Da-Peng Zhang

Roles of the different components of magnesium chelatase in abscisic acid signal transduction

The H subunit of Mg-chelatase (CHLH) was shown to regulate abscisic acid (ABA) signaling and the I subunit (CHLI) was also reported to modulate ABA signaling in guard cells. However, it remains essentially unknown whether and how the Mg-chelatase-catalyzed Mg-protoporphyrin IX-production differs from ABA signaling. Using a newly-developed surface plasmon resonance system, we showed that ABA binds to CHLH, but not to the other Mg-chelatase components/subunits CHLI, CHLD (D subunit) and GUN4. A new rtl1 mutant allele of the CHLH gene in Arabidopsis thaliana showed ABA-insensitive phenotypes in both stomatal movement and seed germination. Upregulation of CHLI1 resulted in ABA hypersensitivity in seed germination, while downregulation of CHLI conferred ABA insensitivity in stomatal response in Arabidopsis. We showed that CHLH and CHLI, but not CHLD, regulate stomatal sensitivity to ABA in tobacco (Nicotiana benthamiana). The overexpression lines of the CHLD gene showed wild-type ABA sensitivity in Arabidopsis. Both the GUN4-RNA interference and overexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses. These findings provide clear evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is distinct from ABA signaling, giving information to understand the mechanism by which the two cellular processes differs at the molecular level.

Crucial roles of the pentatricopeptide repeat protein SOAR1 in Arabidopsis response to drought, salt and cold stresses

Whereas several mitochondrial/chloroplast pentatricopeptide repeat (PPR) proteins have been reported to regulate plant responses to abiotic stresses, no nucleus-localized PPR protein has been found to play role in these processes. In the present experiment, we provide evidence that a cytosol-nucleus dual-localized PPR protein SOAR1, functioning to negatively regulate abscisic acid (ABA) signaling in seed germination and postgermination growth, is a crucial, positive regulator of plant response to abiotic stresses. Downregulation of SOAR1 expression reduces, but upregulation of SOAR1 expression enhances, ABA sensitivity in ABA-induced promotion of stomatal closure and inhibition of stomatal opening, and plant tolerance to multiple, major abiotic stresses including drought, high salinity and low temperature. Interestingly and importantly, the SOAR1-overexpression lines display strong abilities to tolerate drought, salt and cold stresses, with surprisingly high resistance to salt stress in germination and postgermination growth of seeds that are able to potentially germinate in seawater, while no negative effect on plant growth and development was observed. So, the SOAR1 gene is likely useful for improvement of crops by transgenic manipulation to enhance crop productivity in stressful conditions. Further experimental data suggest that SOAR1 likely regulates plant stress responses at least partly by integrating ABA-dependent and independent signaling pathways, which is different from the ABI2/ABI1 type 2C protein phosphatase-mediated ABA signaling. These findings help to understand highly complicated stress and ABA signalling network.

Auto- and Cross-repression of Three Arabidopsis WRKY Transcription Factors WRKY18, WRKY40, and WRKY60 Negatively Involved in ABA Signaling

Some members of the WRKY transcription factor family are known to be involved in ABA signaling. However, it remains unclear how the WRKY transcription factors cooperate to regulate ABA signaling. In the present study, we showed that three Arabidopsis (A. thaliana) WRKY proteins previously identified as ABA signaling regulators, WRKY18, WRKY40, and WRKY60, directly target the W-box regions in various domains of the promoters of all their own encoding genes WRKY18, WRKY40, and WRKY60, which was evidenced by chromatin immunoprecipitation and gel shift assays. Furthermore, we showed that the three WRKY proteins inhibit expression of all three WRKY genes, which was evidenced in both an in vivo assay of coexpression of the WRKY proteins with the three WRKY promoters and expression analysis of the three WRKY genes in various wrky mutants. Additionally and importantly, we provide new evidence, with three different testing systems, that WRKY18, WRKY40, and WRKY60 are negative, not positive, ABA signaling regulators, and that ABA treatment represses all three WRKY genes through a mechanism partly independent of the WRKY proteins, in which the response of the WRKY60 gene to ABA partly requires WRKY18 and WRKY40. These findings describe a mechanism of auto- and cross-repression of the WRKY transcription repressors that suggests a sophisticated mechanism to balance the negative functions of the WRKY transcription repressors in ABA signaling and helps to understand the WRKY-mediated complex events in ABA signaling pathways.

Arabidopsis co-chaperonin CPN20 antagonizes Mg-chelatase H subunit to derepress ABA-responsive WRKY40 transcription repressor

Our previous study demonstrated that a chloroplast co-chaperonin 20 (CPN20), one of the interaction partners of the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR), negatively regulates ABA signaling at the same node with ABAR but upstream of WRKY40 transcription repressor in Arabidopsis thaliana. In the present experiment, we showed that ABA directly inhibits the ABAR-CPN20 interaction, and also represses expression of CPN20, which depends on ABAR. CPN20 inhibits ABAR-WRKY40 interaction by competitively binding to ABAR. ABAR downregulates, but CPN20 upregulates, WRKY40 expression. The cpn20-1 mutation induces downregulation of WRKY40, and suppresses the upregulated level of WRKY40 due to the cch mutation in the ABAR gene. ABA-induced repressive effect of the WRKY40 gene is strengthened by downregulation of CPN20 but reduced by upregulation of CPN20. Together with our previously reported genetic data, we provide evidence that CPN20 functions through antagonizing the ABAR-WRKY40 coupled pathway, and ABA relieves this pathway of repression by inhibiting the ABAR-CPN20 interaction to activate ABAR-WRKY40 interaction.

Cochaperonin CPN20 negatively regulates abscisic acid signaling in Arabidopsis.

Previous study showed that the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR) positively regulates abscisic acid (ABA) signaling. Here, we investigated the functions of a CHLH/ABAR interaction protein, the chloroplast co-chaperonin 20 (CPN20) in ABA signaling in Arabidopsis thaliana. We showed that down-expression of the CPN20 gene increases, but overexpression of the CPN20 gene reduces, ABA sensitivity in the major ABA responses including ABA-induced seed germination inhibition, postgermination growth arrest, promotion of stomatal closure and inhibition of stomatal opening. Genetic evidence supports that CPN20 functions downstream or at the same node of CHLH/ABAR, but upstream of the WRKY40 transcription factor. The other CPN20 interaction partners CPN10 and CPN60 are not involved in ABA signaling. Our findings show that CPN20 functions negatively in the ABAR-WRKY40 coupled ABA signaling independently of its co-chaperonin role, and provide a new insight into the role of co-chaperones in the regulation of plant responses to environmental cues.

Overexpression of an Arabidopsis cysteine-rich receptor-like protein kinase, CRK5, enhances abscisic acid sensitivity and confers drought tolerance

Highlight The cysteine-rich receptor-like protein kinase CRK5 is a potentially positive regulator of ABA signaling in early seedling growth, stomatal movement and plant drought tolerance.

Arabidopsis pentatricopeptide repeat protein SOAR1 plays a critical role in abscisic acid signalling

Summary The authors identify a pentatricopeptide repeat (PPR) protein SOAR1 as a crucial player of ABA signalling, which localizes to both the cytosol and nucleus probably to regulate nuclear gene expression.

Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis

Nuclear factor Y (NF-Y) family proteins are involved in many developmental processes and responses to environmental cues in plants, but whether and how they regulate phytohormone abscisic acid (ABA) signaling need further studies. In the present study, we showed that over-expression of the NF-YC9 gene confers ABA hypersensitivity in both the early seedling growth and stomatal response, while down-regulation of NF-YC9 does not affect ABA response in these processes. We also showed that over-expression of the NF-YC9 gene confers salt and osmotic hypersensitivity in early seedling growth, which is likely to be directly associated with the ABA hypersensitivity. Further, we observed that NF-YC9 physically interacts with the ABA-responsive bZIP transcription factor ABA-INSENSITIVE5 (ABI5), and facilitates the function of ABI5 to bind and activate the promoter of a target gene EM6. Additionally, NF-YC9 up-regulates expression of the ABI5 gene in response to ABA. These findings show that NF-YC9 may be involved in ABA signaling as a positive regulator and likely functions redundantly together with other NF-YC members, and support the model that the NF-YC9 mediates ABA signaling via targeting to and aiding the ABA-responsive transcription factors such as ABI5.

A link between magnesium-chelatase H subunit and sucrose nonfermenting 1 (SNF1)-related protein kinase SnRK2.6/OST1 in Arabidopsis guard cell signalling in response to abscisic acid

Highlight A sucrose nonfermenting 1 (SNF1)-related protein kinase 2, SnRK2.6/ open stomata 1 (OST1), which plays critical role in abscisic acid (ABA) signalling in Arabidopsis guard cells, interacts directly with, and functions downstream of, the magnesium-chelatase H subunit in guard cell signalling in response to ABA.

Approaches to the identification of ABAR as an abscisic acid receptor.

Abscisic acid (ABA) is a vital phytohormone that regulates seed maturation and germination, seedling growth, and adaptation to environmental stresses. ABA functions through a complex network of signaling pathways, where the cell response is initiated by an ABA receptor which triggers downstream signaling cascades to induce the final physiological effects. Two classes of technologies may be used for the isolation of ABA receptors. One is the genetic screening for ABA receptor mutants, and another is the biochemical isolation of ABA-binding proteins that are putative ABA receptors. We implemented biochemical approaches, namely, the purification of ABA-binding proteins to identify a putative ABA receptor; this protein was further characterized by a combination of biochemical and reverse genetic approaches. The identified ABA receptor, called ABAR, mediates the responses of plants to ABA in seed germination, postgerminative growth, and stomatal movement. This protein is the H subunit (CHLH) of the magnesium protoporphyrin-IX chelatase (Mg-chelatase) that also plays a key role in both chlorophyll biosynthesis and plastid-to-nucleus signaling. Here, we describe the experimental procedures for the purification of ABA-binding proteins and the identification of the ABA-binding protein, ABAR/CHLH, as an ABA receptor.