Terrestrial plants are exposed to complex stresses of high salt-induced abscisic acid (ABA) and submergence-induced hypoxia when seawater floods fields. Many studies have investigated plant responses to individual stress conditions, but not so much for coupled or sequentially imposed stresses. We examined molecular regulatory mechanisms of gene expression underlying the cellular responses involved in crosstalk between salt and hypoxia stresses. Salt/ABA- and AtMYC2-dependent induction of a synthetic ABA-responsive element and the native RD22 promoters were utilized in our cell-based functional assays. Such promoter-based reporter induction was largely inhibited by hypoxia and hypoxia-inducible AKIN10 activity. Biochemical analyses showed that AKIN10 negatively modulates AtMYC2 protein accumulation via proteasome activity upon AKIN10 kinase activity-dependent protein modification. Further genetic analysis using transgenic plants expressing AKIN10 provided evidence that AKIN10 activity undermined AtMYC2-dependent salt tolerance. Our findings unravel a novel molecular interaction between the key signalling constituents leading crosstalk between salt and hypoxia stresses in Arabidopsis thaliana under the detrimental condition of submergence in saltwater.

Mitogen-activated protein kinase (MAPK) is activated by various biotic and abiotic stresses. Salt stress induces two well-characterized MAPK activating signaling molecules, phosphatidic acid (PA) via phospholipase D and phospholipase C, and reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. In our previous study, the activity of soybean MAPK, GMK1 was strongly induced within 5 min of 300 mM NaCl treatment and this early activity was regulated by PA. In this study, we focused on the regulation of GMK1 at the later stage of the salt stress, because its activity was strongly persistent for up to 30 min. H(2)O(2) activated GMK1 even in the presence of PA generation inhibitors, but GMK1 activity was greatly decreased in the presence of diphenyleneiodonium, an inhibitor of NADPH-oxidase after 5 min of the treatment. On the contrary, the n-butanol and neomycin reduced GMK1 activity within 5 min of the treatment. Thus, GMK1 activity may be sustained by H(2)O(2) 10 min after the treatment. Further, GMK1 was translocated into the nucleus 60 min after NaCl treatment. In the relationship between GMK1 and ROS generation, ROS generation was reduced by SB202190, a MAPK inhibitor, but was increased in protoplast overexpressing TESD-GMKK1. However, these effects were occurred at prolonged time of NaCl treatment. These data suggest that GMK1 indirectly regulates ROS generation. Taken together, we propose that soybean GMK1 is dually regulated by PA and H(2)O(2) at a time dependant manner and translocated to the nucleus by the salt stress signal.

WRKY proteins play essential roles as negative or positive regulators of pathogen defense. This study explored the roles of different OsWRKY proteins in basal defense and Xa1-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) infection in rice. Assays of disease in OsWRKY10KD and OsWRKY88KD lines following infection with an incompatible Xoo race, which induced Xa1-mediated resistance in wild-type plants, showed that OsWRKY10 and OsWRKY88 were positive regulators of Xa1-mediated resistance. OsWRKY10 also acted as a positive regulator in basal defense by directly or indirectly activating transcription of defense-related genes. OsWRKY10 activated the OsPR1a promoter by binding to specific WRKY binding sites. Two transcriptional regulatory cascades of OsWRKY10 were identified in basal defense and Xa1-mediated resistance. In the first transcriptional regulatory cascade, OsWRKY47 acted downstream of OsWRKY10 whereas OsWRKY51 acted upstream. OsWRKY10 activated OsPR1a in two distinct ways: by binding to its promoter and, at the same time, by indirect activation through OsWRKY47. In the second transcriptional regulatory cascade, OsWRKY47 acted downstream of OsWRKY10, and OsWRKY88 acted upstream. These OsWRKY10 transcriptional regulatory cascades played important roles in basal defense and Xa1-mediated resistance to enable the mounting of a rapid immune response against pathogens.

The R2R3-MYB transcription factor MYB46 functions as a master switch for secondary cell wall biosynthesis, ensuring the exquisite expression of the secondary wall biosynthetic genes in the tissues where secondary walls are critical for growth and development. At the same time, suppression of its function is needed when/where formation of secondary walls is not desirable. Little is known about how this opposing control of secondary cell wall formation is achieved. We used both transient and transgenic expression of MYB46 and mitogen-activated protein kinase 6 (MPK6) to investigate the molecular mechanism of the post-translational regulation of MYB46. We show that MYB46 is phosphorylated by MPK6, leading to site specific phosphorylation-dependent degradation of MYB46 by the ubiquitin-mediated proteasome pathway. In addition, the MPK6-mediated MYB46 phosphorylation was found to regulate in planta secondary wall forming function of MYB46. Furthermore, we provide experimental evidences that MYB83, a paralog of MYB46, is not regulated by MPK6. The coupling of MPK signaling to MYB46 function provides insights into the tissue- and/or condition-specific activity of MYB46 for secondary wall biosynthesis.

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Mitogen-Activated Protein Kinase (MAPK) cascade is one of the main signaling components mediating abiotic and biotic stress and hormone information in plants. Plant MAPK study has been impeded with a genetic approach using a long-term phenotypic analysis in spite of the transient nature of the protein kinase signaling. Arabidopsis leaf mesophyll protoplasts provide a versatile resource for diverse cell-based assays to acquire immediate molecular and biochemical responses with transient expression of MAPK cascade components of interests. Thus, it is an attractive tool for a high-throughput functional analysis of Arabidopsis MAPK cascade signaling. However, transient expression in Arabidopsis mesophyll protoplast (TEAMP) system requires mastered skills for protoplast preparation and handling to achieve steady and stable data. Here, we have described two analytical methods for MAPK cascade signaling using TEAMP system.

Wounding is a constant threat to plant survival throughout their lifespan; therefore, understanding the biological responses to wounds at the cellular level is important. The protoplast system is versatile for molecular biology, however, no wounding studies on this system have been reported. We established a new approach for wounding research using mechanically damaged Arabidopsis mesophyll protoplasts. Wounded protoplasts showed typical wounding responses, such as increased MPK6 kinase activity and upregulated JAZ1 expression. We also assessed expression profiles and protein stability of the basic helix-loop-helix transcription factor MYC2 in wounded protoplasts. Promoter activity, gene expression, and protein stability of MYC2 were compromised, but recovered in the early stage of wounding. In the late stage, the promoter activity and expression of MYC2 were increased, but the protein stability was not changed. According to the results of the present study, this new cell-based approach will be of use in various molecular studies on plant wounding.