| Water stress such as drought, salinity stress is the most important factor affecting plant growth and development, limiting crop yields. One of the most important reactions in plant responses to water stress is rapid accumulation of the plant hormone abscisic acid (ABA). ABA as a stress signal, plays a very important role in regulating plant water balance and inducing stress tolerance. Mitogen-activated protein kinase (MAPK) is a group of serine/threonine-specific protein kinases that are evolutionarily conserved throughout eukaryotes. MAPKs/MPKs act as important signaling components that regulate various cellular activities and respond to various extracellular stimuli, induced by stress, cytokines, plant hormones, growth factors, etc.The Studies about MAPKs/MPKs family on abscisic acid (ABA) and antioxidant defense were not investigated on rice(Oryza sativa.L).To further understand rice OsMPKs family, we used Nipponbare seedlings RNA as a template to investigate the OsMPKs(OsMPK1-17) genes expression by the quantitative and semi-quantitative RT-PCR in rice leaves which were treated with100μM ABA. The study found that OsMPKl,OsMPK5, OsMPK4, OsMPK7and OsMPK14were able to respond to up-regulation in ABA signaling.Rice OsDMI3gene (the role of rice CCaMK,Ca2+/CaM-dependent protein kinase) involved in ABA signaling pathway to induced OsMPKl gene up-regulation and as an upstream factor to regulate OsMPK1.In this study, OsDMI3mutants have been established to study the relationship between OsMPK5, OsMPK4, OsMPK7, OsMPK14and OsDMI3by the quantitative RT-PCR in rice leaves. The results showed that OsMPK5, OsMPK4gene expression was significantly decreased in mutants.There is a slight increased after adding exogenous ABA, but still can not reach the normal level. And we have established a rice seedling protoplast transient expression system to investigate the relationship between them through transforming (35S-OsDMI3-YFP) vector which was constructed by inserting OsDMI3’s CDS into the sites of pXZP008.into rice protoplasts by PEG method. We found that, compared with wild type, the OsMPK5and OsMPK4genes expression were significantly increased with over-expression of OsDMI3. After adding exogenous ABA, the expression was more increased. According to the above results, we further verified that in the ABA-induced signal transduction, OsDMI3regulated OsMPKs.We researched the sensitivity of the mutants of OsDMI3and OsMPKl under the ABA treatment. The wild-type and mutant seeds were germinated for5days and then selected consistent seedlings about5seedlings of each strain, duplicating sets of three parallel experiments which is divided into a control group and the5,10μMABA treated group to process about10days. Finally, take pictures and determine the length of the main root. The results showed that compared with control group, the growth of either the leaf or root, the treated group was significantly suppressed by the ABA, and the higher concentration, the more obvious suppressed. Under the same conditions,comparing wild-type with mutant, whatever in root and leaves,wild-type is more obvious than mutants. Thus proving that the sensitivity of OsDMI3and OsMPK1mutant is lower than the wild-type to ABA.Finally, we selected OsMPKl and OsMPK5from ABA-induced up-regulation five OsMPKs gene to investigate the subcellular localization, we insert CDS of OsMPKl and OsMPK5into the sites of pXZP008. On one hand we transformed the construct pXZ-OsMPK1,pXZ-OsMPK5into rice protoplasts by PEG, on the other hand we incorporated pXZ-OsMPK1,pXZ-OsMPK5into onion (Allium cepa L.)epidermis by particle bombardment. Images of YFP-OsMPKs fusion were then captured with the confocal laser scanning microscope.We initially identified that both OsMPK1, OsMPK5were localized in the cytoplasm and nucleus. |
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