Regulator Of Abscisic Acid Responses Research Articles

Ubiquitin negatively regulates ABA responses by inhibiting SnRK2.2 and SnRK2.3 kinase activity in Arabidopsis.

Abscisic acid (ABA) is an essential phytohormone for plant responses to complex and variable environmental conditions. The molecular basis of the ABA signaling pathway has been well elucidated. SnRK2.2 and SnRK2.3 are key protein kinases participating in ABA responses, and the regulation of their activity plays an important role in signaling. Previous mass spectroscopy analysis of SnRK2.3 suggested that ubiquitin and homologous proteins may bind directly to the kinase. Ubiquitin typically recruits E3 ubiquitin ligase complexes to target proteins, marking them for degradation by the 26S proteasome. Here, we show that SnRK2.2 and SnRK2.3 interact with ubiquitin but are not covalently attached to the protein, resulting in the suppression of their kinase activity. The binding between SnRK2.2, SnRK2.3, and ubiquitin is weakened under prolonged ABA treatment. Overexpression of ubiquitin positively regulated the growth of seedlings exposed to ABA. Our results thus demonstrate a novel function for ubiquitin, which negatively regulates ABA responses by directly inhibiting SnRK2.2 and SnRK2.3 kinase activity.

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.

U-box E3 ubiquitin ligase PUB8 attenuates abscisic acid responses during early seedling growth.

ABSCISIC ACID-INSENSITIVE3 (ABI3) and ABI5 are 2 crucial transcription factors in abscisic acid (ABA) signaling, and their homeostasis at the protein level plays a decisive role in seed germination and subsequent seedling growth. Here, we found that PLANT U-BOX 8 (PUB8), a U-box E3 ubiquitin ligase, physically interacts with ABI3 and ABI5 and negatively regulates ABA responses during early Arabidopsis (Arabidopsis thaliana) seedling growth. Loss-of-function pub8 mutants were hypersensitive to ABA-inhibited cotyledon greening, while lines overexpressing PUB8 with low levels of ABI5 protein abundance were insensitive to ABA. Genetic analyses showed that ABI3 and ABI5 were required for the ABA-sensitive phenotype of pub8, indicating that PUB8 functions upstream of ABI3 and ABI5 to regulate ABA responses. Biochemical analyses showed that PUB8 can associate with ABI3 and ABI5 for degradation through the ubiquitin-mediated 26S proteasome pathway. Correspondingly, loss-of-function of PUB8 led to enhanced ABI3 and ABI5 stability, while overexpression of PUB8 impaired accumulation of ABI3 and ABI5 in planta. Further phenotypic analysis indicated that PUB8 compromised the function of ABI5 during early seedling growth. Taken together, our results reveal the regulatory role of PUB8 in modulating the early seedling growth by controlling the homeostasis of ABI3 and ABI5.

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.

CBF4/DREB1D represses XERICO to attenuate ABA, osmotic and drought stress responses in Arabidopsis.

Water stress can severely impact plant growth, productivity and yield. Consequently, plants have evolved various strategies through which they can respond and adapt to their environment. XERICO (XER) is a stress-responsive RING E3 ubiquitin ligase that modulates abscisic acid (ABA) levels and promotes drought tolerance when overexpressed. To better understand the biological role of XER in stress responses, we characterized a xer-1 hypomorphic mutant and a CRISPR/Cas9-induced xer-2 null mutant in Arabidopsis. Both xer mutant alleles exhibited increased drought sensitivity, supporting the results from overexpression studies. Furthermore, we discovered that both xer mutants have greater stomatal indices and that XER is expressed in epidermal cells, indicating that XER functions in the epidermis to repress stomatal development. To explore XER spatiotemporal and stress-dependent regulation, we conducted a yeast one-hybrid screen and found that CBF4/DREB1D associates with the XER 5' untranslated region (5'-UTR). We generated three cbf4 null mutants with CRISPR/Cas9 and showed that CBF4 negatively regulates ABA responses, promotes stomatal development and reduces drought tolerance, in contrast to the roles shown for XER. CBF4 is induced by ABA and osmotic stress, and localizes to the nucleus where it downregulates XER expression via the DRE element in its 5'-UTR. Lastly, genetic interaction studies confirmed that xer is epistatic to cbf4 in stomatal development and in ABA, osmotic and drought stress responses. We propose that the repression of XER by CBF4 functions to attenuate ABA signaling and stress responses to maintain a balance between plant growth and survival under adverse environmental conditions.

Phosphorylation of DUF1639 protein by osmotic stress/ABA-activated protein kinase 10 regulates abscisic acid-induced antioxidant defense in rice

In rice (Oryza Sativa), Osmotic Stress/ABA-activated Protein Kinase 10 (SAPK10) has been shown to be induced by hyperosmotic stress and abscisic acid (ABA). However, the molecular function of SAPK10 and its downstream targets in ABA-induced antioxidant defense is poorly understood. Here, we identified an unknown function DUF1639 family protein, OsDUF1639.1, which interacts with SAPK10 in vitro and in vivo. OsDUF1639.1 positively regulates ABA responses in seed germination and tolerance to drought stress. We found that SAPK10 directly phosphorylates OsDUF1639.1 at Thr-80 in vitro. The transient expression analysis in combination with mutant analysis in rice protoplasts showed that Thr-80 is essential for ABA-induced stimulation of antioxidant defense by SAPK10. These results suggest that SAPK10 functions upstream of OsDUF1639.1 to regulate the activities of antioxidant enzymes, and Thr-80 phosphorylation of OsDUF1639.1 has a crucial role in ABA-induced antioxidant defense.

Arabidopsis group C Raf-like protein kinases negatively regulate abscisic acid signaling and are direct substrates of SnRK2

The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur.

Arabidopsis cold-regulated plasma membrane protein Cor413pm1 is a regulator of ABA response

Cold-regulated (COR) genes are considered downstream functional genes in the cold-response pathway. However, we identified a plasma membrane-type, AtCor413pm1, as a regulatory gene for the abscisic acid (ABA) response, and found that ABA induced it predominantly in Arabidopsis roots, vasculature, stipules, and guard cells. Differentially expressed genes combined with qPCR analysis revealed the expressions of three ABA-responsive genes (AtDTX50, AtABR1, and AtCIPK20) were significantly altered in the ABA-treated atcor413pm1 mutant, compared to the wild-type. Furthermore, the ABA-induced transient Ca2+ oscillation in the plasma membrane of atcor413pm1 roots was different from that observed in the wild-type. Our results revealed that AtCor413pm1 might play a role in the cross-talk between the ABA and stress response pathways.

MdBZR1 regulates ABA response by modulating the expression of MdABI5 in apple.

MdBZR1 directly binds to the promoter of MdABI5 and suppresses its expression to mediate ABA response. The plant hormones brassinosteroids (BRs) and abscisic acid (ABA) antagonistically regulate various aspects of plant growth and development. However, the association between BR and ABA signaling is less clear. Here, we identified MdBZR1 in apple (Malus domestica) and demonstrated that it was activated by BRs and could respond to ABA treatment. Overexpression of MdBZR1 in apple calli and Arabidopsis reduced ABA-hypersensitive phenotypes, suggesting that MdBZR1 negatively regulates ABA signaling. Subsequently, we found that MdBZR1 directly bound to the promoter region of MdABI5 and suppressed its expression. MdABI5 was significantly induced by ABA treatment. And overexpression of MdABI5 in apple calli increased sensitivity to ABA. Ectopic expression of MdABI5 in Arabidopsis inhibited seed germination and seedling growth. In addition, overexpression of MdBZR1 partially attenuated MdABI5-mediated ABA sensitivity. Taken together, our data indicate that MdBZR1 directly binds to the promoter of MdABI5 and suppresses its expression to antagonistically mediate ABA response. Our work contributes to the functional studies of BZR1 and further broadens the insight into the between BR and ABA signaling.

Rice calcium/calmodulin-dependent protein kinase directly phosphorylates a mitogen-activated protein kinase kinase to regulate abscisic acid responses.

Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment induces phosphorylation in the activation loop of OsMKK1, and the two phosphorylations, in the N-terminus and in the activation loop, are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in activating the MAPK cascade and ABA signaling.

Regulation of the stability and ABA import activity of NRT1.2/NPF4.6 by CEPR2-mediated phosphorylation in Arabidopsis

Abscisic acid (ABA) transport plays an important role in systemic plant responses to environmental factors. However, it remains largely unclear about the precise regulation of ABA transporters in plants. In this study, we show that the C-terminally encoded peptide receptor 2 (CEPR2) directly interacts with the ABA transporter NRT1.2/NPF4.6. Genetic and phenotypic analyses revealed that NRT1.2/NPF4.6 positively regulates ABA response and that NRT1.2/NPF4.6 is epistatically and negatively regulated by CEPR2. Further biochemical assays demonstrated that CEPR2 phosphorylates NRT1.2/NPF4.6 at serine 292 to promote its degradation under normal conditions. However, ABA treatment and non-phosphorylation at serine 292 prevented the degradation of NRT1.2/NPF4.6, indicating that ABA inhibits the phosphorylation of this residue. Transport assays in yeast and Xenopus oocytes revealed that non-phosphorylated NRT1.2/NPF4.6 had high levels of ABA import activity, whereas phosphorylated NRT1.2/NPF4.6 did not import ABA. Analyses of complemented nrt1.2 mutants that mimicked non-phosphorylated and phosphorylated NRT1.2/NPF4.6 confirmed that non-phosphorylated NRT1.2S292A had high stability and ABA import activity in planta. Additional experiments showed that NRT1.2/NPF4.6 was degraded via the 26S proteasome and vacuolar degradation pathways. Furthermore, we found that three E2 ubiquitin-conjugating enzymes, UBC32, UBC33, and UBC34, interact with NRT1.2/NPF4.6 in the endoplasmic reticulum and mediate its ubiquitination. NRT1.2/NPF4.6 is epistatically and negatively regulated by UBC32, UBC33, and UBC34 in planta. Taken together, these results suggest that the stability and ABA import activity of NRT1.2/NPF4.6 are precisely regulated by its phosphorylation and degradation in response to environmental stress.

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.

Abscisic Acid and Flowering Regulation: Many Targets, Different Places.

Plants can react to drought stress by anticipating flowering, an adaptive strategy for plant survival in dry climates known as drought escape (DE). In Arabidopsis, the study of DE brought to surface the involvement of abscisic acid (ABA) in controlling the floral transition. A central question concerns how and in what spatial context can ABA signals affect the floral network. In the leaf, ABA signaling affects flowering genes responsible for the production of the main florigen FLOWERING LOCUS T (FT). At the shoot apex, FD and FD-like transcription factors interact with FT and FT-like proteins to regulate ABA responses. This knowledge will help separate general and specific roles of ABA signaling with potential benefits to both biology and agriculture.

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 Transcription Factor INDUCER OF CBF EXPRESSION1 Interacts with ABSCISIC ACID INSENSITIVE5 and DELLA Proteins to Fine-Tune Abscisic Acid Signaling during Seed Germination in Arabidopsis.

ABSCISIC ACID INSENSITIVE5 (ABI5) is a crucial regulator of abscisic acid (ABA) signaling pathways involved in repressing seed germination and postgerminative growth in Arabidopsis (Arabidopsis thaliana). ABI5 is precisely modulated at the posttranslational level; however, the transcriptional regulatory mechanisms underlying ABI5 and its interacting transcription factors remain largely unknown. Here, we found that INDUCER OF CBF EXPRESSION1 (ICE1) physically associates with ABI5. ICE1 negatively regulates ABA responses during seed germination and directly suppresses ABA-responsive LATE EMBRYOGENESIS ABUNDANT6 (EM6) and EM1 expression. Genetic analysis demonstrated that the ABA-hypersensitive phenotype of the ice1 mutant requires ABI5. ICE1 interferes with the transcriptional activity of ABI5 to mediate downstream regulons. Importantly, ICE1 also interacts with DELLA proteins, which stimulate ABI5 during ABA signaling. Disruption of ICE1 partially restored the ABA-hyposensitive phenotype of the della mutant, gai-t6 rga-t2 rgl1-1 rgl2-1, indicating that ICE1 functions antagonistically with DELLA in ABA signaling. Consistently, DELLA proteins repress ICE1's transcriptional function and the antagonistic effect of ICE1 on ABI5. Collectively, our study demonstrates that ICE1 antagonizes ABI5 and DELLA activity to maintain the appropriate level of ABA signaling during seed germination, providing a mechanistic understanding of how ABA signaling is fine-tuned by a transcriptional complex involving ABI5 and its interacting partners.

Interaction network of ABA receptors in grey poplar.

The plant hormone abscisic acid (ABA) is a key player in responses to abiotic stress. ABA regulates a plant's water status and mediates drought tolerance by controlling stomatal gas exchange, water conductance and differential gene expression. ABA is recognized and bound by the Regulatory Component of ABA Receptors (RCARs)/PYR1/PYL (Pyrabactin Resistance 1/PYR1-like). Ligand binding stabilizes the interaction of RCARs with type 2C protein phosphatases (PP2C), which are ABA co-receptors. While the core pathway of ABA signalling has been elucidated, the large number of different ABA receptors and co-receptors within a plant species generates a complexity of heteromeric receptor complexes that has not functionally been resolved in any plant species to date. In this study, we characterized ABA receptors and co-receptors of grey poplar (Populus x canescens [Ait.] Sm.) and their capacity to regulate ABA responses. We observed a high number of regulatory combinations of holo-receptor complexes, but also some preferential and selective RCAR-PP2C interactions. Poplar and Arabidopsis ABA receptor components revealed a strong structural and functional conservation. Heterologous receptor complexes of poplar and Arabidopsis components showed functionality invitro and regulated ABA-responsive gene expression in cells of both species. ABA-responsive promoters of Arabidopsis were also active in poplar, which was explored to generate poplar reporter lines expressing green fluorescent protein in response to ABA. The study presents a detailed analysis of receptor complexes of a tree species and shows high conservation of ABA receptor components between an annual and a perennial plant.