Endoplasmic reticulum-bound ANAC013 factor is cleaved by RHOMBOID-LIKE 2 during the initial response to hypoxia in Arabidopsis thaliana.

Aerobic reactions are essential to sustain plant growth and development. Impaired oxygen availability due to excessive water availability, e.g., during waterlogging or flooding, reduces plant productivity and survival. Consequently, plants monitor oxygen availability to adjust growth and metabolism accordingly. Despite the identification of central components in hypoxia adaptation in recent years, molecular pathways involved in the very early activation of low-oxygen responses are insufficiently understood. Here, we characterized three endoplasmic reticulum (ER)-anchored Arabidopsis ANAC transcription factors, namely ANAC013, ANAC016, and ANAC017, which bind to the promoters of a subset of hypoxia core genes (HCGs) and activate their expression. However, only ANAC013 translocates to the nucleus at the onset of hypoxia, i.e., after 1.5 h of stress. Upon hypoxia, nuclear ANAC013 associates with the promoters of multiple HCGs. Mechanistically, we identified residues in the transmembrane domain of ANAC013 to be essential for transcription factor release from the ER, and provide evidence that RHOMBOID-LIKE 2 (RBL2) protease mediates ANAC013 release under hypoxia. Release of ANAC013 by RBL2 also occurs upon mitochondrial dysfunction. Consistently, like ANAC013 knockdown lines, rbl knockout mutants exhibit impaired low-oxygen tolerance. Taken together, we uncovered an ER-localized ANAC013-RBL2 module, which is active during the initial phase of hypoxia to enable fast transcriptional reprogramming.

Multiple transcription factors of Arabidopsis thaliana that are activated by LEAFY COTYLEDON 2 regulate triacylglycerol biosynthesis.

Plant triacylglycerols (TAG) are used in food and various industrial feedstocks. LEAFY COTYLEDON 2 (LEC2), a master positive regulator of TAG biosynthesis, regulates a complex network of transcription factors (TFs) during seed development. Aside from WRINKLED1 (WRI1), the TFs regulated by LEC2 related to TAG biosynthesis have not yet been identified. Previously, we identified 25 seed-expressing TFs that were upregulated in Arabidopsis leaves that overexpressed senescence-induced LEC2. In this study, each of the 25 TFs was transiently expressed in the leaves of Nicotiana benthamiana to identify unknown TFs that regulate TAG biosynthesis. The TAG content of the transformed leaves was analyzed using thin layer chromatography and gas chromatography. We observed that five TFs, ARABIDOPSIS RESPONSIVE REGULATOR 21 (ARR21), AINTEGUMENTA-LIKE 6 (AIL6), APETALA2/ETHYLENE RESPONSIVE FACTOR 55 (ERF55), WRKY DNA-BINDING PROTEIN 8 (WRKY8), and ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 38 (ANAC038) increased TAG synthesis in the leaves. Among these, the promoters of AIL6, ERF55, WRKY8, and ANAC038 contain RY motifs, which are LEC2-binding sites activated by LEC2. AIL6 overexpression in Arabidopsis increased the total fatty acid (FA) content in seeds and altered the FA composition, with increases in 16:0, 18:1, and 18:2 and decreases in 18:0, 18:3, and 20:1 compared with those in the wild type (WT). AIL6 overexpression activates several FA and TAG biosynthesis genes. Therefore, our study successfully identified several new TFs regulated by LEC2 in TAG biosynthesis and showed that AIL6 increased the TAG content in seeds.

Arabidopsis SMO2 modulates ribosome biogenesis by maintaining the RID2 abundance during organ growth.

Ribosome biogenesis is a process of making ribosomes that is tightly linked with plant growth and development. Here, through a suppressor screen for the smo2 mutant, we found that lack of a ribosomal stress response mediator, ANAC082 partially restored growth defects of the smo2 mutant, indicating SMO2 is required for the repression of nucleolar stress. Consistently, the smo2 knock-out mutant exhibited typical phenotypes characteristic of ribosome biogenesis mutants, such as pointed leaves, aberrant leaf venation, disrupted nucleolar structure, abnormal distribution of rRNA precursors, and enhanced tolerance to aminoglycoside antibiotics that target ribosomes. SMO2 interacted with ROOT INITIATION DEFECTIVE 2 (RID2), a methyltransferase-like protein required for pre-rRNA processing. SMO2 enhanced RID2 solubility in Escherichia coli and the loss of function of SMO2 in plant cells reduced RID2 abundance, which may result in abnormal accumulation of FIBRILLARIN 1 (FIB1) and NOP56, two key nucleolar proteins, in high-molecular-weight protein complex. Taken together, our results characterized a novel plant ribosome biogenesis factor, SMO2 that maintains the abundance of RID2, thereby sustaining ribosome biogenesis during plant organ growth.

Mitochondrial alternative NADH dehydrogenases NDA1 and NDA2 promote survival of reoxygenation stress in Arabidopsis by safeguarding photosynthesis and limiting ROS generation.

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.

ANAC087 transcription factor positively regulates age-dependent leaf senescence through modulating the expression of multiple target genes in Arabidopsis.

Leaf senescence is the final stage of leaf development and appropriate onset and progression of leaf senescence are critical for reproductive success and fitness. Although great progress has been made in identifying key genes regulating leaf senescence and elucidating the underlining mechanisms in the model plant Arabidopsis, there is still a gap to understanding the complex regulatory network. In this study, we discovered that Arabidopsis ANAC087 transcription factor (TF) positively modulated leaf senescence. Expression of ANAC087 was induced in senescing leaves and the encoded protein acted as a transcriptional activator. Both constitutive and inducible overexpression lines of ANAC087 showed earlier senescence than control plants, whereas T-DNA insertion mutation and dominant repression of the ANAC087 delayed senescence rate. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) profiling showed that the expression of an array of senescence-associated genes was upregulated in inducible ANAC087 overexpression plants including BFN1, NYE1, CEP1, RbohD, SAG13, SAG15, and VPEs, which are involved in programmed cell death (PCD), chlorophyll degradation and reactive oxygen species (ROS) accumulation. In addition, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays demonstrated that ANAC087 directly bound to the canonical NAC recognition sequence (NACRS) motif in promoters of its target genes. Moreover, mutation of two representative target genes, BFN1 or NYE1 alleviated the senescence rate of ANAC087-overexpression plants, suggesting their genetic regulatory relationship. Taken together, this study indicates that ANAC087 serves as an important regulator linking PCD, ROS, and chlorophyll degradation to leaf senescence.

The membrane associated NAC transcription factors ANAC060 and ANAC040 are functionally redundant in the inhibition of seed dormancy in Arabidopsis thaliana.

The NAC family of transcription factors is involved in plant development and various biotic and abiotic stresses. The Arabidopsis thaliana ANAC genes ANAC060, ANAC040, and ANAC089 are highly homologous based on protein and nucleotide sequence similarity. These three genes are predicted to be membrane bound transcription factors (MTFs) containing a conserved NAC domain, but divergent C-terminal regions. The anac060 mutant shows increased dormancy when compared with the wild type. Mutations in ANAC040 lead to higher seed germination under salt stress, and a premature stop codon in ANAC089 Cvi allele results in seeds exhibiting insensitivity to high concentrations of fructose. Thus, these three homologous MTFs confer distinct functions, although all related to germination. To investigate whether the differences in function are caused by a differential spatial or temporal regulation, or by differences in the coding sequence (CDS), we performed swapping experiments in which the promoter and CDS of the three MTFs were exchanged. Seed dormancy and salt and fructose sensitivity analyses of transgenic swapping lines in mutant backgrounds showed that there is functional redundancy between ANAC060 and ANAC040, but not between ANAC060 and ANAC089.

Dual control of MAPK activities by AP2C1 and MKP1 MAPK phosphatases regulates defence responses in Arabidopsis.

Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific Ser/Thr protein phosphatases of the type 2C (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. This work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant, ap2c1 and mkp1, and the ap2c1 mkp1 double mutant displayed enhanced stress-induced activation of the MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants showed an autoimmune-like response, associated with increased levels of the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. This phenotype was reduced in the ap2c1 mkp1 mpk3 and ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MAPK misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MAPK activities, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development.

Genome-wide binding analysis reveals that ANAC060 directly represses sugar-induced transcription of ABI5 in Arabidopsis.

The sugar status of a plant acts as a signal affecting growth and development. The phenomenon by which high levels of sugars inhibit seedling establishment has been widely used to gain insight into sugar-signaling pathways. Natural allelic variation has been identified at the ANAC060 locus. The Arabidopsis Columbia ecotype produces a short ANAC060 protein without a transmembrane domain that is constitutively located to the nucleus, causing sugar insensitivity when overexpressed. In this study, we generated a genome-wide DNA-binding map of ANAC060 via chromatin immunoprecipitation sequencing using transgenic lines that express a functional ANAC060-GFP fusion protein in an anac060 background. A total of 3282 genes associated with ANAC060-binding sites were identified. These genes were enriched in biotic and abiotic stress responses, and the G-box binding motif was highly enriched in ANAC060-bound genomic regions. Expression microarray analysis resulted in the identification of 8350 genes whose activities were altered in the anac060 mutant and upon sugar treatment. Cluster analysis revealed that ANAC060 attenuates sugar-regulated gene expression. Direct target genes of ANAC060 included equivalent numbers of genes that were upregulated or downregulated by ANAC060. The various functions of these target genes indicate that ANAC060 has several functions. Our results demonstrate that ANAC060 directly binds to the promoter of ABI5 and represses the sugar-induced transcription of ABI5. Genetic data indicate that ABI5 is epistatic to ANAC060 in both sugar and abscisic acid responses.

Differential submergence tolerance between juvenile and adult Arabidopsis plants involves the ANAC017 transcription factor.

Plants need to attune their stress responses to the ongoing developmental programmes to maximize their efficacy. For instance, successful submergence adaptation is often associated with a delicate balance between saving resources and their expenditure to activate measures that allow stress avoidance or attenuation. We observed a significant decrease in submergence tolerance associated with ageing in Arabidopsis thaliana, with a critical step between 2 and 3 weeks of post-germination development. This sensitization to flooding was concomitant with the transition from juvenility to adulthood. Transcriptomic analyses indicated that a group of genes related to abscisic acid and oxidative stress response was more highly expressed in juvenile plants than in adult ones. These genes are induced by the endomembrane tethered transcription factor ANAC017 that was in turn activated by submergence-associated oxidative stress. A combination of molecular, biochemical and genetic analyses showed that these genes are located in genomic regions that move towards a heterochromatic state with adulthood, as marked by lysine 4 trimethylation of histone H3. We concluded that, while the mechanisms of flooding stress perception and signal transduction were unaltered between juvenile and adult phases, the sensitivity that these mechanisms set into action is integrated, via epigenetic regulation, into the developmental programme of the plant.

Mitochondrial signalling is critical for acclimation and adaptation to flooding in Arabidopsis thaliana.

Mitochondria have critical functions in the acclimation to abiotic and biotic stresses. Adverse environmental conditions lead to increased demands in energy supply and metabolic intermediates, which are provided by mitochondrial ATP production and the tricarboxylic acid (TCA) cycle. Mitochondria also play a role as stress sensors to adjust nuclear gene expression via retrograde signalling with the transcription factor (TF) ANAC017 and the kinase CDKE1 key components to integrate various signals into this pathway. To determine the importance of mitochondria as sensors of stress and their contribution in the tolerance to adverse growth conditions, a comparative phenotypical, physiological and transcriptomic characterisation of Arabidopsis mitochondrial signalling mutants (cdke1/rao1 and anac017/rao2) and a set of contrasting accessions was performed after applying the complex compound stress of submergence. Our results showed that impaired mitochondrial retrograde signalling leads to increased sensitivity to the stress treatments. The multi-factorial approach identified a network of 702 co-expressed genes, including several WRKY TFs, overlapping in the transcriptional responses in the mitochondrial signalling mutants and stress-sensitive accessions. Functional characterisation of two WRKY TFs (WRKY40 and WRKY45), using both knockout and overexpressing lines, confirmed their role in conferring tolerance to submergence. Together, the results revealed that acclimation to submergence is dependent on mitochondrial retrograde signalling, and underlying transcriptional re-programming is used as an adaptation mechanism.

ANAC019 is required for recovery of reproductive development under drought stress in Arabidopsis.

KEY MESSAGE: Morphological and transcriptomic evidences provide us strong support for the function of ANAC019 in reproductive development under drought stress. Plants are sensitive to drought conditions, particularly at the reproductive stage. Several studies have reported drought effects on crop reproductive development, but the molecular mechanism underlying drought response during reproduction is still unclear. A recent study showed that drought induces in Arabidopsis inflorescence increased expression of many genes, including ANAC019. However, the function of ANAC019 in drought response during reproductive development has not been characterized. Here, we report an investigation of the ANAC019 function in the response to drought during reproduction. ANAC019 is preferentially expressed in the inflorescence compared with the leaf, suggesting possible roles in regulating both stress response and flower development. The anac019 mutant was more sensitive to drought than WT plant, and exhibited a delay in recovery of floral organ development under prolonged drought stress. Moreover, many fewer genes were differentially expressed in the anac019 inflorescence under drought than that of WT, suggesting that the mutant was impaired in drought-induced gene expression. The genes affected by ANAC019 were associated with stress and hormone responses as well as floral development. In particular, the expression levels of several key drought-induced genes, DREB2A, DREB2B, ARF2, MYB21 and MYB24, were dramatically reduced in the absence of ANAC019, suggesting that ANAC019 is an upstream regulator these genes for drought response and flower development. These results provide strong support for the potential function of ANAC019 in reproductive development under drought stress.

The SNAC-A Transcription Factor ANAC032 Reprograms Metabolism in Arabidopsis.

Studies have indicated that the carbon starvation response leads to the reprogramming of the transcriptome and metabolome, and many genes, including several important regulators, such as the group S1 basic leucine zipper transcription factors (TFs) bZIP1, bZIP11 and bZIP53, the SNAC-A TF ATAF1, etc., are involved in these physiological processes. Here, we show that the SNAC-A TF ANAC032 also plays important roles in this process. The overexpression of ANAC032 inhibits photosynthesis and induces reactive oxygen species accumulation in chloroplasts, thereby reducing sugar accumulation and resulting in carbon starvation. ANAC032 reprograms carbon and nitrogen metabolism by increasing sugar and amino acid catabolism in plants. The ChIP-qPCR and transient dual-luciferase reporter assays indicated that ANAC032 regulates trehalose metabolism via the direct regulation of TRE1 expression. Taken together, these results show that ANAC032 is an important regulator of the carbon/energy status that represses photosynthesis to induce carbon starvation.

Overexpression of ANAC046 Promotes Suberin Biosynthesis in Roots of Arabidopsis thaliana.

NAC (NAM (no apical meristem), ATAF1/2, and CUC2 (cup-shaped cotyledon)) proteins are one of the largest families of plant-specific transcription factors, and this family is present in a wide range of land plants. Here, we have investigated the role of ANAC046 in the regulation of suberin biosynthesis and deposition in Arabidopsis. Subcellular localization and transcriptional activity assays showed that ANAC046 localizes in the nucleus, where it functions as a transcription activator. Analysis of the PANAC046:GUS lines revealed that ANAC046 is mainly expressed in the root endodermis and periderm, and is also induced in leaves by wounding. The transgenic lines overexpressing ANAC046 exhibited defective surfaces on the aerial plant parts compared to the wild-type (WT) as characterized by increased permeability for Toluidine blue stain and greater chlorophyll leaching. Quantitative RT-PCR analysis showed that the expression of suberin biosynthesis genes was significantly higher in the roots and leaves of overexpression lines compared to the WT. The biochemical analysis of leaf cuticular waxes showed that the overexpression lines accumulated 30% more waxes than the WT. Concurrently, overexpression lines also deposited almost twice the amount of suberin content in their roots compared with the WT. Taken together, these results showed that ANAC046 is an important transcription factor that promotes suberin biosynthesis in Arabidopsis thaliana roots.

Arabidopsis EARLY FLOWERING3 increases salt tolerance by suppressing salt stress response pathways.

Arabidopsis EARLY FLOWERING3 (ELF3) functions in modulating light input to the circadian clock, as a component of ELF3-ELF4-LUX ARRHYTHMO (LUX) evening complex. However, the role of ELF3 in stress responses remains largely unknown. In this study, we show that ELF3 enhances plants' resilience to salt stress: ELF3-overexpressing (ELF3-OX) plants are salt-tolerant, while elf3 mutants are more sensitive to salt stress. The expressions of many salt stress- and senescence-associated genes are altered in elf3-1 and ELF3-OX plants compared with wild-type. During salt stress, ELF3 suppresses factors that promote salt stress response pathways, mainly GIGANTEA (GI), at the post-translational level, and PHYTOCHROME INTERACTING FACTOR4 (PIF4), at the transcriptional level. To enhance the salt stress response, PIF4 directly downregulates the transcription of JUNGBRUNNEN1 (JUB1/ANAC042), encoding a transcription factor that upregulates the expression of stress tolerance genes, DREB2A and DELLA. Furthermore, PIF4 directly upregulates the transcription of ORESARA1 (ORE1/ANAC092) and SAG29, positive regulators of salt stress response pathways. Based on our results, we propose that ELF3 modulates key regulatory components in salt stress response pathways at the transcriptional and post-translational levels.

Control of proline accumulation under drought via a novel pathway comprising the histone methylase CAU1 and the transcription factor ANAC055.

Proline plays a crucial role in the drought stress response in plants. However, there are still gaps in our knowledge about the molecular mechanisms that regulate proline metabolism under drought stress. Here, we report that the histone methylase encoded by CAU1, which is genetically upstream of P5CS1 (encoding the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase 1), plays a crucial role in proline-mediated drought tolerance. We determined that the transcript level of CAU1 decreased while that of ANAC055 (encoding a transcription factor) increased in wild-type Arabidopsis under drought stress. Further analyses showed that CAU1 bound to the promoter of ANAC055 and suppressed its expression via H4R3sme2-type histone methylation in the promoter region. Thus, under drought stress, a decreased level of CAU1 led to an increased transcript level of ANAC055, which induced the expression of P5CS1 and increased proline level independently of CAS. Drought tolerance and the level of proline were found to be decreased in the cau1 anac055 double-mutant, while proline supplementation restored drought sensitivity in the anac055 mutant. Our results reveal the details of a novel pathway leading to drought tolerance mediated by CAU1.

The Direct Involvement of Dark-Induced Tic55 Protein in Chlorophyll Catabolism and Its Indirect Role in the MYB108-NAC Signaling Pathway during Leaf Senescence in Arabidopsis thaliana.

The chloroplast relies on proteins encoded in the nucleus, synthesized in the cytosol and subsequently transported into chloroplast through the protein complexes Toc and Tic (Translocon at the outer/inner membrane of chloroplasts). A Tic complex member, Tic55, contains a redox-related motif essential for protein import into chloroplasts in peas. However, Tic55 is not crucial for protein import in Arabidopsis. Here, a tic55-II-knockout mutant of Arabidopsis thaliana was characterized for Tic55 localization, its relationship with other translocon proteins, and its association with plant leaf senescence when compared to the wild type. Individually darkened leaves (IDLs) obtained through dark-induced leaf senescence were used to demonstrate chlorophyll breakdown and its relationship with plant senescence in the tic55-II-knockout mutant. The IDLs of the tic55-II-knockout mutant contained higher chlorophyll concentrations than those of the wild type. Our microarray analysis of IDLs during leaf senescence identified seven senescence-associated genes (SAGs) that were downregulated in the tic55-II-knockout mutant: ASP3, APG7, DIN2, DIN11, SAG12, SAG13, and YLS9. Real-time quantitative PCR confirmed the reliability of microarray analysis by showing the same expression patterns with those of the microarray data. Thus, Tic55 functions in dark-induced aging in A. thaliana by indirectly regulating downstream SAGs expression. In addition, the expression of four NAC genes, including ANAC003, ANAC010, ANAC042, and ANAC075 of IDL treated tic55-II-knockout mutant appeared to be downregulated. Yeast one hybrid assay revealed that only ANAC003 promoter region can be bound by MYB108, suggesting that a MYB-NAC regulatory network is involved in dark-stressed senescence.

Arabidopsis ANAC069 binds to C[A/G]CG[T/G] sequences to negatively regulate salt and osmotic stress tolerance.

KEY MESSAGE: ANAC069 binds to the DNA sequence of C[A/G]CG[T/G] to regulate the expression of genes, resulting in decreased ROS scavenging capability and proline biosynthesis, which contribute to increased sensitivity to salt and osmotic stress. NAM-ATAF1/2 and CUC2 (NAC) proteins are plant-specific transcription factors that play important roles in abiotic stress responses. In the present study, we characterized the physiological and regulatory roles of Arabidopsis thaliana ANAC069 in response to abiotic stresses. Arabidopsis plants overexpressing ANAC069 displayed increased sensitivity to abscisic acid, salt, and osmotic stress. Conversely, ANAC069 knockdown plants showed enhanced tolerance to salt and osmotic stress, but no change in ABA sensitivity. Further studies showed that ANAC069 inhibits the expression of SOD, POD, GST, and P5CS genes. Consequently, the transcript level of ANAC069 correlated negatively with the reactive oxygen species (ROS) scavenging ability and the proline level. The genes regulated by ANAC069 were further studied using a gene chip on a genome-wide scale, and 339 and 226 genes up- and downregulated by ANAC069 were identified. Analysis of the promoters of the genes affected by ANAC069 suggested that ANAC069 regulates the expression of genes mainly through interacting with the DNA sequence C[A/G]CG[T/G] in response to abiotic stresses. Collectively, our data suggest that ANAC069 could recognize C[A/G]CG[T/G] sequences to regulate the expression of genes that negatively regulates salt and osmotic stress tolerance by decreasing ROS scavenging capability and proline biosynthesis.

JAZ2 controls stomata dynamics during bacterial invasion.

Coronatine (COR) facilitates entry of bacteria into the plant apoplast by stimulating stomata opening. COR-induced signaling events at stomata remain unclear. We found that the COR and jasmonate isoleucine (JA-Ile) co-receptor JAZ2 is constitutively expressed in guard cells and modulates stomatal dynamics during bacterial invasion We analyzed tissue expression patterns of AtJAZ genes and measured stomata opening and pathogen resistance in loss- and gain-of-function mutants. Arabidopsis jaz2 mutants are partially impaired in pathogen-induced stomatal closing and more susceptible to Pseudomonas. Gain-of-function mutations in JAZ2 prevent stomatal reopening by COR and are highly resistant to bacterial penetration. The JAZ2 targets MYC2, MYC3 and MYC4 directly regulate the expression of ANAC19, ANAC55 and ANAC72 to modulate stomata aperture. Due to the antagonistic interactions between the salicylic acid (SA) and JA defense pathways, efforts to increase resistance to biotrophs result in enhanced susceptibility to necrotrophs, and vice versa. Remarkably, dominant jaz2Δjas mutants are resistant to Pseudomonas syringae but retain unaltered resistance against necrotrophs. Our results demonstrate the existence of a COI1-JAZ2-MYC2,3,4-ANAC19,55,72 module responsible for the regulation of stomatal aperture that is hijacked by bacterial COR to promote infection. They also provide novel strategies for crop protection against biotrophs without compromising resistance to necrotrophs.

Jasmonic acid promotes degreening via MYC2/3/4- and ANAC019/055/072-mediated regulation of major chlorophyll catabolic genes.

Degreening caused by rapid chlorophyll (Chl) degradation is a characteristic event during green organ senescence or maturation. Pheophorbide a oxygenase gene (PAO) encodes a key enzyme of Chl degradation, yet its transcriptional regulation remains largely unknown. Using yeast one-hybrid screening, coupled with in vitro and in vivo assays, we revealed that Arabidopsis MYC2/3/4 basic helix-loop-helix proteins directly bind to PAO promoter. Overexpression of the MYCs significantly enhanced the transcriptional activity of PAO promoter in Arabidopsis protoplasts, and methyl jasmonate (MeJA) treatment greatly induced PAO expression in wild-type Arabidopsis plants, but the induction was abolished in myc2 myc3 myc4. In addition, MYC2/3/4 proteins could promote the expression of another Chl catabolic enzyme gene, NYC1, as well as a key regulatory gene of Chl degradation, NYE1/SGR1, by directly binding to their promoters. More importantly, the myc2 myc3 myc4 triple mutant showed a severe stay-green phenotype, whereas the lines overexpressing the MYCs showed accelerated leaf yellowing upon MeJA treatment. These results suggest that MYC2/3/4 proteins may mediate jasmonic acid (JA)-induced Chl degradation by directly activating these Chl catabolic genes (CCGs). Three NAC family proteins, ANAC019/055/072, downstream from MYC2/3/4 proteins, could also directly promote the expression of a similar set of CCGs (NYE1/SGR1, NYE2/SGR2 and NYC1) during Chl degradation. In particular, anac019 anac055 anac072 triple mutant displayed a severe stay-green phenotype after MeJA treatment. Finally, we revealed that MYC2 and ANAC019 may interact with each other and synergistically enhance NYE1 expression. Together, our study reveals a hierarchical and coordinated regulatory network of JA-induced Chl degradation.

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.