regulates phloem differentiation downstream of GSK3s. Transcriptome analysis using VISUAL confirmed that BES1 promotes bi-directional differentiation of procambial cells into xylem and phloem cells. Genetic analysis of loss-of-function mutants newly generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system revealed that BRASSINAZOLE RESISTANT 1 (BZR1), the closest homolog of BES1, functions in vascular development redundantly with BES1. Notably, BZR1 has a weaker impact on vascular cell differentiation than BES1, suggesting that they contribute differentially to this process. In conclusion, our findings indicate that BES1 and BZR1 are key regulators of both xylem and phloem cell differentiation from vascular stem cells.

CsATAF1 Positively Regulates Drought Stress Tolerance by an ABA-Dependent Pathway and by Promoting ROS Scavenging in Cucumber.

Abstract: The NAC transcription factors play vital roles in responding to drought stress in plants; however, the molecular mechanisms remain largely unknown in cucumber. Suppression of CsATAF1 via RNA interference (RNAi) weakened drought stress tolerance in cucumber due to a higher water loss rate in leaves, a higher level of hydrogen peroxide (H2O2) and superoxide radicals (O2·-), increased malondialdehyde (MDA) content, lower Fv/Fm ratios and lower antioxidant enzyme activity. The analysis of root length and stomatal apertures showed that CsATAF1-RNAi cucumber plants were less responsive to ABA. In contrast, CsATAF1-overexpression (OE) plants showed increased drought stress tolerance and sensitivity to ABA. Quantitative PCR (qPCR) analysis showed that expression of several stress-responsive genes was significantly up-regulated in CsATAF1-OE transformants and down-regulated in CsATAF1-RNAi transformants. CsABI5, CsCu-ZnSOD and CsDREB2C were verified as direct target genes of CsATAF1. Yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) further substantiated that CsATAF1 bound to the promoters of CsABI5, CsCu-ZnSOD and CsDREB2C. Transient expression in tobacco leaves and cucumber protoplasts showed that CsATAF1 directly up-regulated the expression of CsABI5, CsCu-ZnSOD and CsDREB2C. Our results demonstrated that CsATAF1 functioned as a positive regulator in response to drought stress by an ABA-dependent pathway and decreasing reactive oxygen species (ROS) accumulation in cucumber.

Vacuolar Protein Degradation via Autophagy Provides Substrates to Amino Acid Catabolic Pathways as an Adaptive Response to Sugar Starvation in Arabidopsis thaliana

Abstract: The vacuolar lytic degradation of proteins releases free amino acids that plants can use instead of sugars for respiratory energy production. Autophagy is a major cellular process leading to the transport of proteins into the vacuole for degradation. Here, we examine the contribution of autophagy to the amino acid metabolism response to sugar starvation in mature leaves of Arabidopsis thaliana. During sugar starvation arising from the exposure of wild-type (WT) plants to darkness, autophagic transport of chloroplast stroma, which contains most of the proteins in a leaf, into the vacuolar lumen was induced within 2 d. During this time, the level of soluble proteins, primarily Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), decreased and the amount of free amino acid increased. In dark-treated autophagy-defective (atg) mutants, the decrease of soluble proteins was suppressed, which resulted in the compromised release of basic amino acids, branched-chain amino acids (BCAAs) and aromatic amino acids. The impairment of BCAA catabolic pathways in the knockout mutants of the electron transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (etfqo) complex and the electron donor protein isovaleryl-CoA dehydrogenase (ivdh) caused a reduced tolerance to dark treatment similar to that in the atg mutants. The enhanced accumulation of BCAAs in the ivdh and etfqo mutants during the dark treatment was reduced by additional autophagy deficiency. These results indicate that vacuolar protein degradation via autophagy serves as an adaptive response to disrupted photosynthesis by providing substrates to amino acid catabolic pathways, including BCAA catabolism mediated by IVDH and ETFQO.

SAG12, a Major Cysteine Protease Involved in Nitrogen Allocation during Senescence for Seed Production in Arabidopsis thaliana

Abstract: SAG12 is the most widely used senescence-associated reference gene for characterizing leaf senescence, and the increase in SAG12 protein during leaf senescence is remarkable. However, the role of this cysteine protease in N remobilization and the leaf senescence process remains unclear. The role of SAG12 has been poorly investigated and the few reports dealing with this are somewhat controversial. Indeed, sag12 Arabidopsis mutants have not shown any phenotype, while OsSAG12-1 and OsSAG12-2 overexpression in rice moderates senescence progression. Therefore, this study aims at clarifying the role of the SAG12 cysteine protease during the entire plant life span and during leaf senescence. Arabidopsis thaliana plants knocked-out for the SAG12 gene (sag12) did not exhibit any special phenotypic traits when grown under optimal nitrogen supply (HN), suggesting that other cysteine proteases could provide compensatory effects. Moreover, for the first time, this study shows that aspartate protease activity is significantly increased in sag12. Among the putative aspartate proteases involved, a CND41-like aspartate protease has been identified. Under low nitrogen (LN) availability, when inducible proteolytic systems are not sufficient to cope with SAG12 depletion, a decrease in yield is observed. Altogether, these results show that SAG12 (and perhaps also aspartate proteases) could be involved in RuBisCO degradation during the leaf senescence associated with seed filling.

MiR858-Mediated Regulation of Flavonoid-Specific MYB Transcription Factor Genes Controls Resistance to Pathogen Infection in Arabidopsis.

Abstract: MicroRNAs (miRNAs) are a class of short endogenous non-coding small RNAs that direct post-transcriptional gene silencing in eukaryotes. In plants, the expression of a large number of miRNAs has been shown to be regulated during pathogen infection. However, the functional role of the majority of these pathogen-regulated miRNAs has not been elucidated. In this work, we investigated the role of Arabidopsis miR858 in the defense response of Arabidopsis plants to infection by fungal pathogens with necrotrophic (Plectosphaerella cucumerina) or hemibiotrophic (Fusarium oxysporum and Colletotrichum higginsianum) lifestyles. Whereas overexpression of MIR858 enhances susceptibility to pathogen infection, interference with miR858 activity by target mimics (MIM858 plants) results in disease resistance. Upon pathogen challenge, stronger activation of the defense genes PDF1.2 and PR4 occurs in MIM858 plants than in wild-type plants, whereas pathogen infection induced weaker activation of these genes in MIR858 overexpressor plants. Reduced miR858 activity, and concomitant up-regulation of miR858 target genes, in MIM858 plants, also leads to accumulation of flavonoids in Arabidopsis leaves. The antifungal activity of phenylpropanoid compounds, including flavonoids, is presented. Furthermore, pathogen infection or treatment with fungal elicitors is accompanied by a gradual decrease in MIR858 expression in wild-type plants, suggesting that miR858 plays a role in PAMP (pathogen-associated molecular pattern)-triggered immunity. These data support that miR858 is a negative regulator of Arabidopsis immunity and provide new insights into the relevant role of miR858-mediated regulation of the phenylpropanoid biosynthetic pathway in controlling Arabidopsis immunity.

A Phi-Class Glutathione S-Transferase Gene for Verticillium Wilt Resistance in Gossypium arboreum Identified in a Genome-Wide Association Study

Abstract: Verticillium wilt disease is one of the most destructive biotic stresses faced by cotton plants. Here, we performed a genome-wide association study (GWAS) in 215 Chinese Gossypium arboreum accessions inoculated as seedlings with Verticillium dahliae to identify candidate loci involved in wilt resistance. We identified 309 loci that had a significant association with Verticillium wilt resistance and - log(P) values >5.0; the highest signal appeared on Ca3 in a 74 kb haplotype block. Five genes were also located within this haplotype block. One of these genes, CG05, was positioned close to the most significant SNP Ca3_23037225 (14 kb); expression of the gene was induced by V. dahliae or by treatment with salicylic acid (SA). Therefore, we suggest that CG05 may respond to invasion by V. dahliae via an SA-related signaling pathway, and we designated this gene as GaGSTF9. We showed that GaGSTF9 was a positive regulator of Verticillium wilt through the use of virus-induced gene silencing (VIGS) and overexpression in Arabidopsis. In addition, the glutathione S-transferase (GST) mutant gstf9 of Arabidopsis was found to be more susceptible to Verticillium wilt than wild-type plants. The levels of endogenous SA and hydrogen peroxide had a significant effect on Arabidopsis plants that overexpressed GaGSTF9, indicating that GST may regulate reactive oxygen species content via catalytic reduction of the tripeptide glutathione (GSH), and then affect SA content. Our data demonstrated that GaGSTF9 was a key regulator mediating cotton responses to V. dahliae and a potential candidate gene for cotton genetic improvement.

A Sodium Transporter HvHKT1;1 Confers Salt Tolerance in Barley via Regulating Tissue and Cell Ion Homeostasis.

Abstract: Our previous studies showed that high salt tolerance in Tibetan wild barley accessions was associated with HvHKT1;1, a member of the high-affinity potassium transporter family. However, molecular mechanisms of HvHKT1;1 for salt tolerance and its roles in K+/Na+ homeostasis remain to be elucidated. Functional characterization of HvHKT1;1 was conducted in the present study. NaCl-induced transcripts of HvHKT1;1 were significantly higher in the roots of Tibetan wild barley XZ16 relative to other genotypes, being closely associated with its higher biomass and lower tissue Na+ content under salt stress. Heterologous expression of HvHKT1;1 in Saccharomyces cerevisiae (yeast) and Xenopus laevis oocytes showed that HvHKT1;1 had higher selectivity for Na+ over K+ and other monovalent cations. HvHKT1;1 was found to be localized at the cell plasma membrane of root stele and epidermis. Knock-down of HvHKT1;1 in barley led to higher Na+ accumulation in both roots and leaves, while overexpression of HvHKT1;1 in salt-sensitive Arabidopsis hkt1-4 and sos1-12 loss-of-function lines resulted in significantly less shoot and root Na+ accumulation. Additionally, microelectrode ion flux measurements and root elongation assay revealed that the transgenic Arabidopsis plants exhibited a remarkable capacity for regulation of Na+, K+, Ca2+ and H+ homeostasis under salt stress. These results indicate that HvHKT1;1 is critical in radial root Na+ transport, which eventually reduces shoot Na+ accumulation. Additionally, HvHKT1;1 may be indirectly involved in retention of K+ and Ca2+ in root cells, which also improves plant salt tolerance.

Application of CRISPR Interference for Metabolic Engineering of the Heterocyst-Forming Multicellular Cyanobacterium Anabaena sp. PCC 7120.

Abstract: Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that performs nitrogen fixation. This cyanobacterium has been extensively studied as a model for multicellularity in prokaryotic cells. We have been interested in photosynthetic production of nitrogenous compounds using A. 7120. However, the lack of efficient gene repression tools has limited its usefulness. We originally developed an artificial endogenous gene repression method in this cyanobacterium using small antisense RNA. However, the narrow dynamic range of repression of this method needs to be improved. Recently, clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) technology was developed and was successfully applied in some unicellular cyanobacteria. The technology requires expression of nuclease-deficient CRISPR-associated protein 9 (dCas9) and a single guide RNA (sgRNA) that is complementary to a target sequence, to repress expression of the target gene. In this study, we employed CRISPRi technology for photosynthetic production of ammonium through repression of glnA, the only gene encoding glutamine synthetase that is essential for nitrogen assimilation in A. 7120. By strictly regulating dCas9 expression using the TetR gene induction system, we succeeded in fine-tuning the GlnA protein in addition to the level of glnA transcripts. Expression of sgRNA by the heterocyst-specific nifB promoter led to efficient repression of GlnA in heterocysts, as well as in vegetative cells. Finally, we showed that ammonium is excreted into the medium only when inducers of expression of dCas9 were added. In conclusion, CRISPRi enables temporal control of desired products and will be a useful tool for basic science.

SUMO E3 Ligase SlSIZ1 Facilitates Heat Tolerance in Tomato.

Abstract: High temperature has become a major abiotic stress that limits crop productivity. Heat shock transcription factors (HSFs) and heat shock proteins (HSPs) play important roles in enhancing thermotolerance of plants. SUMOylation is an important post-translational modification in regulating cellular functions in eukaryotes. SIZ1, a well-characterized SUMO E3 ligase, mediates the process of SUMOylation. In this study, SUMO conjugations were clearly induced by high temperature. Overexpression of SIZ1 SUMO E3 ligase (SlSIZ1) in tomato could enhance the tolerance to heat stress in tomato. The RNA interference (RNAi) plants were more wilted than the wild type with heat treatment. Under heat stress, SlSIZ1 could decrease the accumulation of reactive oxygen species (ROS) and induce some genes of HSF and HSP transcription. Furthermore, overexpression of SlSIZ1 could increase the level of Hsp70 under high temperature. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that SlSIZ1 could interact with SlHsfA1 to mediate the SUMOylation of SlHsfA1 and consequently enhance thermotolerance of tomato. In conclusion, overexpression of SlSIZ1 enhanced heat tolerance by regulating the activities of HsfA1 and increasing the content Hsp70.

The Vacuolar Transportome of Plant Specialized Metabolites.

Abstract: The plant vacuole is a cellular compartment that is essential to plant development and growth. Often plant vacuoles accumulate specialized metabolites, also called secondary metabolites, which constitute functionally and chemically diverse compounds that exert in planta many essential functions and improve the plant's fitness. These metabolites provide, for example, chemical defense against herbivorous and pathogens or chemical attractants (color and fragrance) to attract pollinators. The chemical composition of the vacuole is dynamic, and is altered during development and as a response to environmental changes. To some extent these alterations rely on vacuolar transporters, which import and export compounds into and out of the vacuole, respectively. During the past decade, significant progress was made in the identification and functional characterization of the transporters implicated in many aspects of plant specialized metabolism. Still, deciphering the molecular players underlying such processes remains a challenge for the future. In this review, we present a comprehensive summary of the most recent achievements in this field.