| Heat shock stress is one of important abiotic factors, high temperature cause plants wilting, impact the development of embryo, and restrict the plant normal growth. The tolerance responses of plants to heat shock stress are conjectured to be controlled by complex gene networks, plant cytoskeleton is a key regulator of cellular responses to extracellular stimuli. Actin cytoskeleton modified to respond the stress rapidly and dramaticly, with assisted of many ABPs. As an actin binding protein, capping protein is composed of a and β subunits, it could stable actin cytoskeleton by binding to F-actin ends to inhibit the subunits add or loss from that end. Recently, the studies of capping protein were mainly in yeast, Drosophila and mammal, the only few reports of plant capping protein have focused on the biochemical functions in vitro, including bounding with actin, inhibiting the rate of F-actin elongation, preventing depolymerization, and nucleation. Until now, the expression patterns and physiological functions of capping protein in planta are poorly understood.In the present study, we have investigated on the physiological functions of a and β subunits of Arabidopsis capping protein in vivo, including their different expression patterns and resistance of heat shock stress. The results are as follows:Firstly, the full length CDS of AtCPA, AtCPBl, and AtCPB2were cloned from Arabidopsis total RNA, respectively, and the prediction that there have not only one β subunit of CP in plant had been inditify. Sequencing analysis indicated that the sequence of5'region of AtCPB1and AtCPB2appeared to be strongly conserved, but3'region showed the highest sequence variability. This result was similar with the studies in animals.Secondly, using realtime PCR and western blot analysis, we showed that, though AtCP a and β subunits were expressed in all tissues, the expression patterns of them were significant different, it seemed that they have a complementary relationship, that was the expression levels of AtCPA in stem and leaf were much higher than in root and flower, but AtCPB were abandent in root and flower than in stem and leaf.Thirdly, we wanted to analysis the different expression of AtCPA and AtCPB in same organ or same tissue by transformation of Arabidopsis with AtCPA and AtCPB promoter tagged with GUS separetly. The analysis of GUS activity showed these two gene could expressed in same organ with different location, it was indicated that they may have their own functions in same organ. In roots, AtCPA was mainly expressed in pericycle cells but none in root hairs, but AtCPB was not only expressed in pericycle cells, but also abundant in root hair cells and cortical cells. In flowers, the expression level AtCPA in anther is slightly higher than in filament, however, we failed to detect the GUS activities of AtCPB in anther but strongly in filament.Fourthly, we observed the subcellular location of AtCPA and AtCPB by expression of yellow fluorescent protein in plant stability, both of the two subunits could co-located with microfilament directly, it was revealed that as actin-bindling proteins, AtCPA and AtCPB exercise their function by interaction with microfilaments.Fifthly, accoding to the Microarray predict analysis (http://www.arabidopsis.org/), using realtime PCR and western blot analysis, we studied the expression levels of AtCPA and AtCPB after heat treatment. We found that both of them could response to heat stress, the expression levels of them were both increased after heat treatment, the highest expression of AtCPA were at3-4h after HS, then the expression declined at6h and12h, however, the transcript level of AtCPB was rapid increased at3h and kept the high level continuously. It was revealed that the changes of expression levels during heat shock stress, and the relationship between AtCP and heat shock stress.Sixthly, in thermotolerance test, seedlings of atcpβ-mutant showed enhanced tolerance than WT and atcpa-mutant, after the lethal temperature treatment, the bleaching rate of atcpβ-mutant was lower than WT and atcpa-mutant, and the survival rate was higher than WT and atcpa-mutant significantly. The result showed the physiological function of AtCPB, it could as a negative regulator during heat shock stress.Finally, we labeled the actin filaments in WT, atcpa-and atcpβ-mutant after45℃treatment and observed under confocol microscopy, the mainly oblique and longitudinal fimaments in atcpβ-mutant were still existed, they could maintain the network of actin cytoskeleton. However. the actin network depolymerization in WT and atcpa-mutant, there were many short cables and aberrantly thick bundles appeared within the cytoplasm. It was said that AtCPB could response to heat shock stress by changing the structure of actin cytoskeleton.In conclusion, AtCPA and AtCPB were both expressed in all tissues of Arabidopsis with different expression patterns, it seemd that they have a complamentary relationship, it was indicated that they could participate in the same cellular progress with different functions. AtCPB loss of function mutant showed enhanced thermotolerance, and the actin filaments were much more complete than WT and AtCPA loss of function mutant, we indicated AtCPB may be as a negative regulator during heat shock stress. It was revealed that the role of AtCP in the process of plant growth, and provided a theoretical basis for further analysis.
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