Abscisic Acid Increases Hydrogen Peroxide in Multiple Subcellular Compartments to Drive Stomatal Closure

Stomatal closure regulates transpiration and gas exchange in response to environmental cues. Drought upregulates ABA signaling, which elevates levels of reactive oxygen species (ROS). However, the subcellular location and identity of these ROS has received limited study. We found that in guard cells, ABA increased fluorescence of the general redox sensor, dichlorofluorescein (DCF), in distinct subcellular locations including chloroplasts, cytosol, nuclei, and cytosolic puncta. These changes were lost in ABA-insensitive quintuple receptor mutant and accentuated in an ABA-hypersensitive mutant. ABA induced ROS accumulation in these subcellular compartments was lost in mutants with defects in genes encoding hydrogen peroxide synthesizing respiratory burst oxidase homolog (RBOH) enzymes and guard cells treated with the RBOH inhibitor VAS2870, while exogenous hydrogen peroxide treatment is sufficient to close guard cells. The hydrogen peroxide-selective probe, peroxy orange1, also showed ABA-dependent increases in chloroplasts and cytosolic puncta. Using the more sensitive genetically-encoded hydrogen peroxide reporter roGFP-Orp1, we also detected significant hydrogen peroxide increases in the cytosol and nucleus. These cytosolic puncta accumulate ROS after ABA treatment show colocalization with Mitotracker and with a mitochondrial targeted mt-roGFP2-Orp1, which also revealed ABA-increased ROS in mitochondria. These results indicate that elevated hydrogen peroxide after ABA treatment in these subcellular compartments is necessary and sufficient to drive stomatal closure.

Interplay between positive and negative regulation by B3-type transcription factors is critical for the accurate expression of the ABA INSENSITIVE 4 gene.

The ABA-INSENSITIVE 4 transcription factor is key for the regulation of diverse aspects of plant development and environmental responses, including proper perception of hormonal and nutritional signals. ABI4 activity is highly regulated at the transcriptional and post-transcriptional levels leading to precise expression mainly in the developing seed and early seedling development. Based on genetic and molecular approaches in the current study we provide new insights into the central mechanism underpinning the transcriptional regulation of ABI4 during both seed and vegetative development. We identified a complex interplay between the LEC2 and ABI3 transcriptional activators and the HSI/VAL repressors that is critical for proper ABI4 expression. Interestingly, the regulation by these proteins relies on the two RY cis-acting motifs present two kb upstream of the ABI4 gene. Our analysis also shows that the chromatin landscape of the ABI4 loci is highly dependent on the LEC2 and HSI2/VAL proteins. LEC2 regulation extends to the vegetative development and the absence of this factor results in ABA- and sugar-insensitive signaling in the developing plant. This regulatory circuit functions as a major control module for the correct spatial-temporal expression of ABI4 and prevents its ectopic accumulation that is harmful to the plant.

Allelic Variations in 5' UTR of TaAFP-B Effecting the Seed Dormancy and Other Agronomic Traits in Transgenic Wheat

BackgroundTaAFP (Triticum aestivum L. ABA insensitive five binding protein) is the homology of AFP of Arabidopsis thaliana which was a negative regulator in ABA signaling and regulated embryo germination and seed dormancy. TaABI5 (Triticum aestivum L. ABA insensitive five) gene was seed-specific, and accumulated during wheat grain maturation and dormancy acquisition, which played an important role in seed dormancy. In our previous study, two allelic variants of TaAFP were identified on chromosome 2BS in common wheat, and designated as TaAFP-B1a and TaAFP-B1b. Sequence analysis showed a 4-bp insertion in the 5’UTR region of TaAFP-B1a compared with TaAFP-B1b, which affected the mRNA transcription level, mRNA decay, translation levels of GUS and tdTomatoER, GUS activity, and was significantly associated with seed dormancy in common wheat. ResultsThe results of transgenic wheats showed that: the genotypes of average GI values, plant height, grain weight of hundred and rough of second and third stem node are all significantly more in pUbi-TaAFP-BaS transformed wheat plants than in pUbi-TaAFP-BbS transformed ones, but transcript expression level. ConclusionAbove all dates indicated that the 4-bp insertion in the 5'UTR of TaAFP-B decreased the transcript expression level of TaAFP-B and the PHS resistance, and increased the plant height, grain weight of hundred and lodging resistance in this system of over expression transgenic wheat.

O-fucosylation of CPN20 by SPINDLY Derepresses Abscisic Acid Signaling During Seed Germination and Seedling Development

SPINDLY is involved in some aspects of plant development. However, the nature of this protein as an O-fucosyltransferase was recently discovered. In this study, we show that SPINDLY (SPY) interacts with CPN20 in yeast two-hybrid and split-luc assays, and the interaction is promoted by ABA. CPN20 is a chloroplast-localized co-chaperonin that negatively regulates ABAR-mediated ABA signaling. By using Electron Transfer Dissociation-MS/MS analysis, two O-fucosylation sites, e.g., 116th and 119th threonines, were detected in ectopically expressed CPN20 in mammalian cells and in Arabidopsis. The O-fucosylation at both threonine residues was confirmed by in vitro peptide O-fucosylation assay. We further show that CPN20 accumulates in the chloroplast of spy mutants, suggesting that SPY negatively regulates CPN20 localization in the chloroplast. In vivo protein degradation assay along with CPN20 localization behavior suggest that import of CPN20 into the chloroplast is negatively regulated by SPY. Genetic analysis shows that ABA insensitive phenotypes of spy-3 in terms of seed germination and early seedling development are partially suppressed by the cpn20 mutation, suggesting that CPN20 acts downstream of SPY in this ABA signaling pathway and that there may exist other pathways in parallel with CPN20. Collectively, the above data support the notion that the O-fucosylation of CPN20 by SPY fine-tunes ABA signaling in Arabidopsis.

CIN-Like TCP13 is Essential For Plant Growth Regulation Under Dehydration Stress

Plants modulate their shape and growth in response to environmental stress. However, regulatory mechanisms underlying the changes in shape and growth under environmental stress remain elusive. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family of transcription factors (TFs) are key regulators for limiting the growth of leaves through negative effect of auxin response. Here, we report that stress-inducible CIN-like TCP13 plays a key role in inducing morphological changes in leaves and growth regulation in leaves and roots that confer dehydration stress tolerance in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing TCP13 ( 35Spro ::TCP13OX ) exhibited leaf rolling, and reduced leaf growth under osmotic stress. The 35Spro:: TCP13OX transgenic leaves showed decreased water loss from leaves, and enhanced dehydration tolerance compared with their control counterparts. Plants overexpressing a chimeric repressor domain SRDX-fused TCP13 ( TCP13pro ::TCP13SRDX ) showed severely serrated leaves and enhanced root growth. Transcriptome analysis of TCP13pro ::TCP13SRDX transgenic plants revealed that TCP13 affects the expression of dehydration- and abscisic acid (ABA)-regulated genes. TCP13 is also required for the expression of dehydration-inducible auxin-regulated genes, INDOLE-3-ACETIC ACID5 ( IAA5 ) and LATERAL ORGAN BOUNDARIES ( LOB) DOMAIN 1 ( LBD1 ). Furthermore, tcp13 knockout mutant plants showed ABA-insensitive root growth and reduced dehydration-inducible gene expression. Our findings provide new insight into the molecular mechanism of CIN-like TCP that is involved in both auxin and ABA response under dehydration stress.

Melatonin Alleviates Low-Temperature Stress via ABI5-Mediated Signals During Seed Germination in Rice (Oryza sativa L.)

With increasing areas of direct sowing, low-temperature (LT) stress drastically affects global rice production. Exogenous applications of melatonin (MT) serve as one of the effective ways to improve seed germination under various stress conditions. In this study, we found that MT treatment greatly improved the LT stress-induced loss of germination percentage and the weak performance of seedlings under LT of constant 20°C (LT20). This was largely dependent on the activated antioxidant system and enhanced activities of storage substance utilization-associated enzymes. Moreover, we also detected that exogenous feeding of MT significantly increased the biosynthesis of gibberellin (GA) and endogenous MT but simultaneously inhibited the accumulation of abscisic acid (ABA) and hydrogen peroxide (H2O2) under LT20 stress. These results suggested that MT had antagonistic effects on ABA and H2O2. In addition, MT treatment also significantly enhanced the expression of CATALYSE 2 (OsCAT2), which was directly regulated by ABA-INSENSITIVE 5 (OsABI5), a core module of ABA-stressed signals, and thus promoting the H2O2 scavenging to reach reactive oxygen species (ROS) homeostasis, which consequently increased GA biosynthesis. However, in abi5 mutants, OsCAT2 failed in response to LT20 stress irrespective of MT treatment, indicating that OsABI5 is essential for MT-mediated seed germination under LT20 stress. Collectively, we now demonstrated that MT showed a synergistic interaction with an ABI5-mediated signal to mediate seed germination, partially through the direct regulation of OsCAT2.

Arabidopsis LSH8 Positively Regulates ABA Signaling by Changing the Expression Pattern of ABA-Responsive Proteins

Phytohormone ABA regulates the expression of numerous genes to significantly affect seed dormancy, seed germination and early seedling responses to biotic and abiotic stresses. However, the function of many ABA-responsive genes remains largely unknown. In order to improve the ABA-related signaling network, we conducted a large-scale ABA phenotype screening. LSH, an important transcription factor family, extensively participates in seedling development and floral organogenesis in plants, but whether its family genes are involved in the ABA signaling pathway has not been reported. Here we describe a new function of the transcription factor LSH8 in an ABA signaling pathway. In this study, we found that LSH8 was localized in the nucleus, and the expression level of LSH8 was significantly induced by exogenous ABA at the transcription level and protein level. Meanwhile, seed germination and root length measurements revealed that lsh8 mutant lines were ABA insensitive, whereas LSH8 overexpression lines showed an ABA-hypersensitive phenotype. With further TMT labeling quantitative proteomic analysis, we found that under ABA treatment, ABA-responsive proteins (ARPs) in the lsh8 mutant presented different changing patterns with those in wild-type Col4. Additionally, the number of ARPs contained in the lsh8 mutant was 397, six times the number in wild-type Col4. In addition, qPCR analysis found that under ABA treatment, LSH8 positively mediated the expression of downstream ABA-related genes of ABI3, ABI5, RD29B and RAB18. These results indicate that in Arabidopsis, LSH8 is a novel ABA regulator that could specifically change the expression pattern of APRs to positively mediate ABA responses.

Genome-wide identification of ZmSnRK2 genes and functional analysis of ZmSnRK2.10 in ABA signaling pathway in maize (Zea mays L)

Background Phytohormone abscisic acid (ABA) is involved in the regulation of a wide range of biological processes. In Arabidopsis, it has been well-known that SnRK2s are the central components of the ABA signaling pathway that control the balance between plant growth and stress response, but the functions of ZmSnRK2 in maize are rarely reported. Therefore, the study of ZmSnRK2 is of great importance to understand the ABA signaling pathways in maize. Results In this study, 14 ZmSnRK2 genes were identified in the latest version of maize genome database. Phylogenetic analysis revealed that ZmSnRK2s are divided into three subclasses based on their diversity of C-terminal domains. The exon-intron structures, phylogenetic, synteny and collinearity analysis indicated that SnRK2s, especially the subclass III of SnRK2, are evolutionally conserved in maize, rice and Arabidopsis. Subcellular localization showed that ZmSnRK2 proteins are localized in the nucleus and cytoplasm. The RNA-Seq datasets and qRT-PCR analysis showed that ZmSnRK2 genes exhibit spatial and temporal expression patterns during the growth and development of different maize tissues, and the transcript levels of some ZmSnRK2 genes in kernel are significantly induced by ABA and sucrose treatment. In addition, we found that ZmSnRK2.10, which belongs to subclass III, is highly expressed in kernel and activated by ABA. Overexpression of ZmSnRK2.10 partially rescued the ABA-insensitive phenotype of snrk2.2/2.3 double and snrk2.2/2.3/2.6 triple mutants and led to delaying plant flowering in Arabidopsis. Conclusion The SnRK2 gene family exhibits a high evolutionary conservation and has expanded with whole-genome duplication events in plants. The ZmSnRK2s expanded in maize with whole-genome and segmental duplication, not tandem duplication. The expression pattern analysis of ZmSnRK2s in maize offers important information to study their functions. Study of the functions of ZmSnRK.10 in Arabidopsis suggests that the ABA-dependent members of SnRK2s are evolutionarily conserved in plants. Our study elucidated the structure and evolution of SnRK2 genes in plants and provided a basis for the functional study of ZmSnRK2s protein in maize.

Arabidopsis ABIG1 Functions in Laminar Growth and Polarity Formation through Regulation by REVOLUTA and KANADI

Leaf laminar growth and adaxial-abaxial boundary formation are fundamental outcomes of plant development. Boundary and laminar growth coordinate the further patterning and growth of the leaf, directing the differentiation of cell types within the top and bottom domains and promoting initiation of lateral organs along their adaxial/abaxial axis. Leaf adaxial-abaxial polarity specification and laminar out-growth are regulated by two transcription factors, REVOLUTA (REV) and KANADI (KAN). ABA INSENSITIVE TO GROWTH 1 (ABIG1) is a HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) Class II transcription factor and is a direct target of the adaxial-abaxial regulators REV and KAN. To investigate the role of ABIG1 in the leaf development and establishment of polarity, we examined the phenotypes of both gain-of-function and loss-of-function mutants. Through genetic interaction analysis with REV and KAN mutants, we have determined that ABIG1 plays a role in leaf laminar-growth as well as in adaxial-abaxial polarity establishment. Genetic and physical interaction assays showed that ABIG1 interacts with the transcriptional corepressor TOPLESS (TPL). This study provides new evidence that another HD-ZIP II gene, ABIG1, facilitates growth through the corepressor TPL.