Abscisic Acid Responses In Arabidopsis Research Articles

SUPPRESSOR OF MAX2 LIKE 6, 7, and 8 Interact with DDB1 BINDING WD REPEAT DOMAIN HYPERSENSITIVE TO ABA DEFICIENT 1 to Regulate the Drought Tolerance and Target SUCROSE NONFERMENTING 1 RELATED PROTEIN KINASE 2.3 to Abscisic Acid Response in Arabidopsis.

SUPPRESSOR OF MAX2-LIKE 6, 7, and 8 (SMXL6,7,8) function as repressors and transcription factors of the strigolactone (SL) signaling pathway, playing an important role in the development and stress tolerance in Arabidopsis thaliana. However, the molecular mechanism by which SMXL6,7,8 negatively regulate drought tolerance and ABA response remains largely unexplored. In the present study, the interacting protein and downstream target genes of SMXL6,7,8 were investigated. Our results showed that the substrate receptor for the CUL4-based E3 ligase DDB1-BINDING WD-REPEAT DOMAIN (DWD) HYPERSENSITIVE TO ABA DEFICIENT 1 (ABA1) (DWA1) physically interacted with SMXL6,7,8. The degradation of SMXL6,7,8 proteins were partially dependent on DWA1. Disruption of SMXL6,7,8 resulted in increased drought tolerance and could restore the drought-sensitive phenotype of the dwa1 mutant. In addition, SMXL6,7,8 could directly bind to the promoter of SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASE 2.3 (SnRK2.3) to repress its transcription. The mutations in SnRK2.2/2.3 significantly suppressed the hypersensitivity of smxl6/7/8 to ABA-mediated inhibition of seed germination. Conclusively, SMXL6,7,8 interact with DWA1 to negatively regulate drought tolerance and target ABA-response genes. These data provide insights into drought tolerance and ABA response in Arabidopsis via the SMXL6,7,8-mediated SL signaling pathway.

BIC2, a Cryptochrome Function Inhibitor, Is Involved in the Regulation of ABA Responses in Arabidopsis.

The plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via regulating the expression of ABA response genes. BIC1 (Blue-light Inhibitor of Cryptochromes 1) and BIC2 have been identified as the inhibitors of plant cryptochrome functions, and are involved in the regulation of plant development and metabolism in Arabidopsis . In this study, we report the identification of BIC2 as a regulator of ABA responses in Arabidopsis . RT-PCR (Reverse Transcription-Polymerase Chain Reaction) results show that the expression level of BIC1 remained largely unchanged, but that of BIC2 increased significantly in response to ABA treatment. Transfection assays in Arabidopsis protoplasts show that both BIC1 and BIC2 were mainly localized in the nucleus, and were able to activate the expression of the co-transfected reporter gene. Results in seed germination and seedling greening assays show that ABA sensitivity was increased in the transgenic plants overexpressing BIC2, but increased slightly, if any, in the transgenic plants overexpressing BIC1. ABA sensitivity was also increased in the bic2 single mutants in seedling greening assays, but no further increase was observed in the bic1 bic2 double mutants. On the other hand, in root elongation assays, ABA sensitivity was decreased in the transgenic plants overexpressing BIC2, as well as the bic2 single mutants, but no further decrease was observed in the bic1 bic2 double mutants. By using qRT-PCR (quantitative RT-PCR), we further examined how BIC2 may regulate ABA responses in Arabidopsis , and found that inhibition of ABA on the expression of the ABA receptor genes PYL4 (PYR1-Like 4) and PYL5 were decreased, but promotion of ABA on the expression of the protein kinase gene SnRK2.6 (SNF1-Related Protein Kinases 2.6) was enhanced in both the bic1 bic2 double mutants and 35S:BIC2 overexpression transgenic plants. Taken together, our results suggest that BIC2 regulates ABA responses in Arabidopsis possibly by affecting the expression of ABA signaling key regulator genes.

ASR1 and ASR2, Two Closely Related ABA-Induced Serine-Rich Transcription Repressors, Function Redundantly to Regulate ABA Responses in Arabidopsis.

The plant hormone abscisic acid (ABA) is able to regulate the expression of ABA-responsive genes via signaling transduction, and thus plays an important role in regulating plant responses to abiotic stresses. Hence, characterization of unknown ABA response genes may enable us to identify novel regulators of ABA and abiotic stress responses. By using RT-PCR analysis, we found that the expression levels of ABA-induced Serine-rich Repressor 1 (ASR1)and ASR2, two closely related unknown function genes, were increased in response to ABA treatment. Amino acid sequence analyses show that ASR1 contains an L×L×L motif and both ASR1 and ASR2 are enriched in serine. Transfection assays in Arabidopsis leaf protoplasts show that ASR1 and ASR2 were predominantly localized in the nucleus and were able to repress the expression of the reporter gene. The roles of ASRs in regulating ABA responses were examined by generating transgenic Arabidopsis plants over-expressing ASR1 and ASR2, respectively, and CRISPR/Cas9 gene-edited single and double mutants for ASR1 and ASR2. In both the seed germination and cotyledon greening assays, ABA sensitivity remained largely unchanged in the over-expression transgenic plants and the single mutants of ASR1 and ASR2, but greatly increased ABA sensitivity was observed in the asr1 asr2 double mutants. In root elongation assays, however, decreased ABA sensitivity was observed in the 35S:ASR1 and 35S:ASR2 transgenic plants, whereas increased ABA sensitivity was observed in the asr1 and asr2 single mutants, and ABA sensitivity was further increased in the asr1 asr2 double mutants. Transcriptome analysis show that the differentially expressed genes (DEGs) down-regulated in the 35S:ASR1 transgenic plant seedlings, but up-regulated in the asr1 asr2 double mutant seedlings were highly enriched in processes including responses to plant hormones and stress stimuli. Taken together, our results show that ASR1 and ASR2 are closely related ABA response genes, ASR1 and ASR2 are serine-rich novel transcription repressors, and they negatively regulate ABA responses in Arabidopsis in a redundant manner.

Calcium-dependent protein kinase CDPK16 phosphorylates serine-856 of glutamate receptor-like GLR3.6 protein leading to salt-responsive root growth in Arabidopsis.

Calcium-permeable channels in the plasma membrane play vital roles in plant growth, development, and response to environmental stimuli. Arabidopsis possesses 20 glutamate receptor-like proteins that share similarities with animal ionotropic glutamate receptors and mediate Ca2+ influx in plants. Calcium-dependent protein kinases (CDPKs) phosphorylate serine (Ser)-860 of glutamate receptor-like (GLR)3.7 protein, which interacts with 14-3-3ω and plays an essential role in salt and abscisic acid response in Arabidopsis by modulating Ca2+ signaling. However, the significance of CDPK- mediated phosphorylation status of Ser residues of GLR3.6 with regard to the functioning of GLR3.6 remains to be elucidated. In this study, we performed an in vitro kinase assay using CDPK16 and peptides containing the 14-3-3ω interacting domain of GLR3.6. We showed that Ser861/862 of GLR3.6 are required for the interaction with 14-3-3ω and that Ser856 of GLR3.6 is specifically phosphorylated by CDPK16 but not by CDPK3 and CDPK34. In addition, the expression of GLR3.6 was quickly downregulated by salt stress, and plants of glr3.6 mutants and GLR3.6-overexpression lines presented shorter and longer root lengths, respectively, under normal growth conditions than Col. Overexpression of the GLR3.6-Ser856 to Ala mutation resulted in a less sensitive phenotype in response to salt stress similar to glr3.6. Our results indicated that the Ser861/862 residues of GLR3.6 are required for interaction with 14-3-3ω. Additionally, the phosphorylation status of Ser856 residue of GLR3.6, which is mediated specifically by CDPK16, regulates root growth in normal and salt stress and conditions.

The RNA N6 -methyladenosine demethylase ALKBH9B modulates ABA responses in Arabidopsis.

The mRNA modification N6 -methyladenosine (m6 A) plays vital roles in plant development and biotic and abiotic stress responses. The RNA m6 A demethylase ALKBH9B can remove m6 A in alfalfa mosaic virus RNA and plays roles in alfalfa mosaic virus infection in Arabidopsis. However, it is unknown whether ALKBH9B also exhibits demethylation activity and has a biological role in endogenous plant mRNA. We demonstrated here that mRNA m6 A modification is induced by the phytohormone abscisic acid (ABA) and that ALKBH9B has m6 A demethylation activity on endogenous mRNA. Knocking out ALKBH9B led to hypersensitivity to ABA treatment during seed germination and early seedling development. We further showed that ALKBH9B removes the m6 A modification in the ABA INSENSITIVE 1 (ABI1) and BRI1-EMS-SUPPRESSOR 1 (BES1) transcripts following ABA treatment, affecting the stability of these mRNAs. Furthermore, we determined that ALKBH9B acts genetically upstream of the transcription factors ABI3 and ABI5, and its regulatory function in ABA responses depended on ABI3 and ABI5. Our findings reveal the important roles of the m6 A modification in ABA responses and highlight the role of ALKBH9B-mediated m6 A demethylation in regulating ABA responses post-transcriptionally.

AtbZIP62 Acts as a Transcription Repressor to Positively Regulate ABA Responses in Arabidopsis.

The basic region/leucine zipper (bZIP) transcription factor AtbZIP62 is involved in the regulation of plant responses to abiotic stresses, including drought and salinity stresses, NO3 transport, and basal defense in Arabidopsis. It is unclear if it plays a role in regulating plant responses to abscisic acid (ABA), a phytohormone that can regulate plant abiotic stress responses via regulating downstream ABA-responsive genes. Using RT-PCR analysis, we found that the expression level of AtbZIP62 was increased in response to exogenously applied ABA. Protoplast transfection assays show that AtbZIP62 is predominantly localized in the nucleus and functions as a transcription repressor. To examine the roles of AtbZIP62 in regulating ABA responses, we generated transgenic Arabidopsis plants overexpressing AtbZIP62 and created gene-edited atbzip62 mutants using CRISPR/Cas9. We found that in both ABA-regulated seed germination and cotyledon greening assays, the 35S:AtbZIP62 transgenic plants were hypersensitive, whereas atbzip62 mutants were hyposensitive to ABA. To examine the functional mechanisms of AtbZIP62 in regulating ABA responses, we generated Arabidopsis transgenic plants overexpressing 35S:AtbZIP62-GR, and performed transcriptome analysis to identify differentially expressed genes (DEGs) in the presence and absence of DEX, and found that DEGs are highly enriched in processes including response to abiotic stresses and response to ABA. Quantitative RT-PCR results further show that AtbZIP62 may regulate the expression of several ABA-responsive genes, including USP, ABF2, and SnRK2.7. In summary, our results show that AtbZIP62 is an ABA-responsive gene, and AtbZIP62 acts as a transcription repressor to positively regulate ABA responses in Arabidopsis.

AtEAU1 and AtEAU2, Two EAR Motif-Containing ABA Up-Regulated Novel Transcription Repressors Regulate ABA Response in Arabidopsis.

EAR (Ethylene-responsive element binding factor-associated Amphiphilic Repression) motif-containing transcription repressors have been shown to regulate plant growth and development, and plant responses to plant hormones and environmental stresses including biotic and abiotic stresses. However, the functions of most EAR-motif-containing proteins remain largely uncharacterized. The plant hormone abscisic acid (ABA) also plays important roles in regulating plant responses to abiotic stresses via activation/repression of ABA-responsive genes. We report here the identification and functional characterization of two ABA-responsive EAR motif-containing protein genes, AtEAU1 (Arabidopsis thaliana EAR motif-containing ABA Up-regulated 1) and AtEAU2. Quantitative RT-PCR results show that the expressions of AtEAU1 and AtEAU2 were increased by ABA treatment, and were decreased in the ABA biosynthesis mutant aba1-5. Assays in transfected Arabidopsis protoplasts show that both AtEAU1 and AtEAU2 were specifically localized in the nucleus, and when recruited to the promoter region of the reporter gene by a fused DNA binding domain, repressed reporter gene expression. By using T-DNA insertion mutants and a gene-edited transgene-free mutant generated by CRISPR/Cas9 gene editing, we performed ABA sensitivity assays, and found that ABA sensitivity in the both ateau1 and ateau2 single mutants was increased in seedling greening assays. ABA sensitivity in the ateau1 ateau2 double mutants was also increased, but was largely similar to the ateau1 single mutants. On the other hand, all the mutants showed a wild type response to ABA in root elongation assays. Quantitative RT-PCR results show that the expression level of PYL4, an ABA receptor gene was increased, whereas that of ABI2, a PP2C gene was decreased in the ateau1 and ateau1 single, and the ateau1 ateau2 double mutants. In summary, our results suggest that AtEAU1 and AtEAU2 are ABA-response genes, and AtEAU1 and AtEAU2 are novel EAR motif-containing transcription repressors that negatively regulate ABA responses in Arabidopsis, likely by regulating the expression of some ABA signaling key regulator genes.

The R2R3 MYB Transcription Factor MYB71 Regulates Abscisic Acid Response in Arabidopsis.

Abscisic acid (ABA) regulates plant responses to abiotic stresses via regulating the expression of downstream genes, yet the functions of many ABA responsive genes remain unknown. We report here the characterization of MYB71, a R2R3 MYB transcription factor in regulating ABA responses in Arabidopsis. RT-PCR results show that the expression level of MYB71 was increased in response to ABA treatment. Arabidopsis protoplasts transfection results show that MYB71 was specifically localized in nucleus and it activated the Gal4:GUS reporter gene when recruited to the Gal4 promoter by a fused DNA binding domain GD. Roles of MYB71 in regulating plant response to ABA were analyzed by generating Arabidopsis transgenic plants overexpression MYB71 and gene edited mutants of MYB71. The results show that ABA sensitivity was increased in the transgenic plants overexpression MYB71, but decreased in the MYB71 mutants. By using a DEX inducible system, we further identified genes are likely regulated by MYB71, and found that they are enriched in biological process to environmental stimuli including abiotic stresses, suggesting that MYB71 may regulate plant response to abiotic stresses. Taken together, our results suggest that MYB71 is an ABA responsive gene, and MYB71 functions as a transcription activator and it positively regulates ABA response in Arabidopsis.

Monomerization of abscisic acid receptors through CARKs-mediated phosphorylation.

Cytosolic ABA Receptor Kinases (CARKs) play a pivotal role in abscisic acid (ABA)-dependent pathway in response to dehydration, but their regulatory mechanism in ABA signaling remains unexplored. In this study, we showed that CARK4/5 of CARK family physically interacted with ABA receptors (RCARs/PYR1/PYLs), including RCAR3, RCAR11-RCAR14, while CARK2/7/11 only interacted with RCAR11-RCAR14, but not RCAR3. It indicates that the members in CARK family function redundantly and differentially in ABA signaling. RCAR12 can form heterodimer with RCAR3 in vitro and in vivo. Moreover, the members of CARK family can form homodimer or heterodimer in a kinase activity dependent manner. ITC (isothermal titration calorimetry) analysis demonstrated that the phosphorylation of RCAR12 by CARK1 enhanced the ABA binding affinity. The phosphor-mimic RCAR12T105D significantly displayed ABA-induced inhibition of the phosphatase ABI1 (ABA insensitive 1) activity, leading to upregulation of ABA-responsive genes RD29A and RD29B in cark157:RCAR12T105D transgenic plants, which exhibited ABA hypersensitive phenotype. The transcription factor ABI5 (ABA insensitive 5) activates the transcriptions of CARK1 and CARK3 by binding to ABA-response elements (ABREs) of their promoters. Collectively, our data imply that the dimeric CARKs phosphorylate homodimer or heterodimer ABA receptors, leading to monomerization for triggering ABA responses in Arabidopsis.

Arabidopsis glutamate receptor GLR3.7 is involved in abscisic acid response

ABSTRACT The ionotropic glutamate receptor (iGluR) plays an important role in neuronal signaling in animal cells. There are at least 20 glutamate receptor-like (GLR) genes in Arabidopsis thaliana. These genes are involved in seed germination, root growth, wounding response, stomata closure, etc. A recent study showed that Arabidopsis clade III glutamate receptor GLR3.7 is involved in salt stress response. We tested whether GLR3.7 is involved in abscisic acid (ABA) response. In the present study, we found that the expression of GLR3.7 was reduced by ABA treatment. Under ABA-treated condition, GLR3.7 overexpression lines exhibited significantly higher seed germination rate at 60, 72 and 84 h under ABA-treated condition. A point mutation in 14-3-3 binding site of GLR3.7 in GLR3.7-S860A overexpression lines exhibited higher seed germination inhibition under ABA-treated conditions. Our results support that GLR3.7 is involved in ABA response in Arabidopsis. In addition, Ser-860 of GLR3.7 appears to be important in ABA response.

Upstream Open Reading Frame Mediated Translation of WNK8 Is Required for ABA Response in Arabidopsis.

With no lysine (K) (WNK) kinases comprise a family of serine/threonine kinases belonging to an evolutionary branch of the eukaryotic kinome. These special kinases contain a unique active site and are found in a wide range of eukaryotes. The model plant Arabidopsis has been reported to have 11 WNK members, of which WNK8 functions as a negative regulator of abscisic acid (ABA) signaling. Here, we found that the expression of WNK8 is post-transcriptionally regulated through an upstream open reading frame (uORF) found in its 5′ untranslated region (5′-UTR). This uORF has been predicted to encode a conserved peptide named CPuORF58 in both monocotyledons and dicotyledons. The analysis of the published ribosome footprinting studies and the study of the frameshift CPuORF58 peptide with altered repression capability suggested that this uORF causes ribosome stalling. Plants transformed with the native WNK8 promoter driving WNK8 expression were comparable with wild-type plants, whereas the plants transformed with a similar construct with mutated CPuORF58 start codon were less sensitive to ABA. In addition, WNK8 and its downstream target RACK1 were found to synergistically coordinate ABA signaling rather than antagonistically modulating glucose response and flowering in plants. Collectively, these results suggest that the WNK8 expression must be tightly regulated to fulfill the demands of ABA response in plants.

MIR156-Targeted SPL9 Is Phosphorylated by SnRK2s and Interacts With ABI5 to Enhance ABA Responses in Arabidopsis.

The miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors play key roles in regulating plant development, but little is known about their function in abscisic acid (ABA) signaling. Here, we report that the miR156-targeted SPLs enhance ABA responses and contribute to the inhibition of pre-harvest sprouting. We find that SPL9 directly activates the expression of ABA responsive genes through binding to their promoters. SPL9 was further shown to physically interact with ABSCISIC ACID INSENSITIVE 5 (ABI5), a master transcription factor in ABA signaling, thus promoting its association with the promoters of ABA responsive genes. Furthermore, we reveal that the protein kinases SnRK2s interact with and phosphorylate SPL9, which is essential for its role in the activation of ABA responses. Together, our results disclose a SnRK2s-SPLs-ABI5 regulatory module in ABA signaling in Arabidopsis.

Hydrogen sulfide-linked persulfidation of ABI4 controls ABA responses through the transactivation of MAPKKK18 in Arabidopsis

Hydrogen sulfide (H2S) is a signaling molecule that regulates plant hormone and stress responses. The phytohormone abscisic acid (ABA) plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1 (DES1) and the production of H2S in guard cells. However, it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals. In this study, we discovered that ABSCISIC ACID INSENSITIVE 4 (ABI4) acts downstream of DES1 in the control of ABA responses in Arabidopsis. ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA, and loss of DES1 function impairs this process. Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination, seedling establishment, and stomatal closure, which are abolished in the ABI4Cys250Ala mutated variant. Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA. Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18 (MAPKKK18), which propagates the MAPK signaling cascade induced by ABA. Furthermore, the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18, and ABI4 can bind the DES1 promoter, forming a regulatory loop. Taken together, these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.

AITRL, an evolutionarily conserved plant specific transcription repressor regulates ABA response in Arabidopsis

Expression of stress response genes can be regulated by abscisic acid (ABA) dependent and ABA independent pathways. Osmotic stresses promote ABA accumulation, therefore inducing the expression of stress response genes via ABA signaling. Whereas cold and heat stresses induce the expression of stress response genes via ABA independent pathway. ABA induced transcription repressors (AITRs) are a family of novel transcription factors that play a role in ABA signaling, and Drought response gene (DRG) has previously been shown to play a role in regulating plant response to drought and freezing stresses. We report here the identification of DRG as a novel transcription factor and a regulator of ABA response in Arabidopsis. We found that the expression of DRG was induced by ABA treatment. Homologs searching identified AITR5 as the most closely related Arabidopsis protein to DRG, and homologs of DRG, including the AITR-like (AITRL) proteins in bryophytes and gymnosperms, are specifically presented in embryophytes. Therefore we renamed DRG as AITRL. Protoplast transfection assays show that AITRL functioned as a transcription repressor. In seed germination and seedling greening assays, the aitrl mutants showed an increased sensitivity to ABA. By using qRT-PCR, we show that ABA responses of some ABA signaling component genes including some PYR1-likes (PYLs), PROTEIN PHOSPHATASE 2Cs (PP2Cs) and SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2s (SnRK2s) were reduced in the aitrl mutants. Taken together, our results suggest that AITRLs are a family of novel transcription repressors evolutionally conserved in embryophytes, and AITRL regulates ABA response in Arabidopsis by affecting ABA response of some ABA signaling component genes.

NAC103, a NAC family transcription factor, regulates ABA response during seed germination and seedling growth in Arabidopsis.

The Arabidopsis transcription factor NAC103 is up-regulated and its encoding protein is stabilized by ABA treatment, which positively regulates several ABA-responsive downstream genes during seed germination and seedlings growth. The Arabidopsis transcription factor NAC103 was previously found to be involved in endoplasmic reticulum (ER) stress and DNA damage responses. In this study, we report the new biological function of NAC103 in abscisic acid (ABA) response during seed germination and seedling growth in Arabidopsis. The expression of NAC103 was up-regulated and the NAC103 protein was stabilized by ABA treatment. Both the loss-of-function mutants of NAC103, created by targeted gene-editing, and the over-expression plants of NAC103 have no obvious germination-related phenotype under normal growth conditions. However, under exogenous ABA treatment conditions, the NAC103 mutants were less sensitive to ABA during seed germination; in contrast, the NAC103 over-expression plants were more sensitive to ABA during seed germination and young seedling growth. Further, NAC103 regulated several ABA-responsive downstream genes including MYB78, MYB3, PLP3, AMY1, and RGL2. These results demonstrate that NAC103 positively regulates ABA response in Arabidopsis.

The RING finger E3 ligases PIR1 and PIR2 mediate PP2CA degradation to enhance abscisic acid response in Arabidopsis.

Recent work has established a core ABA signaling pathway in which A-type PP2C protein phosphatases act as central negative modulators. Although ABA signaling inhibits PP2C activity through ABA-receptor complex, it remains unknown if other mechanisms exist to modulate the level of PP2Cs. Here, we identified a RING domain ubiquitin E3 ligase, PIR1 (PP2CA interacting RING finger protein 1), that interacted with PP2CA. Of the two splicing isoforms, PIR1.2 was isolated from leaf tissue. The PIR1.2 exhibited E3 ligase activity and determined PP2CA stability in the presence of ABA. Consistent with the conclusion that PIR1 promotes ABA signaling by removing PP2CA, a negative modulator, the pir1 knockout mutant displayed an ABA-hyposensitive phenotype. We further showed that PIR2, the closest homologue of PIR1.2, also interacted with PP2CA. Although the pir2 knockout mutant did not display altered ABA response, the pir1-1/pir2 double mutant became more insensitive to ABA than the wild-type or pir1-1 and pir2 single mutants. Using a cell-free degradation assay, ABA promoted degradation of PP2CA, however, such degradation was delayed when incubated with protein extract prepared from the pir1-1/pir2 double mutant. Our data suggest that PIR1 and PIR2 positively modulate ABA signaling by targeting PP2CA for degradation.

Calmodulin 1 Regulates Senescence and ABA Response in Arabidopsis.

Cellular calcium acts as a second messenger and regulates diverse developmental events and stress responses. Cytosolic calcium has long been considered as an important regulator of senescence, however, the role of Ca2+ in plant senescence has remained elusive. Here we show that the Calmodulin 1 (CaM1) gene, which encodes Ca2+-binding protein calmodulin 1, positively regulates leaf senescence in Arabidopsis. Yellowing of leaves, accumulation of reactive oxygen species (ROS), and expression of the senescence-associated gene 12 (SAG12) were significantly enhanced in CaM1 overexpression plants. In contrast, abscisic acid (ABA)-triggered ROS production and stomatal closure were reduced in amiRNA-CaM1 plants. We found a positive-feedback regulation loop among three signaling components, CaM1, RPK1, and RbohF, which physically associate with each other. RPK1 positively regulates the expression of the CaM1 gene, and the CaM1 protein, in turn, up-regulates RbohF gene expression. Interestingly, the expression of CaM1 was down-regulated in rbohD, rbohF, and rbohD/F mutants. We show that CaM1 positively regulates ROS production, leaf senescence, and ABA response in Arabidopsis.

The Asparagine-Rich Protein NRP Facilitates the Degradation of the PP6-type Phosphatase FyPP3 to Promote ABA Response in Arabidopsis

The phytohormone abscisic acid (ABA) plays critical roles in abiotic stress responses and plant development. In germinating seeds, the phytochrome-associated protein phosphatase, FyPP3, negatively regulates ABA signaling by dephosphorylating the transcription factor ABI5. However, whether and how FyPP3 is regulated at the posttranscriptional level remains unclear. Here, we report that an asparagine-rich protein, NRP, interacts with FyPP3 and tethers FyPP3 to SYP41/61-positive endosomes for subsequent degradation in the vacuole. Upon ABA treatment, the expression of NRP was induced and NRP-mediated FyPP3 turnover was accelerated. Consistently, ABA-induced FyPP3 turnover was abolished in an nrp null mutant. On the other hand, FyPP3 can dephosphorylate NRP in vitro, and overexpression of FyPP3 reduced the half-life of NRP in vivo. Genetic analyses showed that NRP has a positive role in ABA-mediated seed germination and gene expression, and that NRP is epistatic to FyPP3. Taken together, our results identify a new regulatory circuit in the ABA signaling network, which links the intracellular trafficking with ABA signaling.

Actin depolymerization-induced changes in proteome of Arabidopsis roots

Actin cytoskeleton is a vital cellular structure primarily known for controlling cell integrity, division and expansion. Here we present a proteomic dissection of Arabidopsis roots treated by actin depolymerizing agent latrunculin B. Pharmacological disintegration of the actin cytoskeleton by latrunculin B caused downregulation of several proteins involved in the actin organization and dynamics. Moreover, this approach helped to identify new protein candidates involved in gene transcription, due to the altered abundance of proteins involved in mRNA nuclear export. Finally, latrunculin B negatively affected the abundance of abscisic acid (ABA) responsive proteins. SignificanceThis article substantially contributes to the current knowledge about the importance of actin organization and dynamics in proteome remodelling. We employed gel based and gel free proteomic analyses and identified several new protein candidates and protein networks linking actin dynamics to the gene transcription and to the ABA response in Arabidopsis.

An ABA down-regulated bHLH transcription repressor gene, bHLH129 regulates root elongation and ABA response when overexpressed in Arabidopsis.

Plant hormone abscisic acid (ABA) plays a crucial role in modulating plant responses to environmental stresses. Basic helix-loop-helix (bHLH) transcription factors are one of the largest transcription factor families that regulate multiple aspects of plant growth and development, as well as of plant metabolism in Arabidopsis. Several bHLH transcription factors have been shown to be involved in the regulation of ABA signaling. We report here the characterization of bHLH129, a bHLH transcription factor in Arabidopsis. We found that the expression level of bHLH129 was reduced in response to exogenously applied ABA, and elevated in the ABA biosynthesis mutant aba1-5. Florescence observation of transgenic plants expressing bHLH129-GFP showed that bHLH129 was localized in the nucleus, and transient expression of bHLH129 in protoplasts inhibited reporter gene expression. When expressed in Arabidopsis under the control of the 35S promoter, bHLH129 promoted root elongation, and the transgenic plants were less sensitivity to ABA in root elongation assays. Quantitative RT-PCR results showed that ABA response of several genes involved in ABA signaling, including ABI1, SnRK2.2, SnRK2.3 and SnRK2.6 were altered in the transgenic plants overexpressing bHLH129. Taken together, our study suggests that bHLH129 is a transcription repressor that negatively regulates ABA response in Arabidopsis.