Cytosolic calcium signaling is critical for regulating downstream responses in plants encountering unfavorable environmental conditions. In a genetic screen for Arabidopsis thaliana mutants defective in stress-induced cytosolic free Ca ([Ca ] ) elevations, we identified the R2R3-MYB transcription factor MYB30 as a regulator of [Ca ] in response to H O and heat stresses. Plants lacking MYB30 protein exhibited greater elevation of [Ca ] in response to oxidative and heat stimuli. Real-time reverse transcription-polymerase chain reaction (RT-PCR) results indicated that the expression of a number of ANNEXIN (ANN) genes, which encode Ca -regulated membrane-binding proteins modulating cytosolic calcium signatures, were upregulated in myb30 mutants. Further analysis showed that MYB30 bound to the promoters of ANN1 and ANN4 and repressed their expression. myb30 mutants were sensitive to methyl viologen (MV) and heat stresses. The H O - and heat-induced abnormal [Ca ] in myb30 was dependent on the function of ANN proteins. Moreover, the MV and heat sensitivity of myb30 was suppressed in mutants lacking ANN function or by application of LaCl , a calcium channel blocker. These results indicate that MYB30 regulates oxidative and heat stress responses through calcium signaling, which is at least partially mediated by ANN1 and ANN4.

Cytoplasmic Ca2+ ([Ca2+]cyt) elevation induced by various signals is responsible for appropriate downstream responses. Through a genetic screen of Arabidopsis thaliana mutants defective in stress-induced [Ca2+]cyt elevation, the glycosyltransferase QUASIMODO1 (QUA1) was identified as a regulator of [Ca2+]cyt in response to salt stress. Compared with the wild type, the qua1-4 mutant exhibited a dramatically greater increase in [Ca2+]cyt under NaCl treatment. Functional analysis showed that QUA1 is a novel chloroplast protein that regulates cytoplasmic Ca2+ signaling. QUA1 was detected in chloroplast thylakoids, and the qua1-4 mutant exhibited irregularly stacked grana. The observed greater increase in [Ca2+]cyt was inhibited upon recovery of chloroplast function in the qua1-4 mutant. Further analysis showed that CAS, a thylakoid-localized calcium sensor, also displayed irregularly stacked grana, and the chloroplasts of the qua1-4 cas-1 double mutant were similar to those of cas-1 plants. In QUA1-overexpressing plants, the protein level of CAS was decreased, and CAS was readily degraded under osmotic stress. When CAS was silenced in the qua1-4 mutant, the large [Ca2+]cyt increase was blocked, and the higher expression of PLC3 and PLC4 was suppressed. Under osmotic stress, the qua1-4 mutant showed an even greater elevation in [Ca2+]cyt and was hypersensitive to drought stress. However, this sensitivity was inhibited when the increase in [Ca2+]cyt was repressed in the qua1-4 mutant. Collectively, our data indicate that QUA1 may function in chloroplast-dependent calcium signaling under salt and drought stresses. Additionally, CAS may function downstream of QUA1 to mediate these processes.

The phytohormone abscisic acid (ABA) is critical for plants encountering abiotic stress. We reported previously that the Arabidopsis () transcription factor MYB30 participates in ABA responses via SUMO ligase SAP-MIZ Domain-Containing SIZ1-mediated sumoylation. Here, we show that the RING-type ubiquitin E3 ligase RHA2b, which positively regulates ABA signaling, interacted with and ubiquitinated MYB30 to modulate MYB30 stability through the 26S proteasome pathway. The degradation rate of MYB30 was repressed significantly in the mutant. Phenotypic analyses showed that acts genetically downstream of in ABA signaling. Substitutions of lysine-283 (K283) and K165 blocked ubiquitination, suggesting that these residues are sites of ubiquitination. K283 residue substitution significantly inhibited the degradation of MYB30 induced by ABA. The K165 site functioned additively with K283 in ABA-induced MYB30 degradation and ABA responses. At the same time, sumoylation protected MYB30 from degradation under cycloheximide and ABA treatment. Compared with MYB30, overexpression of MYB30-SUMO1 partially recovered the ABA sensitivity of But MYB30-SUMO1 exhibited similar localization with MYB30 in nuclei. Overall, our results suggest that RHA2b targets MYB30 for degradation to modulate ABA signaling. Considering that the K283 residue also is the major site for sumoylation, we propose that sumoylation and ubiquitination act antagonistically in the ABA response to regulate the stability of MYB30 by occupying the same residue.