Chimeric Repressor Research Articles

Splicing repression is a major function of TDP-43 in motor neurons.

Nuclear depletion of TDP-43, an essential RNA binding protein, may underlie neurodegeneration in amyotrophic lateral sclerosis (ALS). As several functions have been ascribed to this protein, the critical role(s) of TDP-43 in motor neurons that may be compromised in ALS remains unknown. We show here that TDP-43 mediated splicing repression, which serves to protect the transcriptome by preventing aberrant splicing, is central to the physiology of motor neurons. Expression in Drosophila TDP-43 knockout models of a chimeric repressor, comprised of the RNA recognition domain of TDP-43 fused to an unrelated splicing repressor, RAVER1, attenuated motor deficits and extended lifespan. Likewise, AAV9-mediated delivery of this chimeric rescue repressor to mice lacking TDP-43 in motor neurons delayed the onset, slowed the progression of motor symptoms, and markedly extended their lifespan. In treated mice lacking TDP-43 in motor neurons, aberrant splicing was significantly decreased and accompanied by amelioration of axon degeneration and motor neuron loss. This AAV9 strategy allowed long-term expression of the chimeric repressor without any adverse effects. Our findings establish that splicing repression is a major function of TDP-43 in motor neurons and strongly support the idea that loss of TDP-43-mediated splicing fidelity represents a key pathogenic mechanism underlying motor neuron loss in ALS.

PuHSFA4a Enhances Tolerance To Excess Zinc by Regulating Reactive Oxygen Species Production and Root Development in Populus.

Zinc (Zn) is an essential micronutrient but in excess is highly toxic to plants. Plants regulate Zn homeostasis and withstand excess Zn through various pathways; these pathways are generally tightly regulated by a specific set of genes. However, the transcription factors involved in excess Zn tolerance have yet to be identified. Here, we characterized a Populus ussuriensis heat shock transcription factor A4a (PuHSFA4a) that acts as a positive regulator of excess Zn tolerance in P ussuriensis We used overexpression (PuHSFA4a-OE) and chimeric dominant repressor (PuHSFA4a-SRDX) lines to identify the targets of PuHSFA4a PuHSFA4a transcription is specifically induced in roots by high Zn. Overexpression of PuHSFA4a conferred excess Zn tolerance and a dominant repressor version of PuHSFA4a increased excess Zn sensitivity in P ussuriensis by regulating the antioxidant system in roots. PuHSFA4a coordinately activates genes related to abiotic stress responses and root development and directly binds to the promoter regions of glutathione-s-transferase U17 (PuGSTU17) and phospholipase A2 (PuPLA2 ). PuGSTU17 overexpression significantly increased GST activity and reduced reactive oxygen species levels in roots while PuGSTU17-RNA interference lines exhibited the opposite phenotype. Furthermore, PuPLA2 overexpression promoted root growth under high Zn stress. Taken together, we provide evidence that PuHSFA4a coordinately activates the antioxidant system and root development-related genes and directly targets PuGSTU17 and PuPLA, thereby promoting excess Zn tolerance in P ussuriensis roots.

A Chimeric IDD4 Repressor Constitutively Induces Immunity in Arabidopsis via the Modulation of Salicylic Acid and Jasmonic Acid Homeostasis.

INDETERMINATE DOMAIN (IDD)/BIRD proteins belong to a highly conserved plant-specific group of transcription factors with dedicated functions in plant physiology and development. Here, we took advantage of the chimeric repressor gene-silencing technology (CRES-T, SRDX) to widen our view on the role of IDD4/IMPERIAL EAGLE and IDD family members in plant immunity. The hypomorphic idd4SRDX lines are compromised in growth and show a robust autoimmune phenotype. Hormonal measurements revealed the concomitant accumulation of salicylic acid and jasmonic acid suggesting that IDDs are involved in regulating the metabolism of these biotic stress hormones. The analysis of immunity-pathways showed enhanced activation of immune MAP kinase-signaling pathways, the accumulation of hydrogen peroxide and spontaneous programmed cell death. The transcriptome of nonelicited idd4SRDX lines can be aligned to approximately 40% of differentially expressed genes (DEGs) in flg22-treated wild-type plants. The pattern of DEGs implies IDDs as pivotal repressors of flg22-dependent gene induction. Infection experiments showed the increased resistance of idd4SRDX lines to Pseudomonas syringae and Botrytis cinerea implying a function of IDDs in defense adaptation to hemibiotrophs and necrotrophs. Genome-wide IDD4 DNA-binding studies (DAP-SEQ) combined with DEG analysis of idd4SRDX lines identified IDD4-regulated functional gene clusters that contribute to plant growth and development. In summary, we discovered that the expression of idd4SRDX activates a wide range of defense-related traits opening up the possibility to apply idd4SRDX as a powerful tool to stimulate innate immunity in engineered crops.

The R2R3-MYB Transcription Factor MYB49 Regulates Cadmium Accumulation.

Abscisic acid (ABA) reduces accumulation of potentially toxic cadmium (Cd) in plants. How the ABA signal is transmitted to modulate Cd uptake remains largely unclear. Here, we report that the basic region/Leu zipper transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5), a central ABA signaling molecule, is involved in ABA-repressed Cd accumulation in plants by physically interacting with a previously uncharacterized R2R3-type MYB transcription factor, MYB49. Overexpression of the Cd-induced MYB49 gene in Arabidopsis (Arabidopsis thaliana) resulted in a significant increase in Cd accumulation, whereas myb49 knockout plants and plants expressing chimeric repressors of MYB49:ERF-associated amphiphilic repression motif repression domain (SRDX49) exhibited reduced accumulation of Cd. Further investigations revealed that MYB49 positively regulates the expression of the basic helix-loop-helix transcription factors bHLH38 and bHLH101 by directly binding to their promoters, leading to activation of IRON-REGULATED TRANSPORTER1, which encodes a metal transporter involved in Cd uptake. MYB49 also binds to the promoter regions of the heavy metal-associated isoprenylated plant proteins (HIPP22) and HIPP44, resulting in up-regulation of their expression and subsequent Cd accumulation. On the other hand, as a feedback mechanism to control Cd uptake and accumulation in plant cells, Cd-induced ABA up-regulates the expression of ABI5, whose protein product interacts with MYB49 and prevents its binding to the promoters of downstream genes, thereby reducing Cd accumulation. Our results provide new insights into the molecular feedback mechanisms underlying ABA signaling-controlled Cd uptake and accumulation in plants.

NINJA-associated ERF19 negatively regulates Arabidopsis pattern-triggered immunity

Recognition of microbe-associated molecular patterns (MAMPs) derived from invading pathogens by plant pattern recognition receptors (PRRs) initiates a subset of defense responses known as pattern-triggered immunity (PTI). Transcription factors (TFs) orchestrate the onset of PTI through complex signaling networks. Here, we characterized the function of ERF19, a member of the Arabidopsis thaliana ethylene response factor (ERF) family. ERF19 was found to act as a negative regulator of PTI against Botrytis cinerea and Pseudomonas syringae. Notably, overexpression of ERF19 increased plant susceptibility to these pathogens and repressed MAMP-induced PTI outputs. In contrast, expression of the chimeric dominant repressor ERF19-SRDX boosted PTI activation, conferred increased resistance to the fungus B. cinerea, and enhanced elf18-triggered immunity against bacteria. Consistent with a negative role for ERF19 in PTI, MAMP-mediated growth inhibition was weakened or augmented in lines overexpressing ERF19 or expressing ERF19-SRDX, respectively. Using biochemical and genetic approaches, we show that the transcriptional co-repressor Novel INteractor of JAZ (NINJA) associates with and represses the function of ERF19. Our work reveals ERF19 as a novel player in the mitigation of PTI, and highlights a potential role for NINJA in fine-tuning ERF19-mediated regulation of Arabidopsis innate immunity.

Arabidopsis thaliana NGATHA1 transcription factor induces ABA biosynthesis by activating NCED3 gene during dehydration stress

The plant hormone abscisic acid (ABA) is accumulated after drought stress and plays critical roles in the responses to drought stress in plants, such as gene regulation, stomatal closure, seed maturation, and dormancy. Although previous reports revealed detailed molecular roles of ABA in stress responses, the factors that contribute to the drought-stress responses-in particular, regulation of ABA accumulation-remain unclear. The enzyme NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) is essential for ABA biosynthesis during drought stress, and the NCED3 gene is highly induced by drought stress. In the present study, we isolated NGATHAs (NGAs) as candidate transcriptional regulators of NCED3 through a screen of a plant library harboring the transcription factors fused to a chimeric repressor domain, SRDX. The NGA proteins were directly bound to a cis-element NGA-binding element (NBE) in the 5' untranslated region (5' UTR) of the NCED3 promoter and were suggested to be transcriptional activators of NCED3 Among the single-knockout mutants of four NGA family genes, we found that the NGATHA1 (NGA1) knockout mutant was drought-stress-sensitive with a decreased expression level of NCED3 during dehydration stress. These results suggested that NGA1 essentially functions as a transcriptional activator of NCED3 among the NGA family proteins. Moreover, the NGA1 protein was degraded under nonstressed conditions, and dehydration stress enhanced the accumulation of NGA1 proteins, even in ABA-deficient mutant plants, indicating that there should be ABA-independent posttranslational regulations. These findings emphasize the regulatory mechanisms of ABA biosynthesis during early drought stress.

Overexpression of OsAP2 and OsWRKY24 in Arabidopsis results in reduction of plant size.

Recently, two rice genes, OsAPETALA2 (OsAP2) and OsWRKY24 have been reported to be positive regulators involved in increased lamina inclination and grain size through cell elongation. Here, we found that the two genes have tightly linked expression patterns and functional convergence in rice, and are also likely to play an opposite role in Arabidopsis. Overexpression of the two rice transcription factors in Arabidopsis caused smaller plant size with reduced cell size, and the expression of a series of genes encoding expansins and xyloglucan endotransglucosylase/hydrolases (XTHs) involved in cell elongation was reduced. However, transgenic Arabidopsis expressing OsWRKY24-SRDX as a synthetic chimeric repressor displayed indistinguishable phenotypes from wild-type plants. Moreover, the subcellular localization pattern of OsWRKY24 in Arabidopsis was different from that in rice. Thus, we demonstrate an example of transcription factors from one species playing distinct roles in different plant species.

CaWRKY40b in Pepper Acts as a Negative Regulator in Response to Ralstonia solanacearum by Directly Modulating Defense Genes Including CaWRKY40.

WRKY transcription factors (TFs) have been implicated in plant growth, development, and in response to environmental cues; however, the function of the majority of pepper WRKY TFs remains unclear. In the present study, we functionally characterized CaWRKY40b, a homolog of AtWRKY40, in pepper immunity. Ralstonia solanacearum inoculation (RSI) in pepper plants resulted in downregulation of CaWRKY40b transcript, and green fluorescent protein (GFP)-tagged CaWRKY40b was localized to the nuclei when transiently overexpressed in the leaves of Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of CaWRKY40b significantly decreased pepper’ susceptibility to RSI. Consistently, the transient over-expression of CaWRKY40b-SRDX (chimeric repressor version of CaWRKY40b) triggered cell death, as indicated by darker trypan blue and DAB staining. CaWRKY40b targets a number of immunity-associated genes, including CaWRKY40 JAR, RLK1, EIN3, FLS2, CNGIC8, CDPK13, and heat shock cognate protein 70 (HSC70), which were identified by ChIP-seq and confirmed using ChIP-real time PCR. Among these target genes, the negative regulator HSC70 was upregulated by transient overexpression of CaWRKY40b and downregulated by silencing of CaWRKY40b, whereas other positive regulators as well as two non-target genes, CaNPR1 and CaDEF1, were downregulated by the transient overexpression of CaWRKY40b and upregulated by CaWRKY40b silencing or transient overexpression of CaWRKY40b-SRDX. In addition, CaWRKY40b exhibited a positive feedback regulation at transcriptional level by directly targeting the promoter of itself. In conclusion, the findings of the present study suggest that CaWRKY40b acts as a negative regulator in pepper immunity against R. solanacearum by transcriptional modulation of a subset of immunity-associated genes; it also represses immunity in the absence of a pathogen, and derepresses immunity upon pathogen challenge.

Improvement of cell wall digestibility in tall fescue by Oryza sativa SECONDARY WALL NAC DOMAIN PROTEIN2 chimeric repressor

Forage digestibility is one of the most important factors in livestock performance. As grasses grow and mature, dry matter increases but they become fibrous with secondary cell wall deposition and lignification of sclerenchyma cells, and forage quality drops. In rice (Oryza sativa), the SECONDARY WALL NAC DOMAIN PROTEIN2 fused with the modified EAR-like motif repression domain (OsSWN2-SRDX) reduces secondary cell wall thickening in sclerenchyma cells. We introduced OsSWN2-SRDX under the control of the OsSWN1 promoter into tall fescue (Festuca arundinacea Schreb.) to increase cell wall digestibility. Of 23 transgenic plants expressing OsSWN2-SRDX, nine had brittle internodes that were easily broken by bending. Their secondary cell walls were significantly thinner than those of the wild type in interfascicular fibers of internodes and in cortical fiber cells between leaf epidermal cells and vascular bundles. The dry matter digestibility increased by 11.8% in stems and by 6.8% in leaves compared with the wild type, and therefore forage quality was improved. In stem interfascicular fibers, acid detergent fiber and acid insoluble lignin were greatly reduced. Thus, the reduction of indigestible fiber composed of cellulose and lignin increased the degradability of sclerenchyma cell walls. OsSWN2-SRDX plants offer great potential in the genetic improvement of forage digestibility.

Effects of GhWUS from upland cotton (Gossypium hirsutum L.) on somatic embryogenesis and shoot regeneration

The WUSCHEL (WUS) gene encodes a plant-specific homeodomain-containing transcriptional regulator, which plays important roles during embryogenesis, as well as in the formation of shoot and flower meristems. Here, we isolated two homologues of Arabidopsis thaliana WUS (AtWUS), GhWUS1a_At and GhWUS1b_At, from upland cotton (Gossypium hirsutum). Domain analysis suggested that the two putative GhWUS proteins contained a highly conserved DNA-binding HOX domain and a WUS-box. Expression profile analysis showed that GhWUSs were predominantly expressed during the embryoid stage. Ectopic expression of GhWUSs in Arabidopsis could induce somatic embryo and shoot formation from seedling root tips. Furthermore, in the absence of exogenous hormone, overexpression of GhWUSs in Arabidopsis could promote shoot regeneration from excised roots, and in the presence of exogenous auxin, excised roots expressing GhWUS could be induced to produce somatic embryo. In addition, expression of the chimeric GhWUS repressor in cotton callus inhibited embryogenic callus formation. Our results show that GhWUS is an important regulator of somatic embryogenesis and shoot regeneration.

OsWRKY24, a blast-disease responsive transcription factor, positively regulates rice disease resistance

WRKYs are plant-specific transcription factors that play important roles in disease response. Here, we showed that a WRKY gene in rice, OsWRKY24, was upregulated in response to inoculation with rice blast fungus Pyricularia oryzae. Its expression was also induced by wounding. Green fluorescent protein-fused OsWRKY24 localized to nuclei in rice epidermal cells, and OsWRKY24 had transactivation activity in yeast cells. In addition, functional repression of OsWRKY24 through a chimeric repressor (OsWRKY24-SRDX) greatly increased susceptibility to P. oryzae. These results indicate that OsWRKY24 is a transcriptional activator that positively regulates disease resistance in rice.

Utilization of transcription factors for controlling floral morphogenesis in horticultural plants.

Transcription factors play important roles not only in the development of floral organs but also in the formation of floral characteristics in various plant species. Therefore, transcription factors are reasonable targets for modifying these floral traits and generating new flower cultivars. However, it has been difficult to control the functions of transcription factors because most plant genes, including those encoding transcription factors, exhibit redundancy. In particular, it has been difficult to understand the functions of these redundant genes by genetic analysis. Thus, a breakthrough silencing method called chimeric repressor gene silencing technology (CRES-T) was developed specifically for plant transcription factors. This method transforms transcriptional activators into dominant repressors, and the artificial chimeric repressors suppress the function of transcription factors regardless of their redundancy. Among these chimeric repressors, some were found to be inappropriate for expression throughout the plant body because they resulted in deformities. For these chimeric repressors, utilization of floral organ-specific promoters overcomes this problem by avoiding expression throughout the plant body. In contrast, attachment of viral activation domain VP16 to transcriptional repressors effectively alters into transcriptional activators. This review presents the importance of transcription factors for characterizing floral traits, describes techniques for controlling the functions of transcription factors.

The NAC Transcription Factor Gene OsY37 (ONAC011) Promotes Leaf Senescence and Accelerates Heading Time in Rice.

Leaf senescence is an important physiological process involving the degradation of a number of metabolites and their remobilization to new reproductive and storage organs. NAC (NAM, ATAF, and CUC) transcription factors are reported as important regulators of the senescence process. Here, we describe the identification and functional characterization of the NAC transcription factor gene, OsY37 (Oryza sativa Yellow37, ONAC011) obtained from Oryza sativa cv. indica, and japonica. We created transgenic plants expressing the OsY37 gene under the control of a strong and constitutive CaMV35S promoter. The resulting transgenic plants overexpressing OsY37 gene showed early heading and precocious senescence phenotype of flag leaves compared with wild-type plants. By contrast, blocking the function of this gene via RNAi (RNA interference) and CRES-T (Chimeric Repressor Silencing Technology) technology, delayed both heading time and leaf senescence. Furthermore, knockdown of OsY37 expression caused dwarfism and high accumulation of chlorophyll during the vegetative phase. Irrespective of early or delayed senescence, transgenic plants showed reduced grain yields. Our results indicate that OsY37 acts as a positive regulator of heading and senescence during the reproductive phase in rice. In addition, OsY37 may be involved in plant development and grain yield.

Combination of Cyclamen persicum Mill. floral gene promoters and chimeric repressors for the modification of ornamental traits in Torenia fournieri Lind.

Although chimeric repressors such as the Arabidopsis TCP3 repressor are known to have significant effects on flower morphology and color, their cellular-level effects on flower petals are not understood. The promoter sequences of the genes expressed in the flowers of cyclamen, a representative potted flower grown during the winter season, are also unknown. Here, we isolated eight promoters from cyclamen genes that are reportedly expressed in the petals. These promoters were then fused to four chimeric repressors and introduced into the model flower torenia to screen for effective combinations of promoters and repressors for flower breeding. As expected, some of the constructs altered flower phenotypes upon transformation. We further analyzed the effects of chimeric repressors at the cellular level. We observed that complicated petal and leaf serrations were accompanied by excessive vascular branching. Dichromatism in purple anthocyanin was inferred to result in bluish flowers, and imbalanced cell proliferation appeared to result in epinastic flowers. Thus, the genetic constructs and phenotypic changes described in this report will benefit the future breeding and characterization of ornamental flowers.

Photoperiod-insensitive floral transition in chrysanthemum induced by constitutive expression of chimeric repressor CsLHY-SRDX

A wide variety of physiological processes including flowering are controlled by the circadian clock in plants. In Arabidopsis, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) constitute the central oscillator, and their gain of function and loss of function disrupt the circadian clock and affect flowering time through FLOWERING LOCUS T (FT), a gene encoding a florigen. Chrysanthemum is a typical short-day (SD) plant and responds to shortening of day length by the transition from the vegetative to reproductive phase. We identified FLOWERING LOCUS T-LIKE 3 (FTL3) and ANTI-FLORIGENIC FT/TFL1 FAMILY PROTEIN (AFT) as a florigen and antiflorigen, respectively, in a wild diploid chrysanthemum (Chrysanthemum seticuspe f. boreale). CsFTL3 and CsAFT are induced under SD or a noninductive photoperiod, respectively, and their balance determines the floral transition and anthesis. Meanwhile, the time-keeping mechanism that regulates the photoperiodic flowering in chrysanthemum is poorly understood. Here, we focused on a LHY/CCA1-like gene called CsLHY in chrysanthemum. We fused CsLHY to a gene encoding short transcriptional repressor domain (SRDX) and constitutively expressed it in chrysanthemum. Although the transcription of clock-related genes was conditionally affected, circadian rhythm was not completely disrupted in CsLHY-SRDX transgenic plants. These plants formed almost the same number of leaves before floral transition under SD and long-day conditions. Thus, CsLHY-SRDX chrysanthemum showed photoperiod-insensitive floral transition, but further development of the capitulum was arrested, and anthesis was not observed. Simultaneously with the flowering phenotype, CsFTL3 and CsAFT were downregulated in CsLHY-SRDX transgenic plants. These results suggest that CsLHY-SRDX affects CsFTL3 and CsAFT expression and causes photoperiod-insensitive floral transition without a severe defect in the circadian clock.

Overexpression of MsDREB6.2 results in cytokinin-deficient developmental phenotypes and enhances drought tolerance in transgenic apple plants.

Dehydration-responsive element binding factors (DREBs) play important roles in plant growth, development, and stress signaling pathways in model plants. However, little is known about the function of DREBs in apple (Malus × domestica), a widely cultivated crop that is frequently threatened by drought. We isolated a DREB gene from Malus sieversii (Ledeb.) Roem., MsDREB6.2, and investigated its functions using overexpression analysis and chimeric repressor gene-silencing technology (CRES-T). We identified possible target genes of the protein encoded by MsDREB6.2 using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP). Overexpression of MsDREB6.2 increased the expression of a key cytokinin (CK) catabolism gene, MdCKX4a, which led to a significant reduction in endogenous CK levels, and caused a decrease in shoot:root ratio in transgenic apple plants. Overexpression of MsDREB6.2 resulted in a decrease in stomatal aperture and density and an increase in root hydraulic conductance (L0 ), and thereby enhanced drought tolerance in transgenic plants. Furthermore, manipulating the level of MsDREB6.2 expression altered the expression of two aquaporin (AQP) genes. The effect of the two AQP genes on L0 was further characterized using the AQP inhibitor HgCl2 . Based on these observations, we conclude that MsDREB6.2 enhances drought tolerance and that its function may be due, at least in part, to its influence on stomatal opening, root growth, and AQP expression. These results may have applications in apple rootstock breeding programs aimed at developing drought-resistant apple varieties.

The chimeric repressor for the GATA4 transcription factor improves tolerance to nitrogen deficiency in Arabidopsis.

Nitrogen limits crop yield, but application of nitrogen fertilizer can cause environmental problems and much fertilizer is lost without being absorbed by plants. Increasing nitrogen use efficiency in plants may help overcome these problems and is, therefore, an important and active subject of agricultural research. Here, we report that the expression of the chimeric repressor for the GATA4 transcription factor (35S:GATA4-SRDX) improved tolerance to nitrogen deficiency in Arabidopsis thaliana. 35S:GATA4-SRDX seedlings were significantly larger than wild type under nitrogen-sufficient and -deficient conditions (10 and 0.5 mM NH4NO3, respectively). 35S:GATA4-SRDX plants exhibited shorter primary roots, fewer lateral roots, and higher root hair density compared with wild type. The expression levels of NITRATE TRANSPORTER 2.1, ASPARAGINE SYNTHETASE and NITRATE REDUCTASE 1 were significantly higher in roots of 35S:GATA4-SRDX plants than in wild type under nitrogen-sufficient conditions. Under nitrogen-deficient conditions, the expression of genes for cytosolic glutamine synthetases was upregulated in shoots of 35S:GATA4-SRDX plants compared with wild type. This upregulation of nitrogen transporter and nitrogen assimilation-related genes might confer tolerance to nitrogen deficiency in 35S:GATA4-SRDX plants.

Root avoidance of toxic metals requires the GeBP-LIKE 4 transcription factor in Arabidopsis thaliana.

Summary Plants reorganize their root architecture to avoid growth into unfavorable regions of the rhizosphere. In a screen based on chimeric repressor gene‐silencing technology, we identified the Arabidopsis thaliana GeBP‐LIKE 4 (GPL4) transcription factor as an inhibitor of root growth that is induced rapidly in root tips in response to cadmium (Cd).We tested the hypothesis that GPL4 functions in the root avoidance of Cd by analyzing root proliferation in split medium, in which only half of the medium contained toxic concentrations of Cd.The wild‐type (WT) plants exhibited root avoidance by inhibiting root growth in the Cd side but increasing root biomass in the control side. By contrast, GPL4‐suppression lines exhibited nearly comparable root growth in the Cd and control sides and accumulated more Cd in the shoots than did the WT. GPL4 suppression also altered the root avoidance of toxic concentrations of other essential metals, modulated the expression of many genes related to oxidative stress, and consistently decreased reactive oxygen species concentrations.We suggest that GPL4 inhibits the growth of roots exposed to toxic metals by modulating reactive oxygen species concentrations, thereby allowing roots to colonize noncontaminated regions of the rhizosphere.

The Arabidopsis Transcription Factor ANAC032 Represses Anthocyanin Biosynthesis in Response to High Sucrose and Oxidative and Abiotic Stresses.

Production of anthocyanins is one of the adaptive responses employed by plants during stress conditions. During stress, anthocyanin biosynthesis is mainly regulated at the transcriptional level via a complex interplay between activators and repressors of anthocyanin biosynthesis genes. In this study, we investigated the role of a NAC transcription factor, ANAC032, in the regulation of anthocyanin biosynthesis during stress conditions. ANAC032 expression was found to be induced by exogenous sucrose as well as high light (HL) stress. Using biochemical, molecular and transgenic approaches, we show that ANAC032 represses anthocyanin biosynthesis in response to sucrose treatment, HL and oxidative stress. ANAC032 was found to negatively affect anthocyanin accumulation and the expression of anthocyanin biosynthesis (DFR, ANS/LDOX) and positive regulatory (TT8) genes as demonstrated in overexpression line (35S:ANAC032) compared to wild-type under HL stress. The chimeric repressor line (35S:ANAC032-SRDX) exhibited the opposite expression patterns for these genes. The negative impact of ANAC032 on the expression of DFR, ANS/LDOX and TT8 was found to be correlated with the altered expression of negative regulators of anthocyanin biosynthesis, AtMYBL2 and SPL9. In addition to this, ANAC032 also repressed the MeJA- and ABA-induced anthocyanin biosynthesis. As a result, transgenic lines overexpressing ANAC032 (35S:ANAC032) produced drastically reduced levels of anthocyanin pigment compared to wild-type when challenged with salinity stress. However, transgenic chimeric repressor lines (35S:ANAC032-SRDX) exhibited the opposite phenotype. Our results suggest that ANAC032 functions as a negative regulator of anthocyanin biosynthesis in Arabidopsis thaliana during stress conditions.

ANAC032 Positively Regulates Age-Dependent and Stress-Induced Senescence in Arabidopsis thaliana.

Members of the NAC transcription factor family have been implicated in the regulation of different processes of plant development including senescence. In this study, the role of ANAC032 is analyzed in Arabidopsis thaliana (Col-0). ANAC032 is shown to act as a transcriptional activator and its expression is induced in senescing leaves as well as in dark-treated detached leaves. Analysis of transgenic overexpressors (OXs) and chimeric repressors (SRDXs) of ANAC032 indicates that ANAC032 positively regulates age-dependent and dark-induced leaf senescence. Quantitative real-time PCR analysis showed that ANAC032 regulates leaf senescence mainly through the modulation of expression of the senescence-associated genes AtNYE1, SAG113 and SAUR36/SAG201, which are involved in Chl degradation, and ABA and auxin promotion of senescence, respectively. In addition, ANAC032 expression is induced by a range of oxidative and abiotic stresses. As a result, ANAC032 overexpression lines exhibited enhanced leaf senescence when challenged with different oxidative (3-aminotriazole, fumonisin B1 and high light) and abiotic stress (osmotic and salinity) conditions compared with the wild type. In contrast, ANAC032 SRDX lines displayed the opposite phenotype. ANAC032 transgenic lines showed altered 2,4-D-mediated root tip swelling and root inhibition responses when compared with the wild type. The altered response to auxin, oxidative and abiotic stress treatments in ANAC032 transgenic lines involves differential accumulation of H2O2 compared with the wild type. Taken together, these results indicate that ANAC032 is an important transcription factor that positively regulates age-dependent and stress-induced senescence in A. thaliana by modulating reactive oxygen species production.