The Relationship Between Brassinosteroid (BR) And Ca2+/CaM Signal Transduction Network

Brassinosteroids (BRs) were steroidal hormones that play an important role in the growth and development of plants, which could regulate the agronomic characters of plant (including plant height, leaf angle, fertility, etc.) to improve plants’anti-stress ability (cold, salt, drought, high temperature, hypoxia, etc) and had important application value in agricultural production. It is not clear that how BR (as an extracellular signal) is transfered into intracellular during regulation of plant growth and development. Ca2+was recognized as the second messenger in plants, and also was the major component of the cell. The change of the level of Ca2+in the cytoplasmand and the change of the level of Ca2+in cytosolic were the initial situation of the cells which responded to various stimuli signals, and then inducing a series events of signal transduction downstream. To study the influence of the BRs act on the Ca2+messenger system, we used Arabidopsis and maize as materials to research the effect of BR treatment on the location of Ca2+in cell by potassium pyruantimonatc technique, and to analyze genes expression level which coded Ca2+-ATPase genes located in the membrane, vacuole, endoplasmic reticulum and coded Ca2+channel genes located in membrane, vacuole, lysosome, which controlled intracellular Ca2+level by Real-Time PCR technique; in order to further elucidate the effect of BR on Ca2+/CaM signal transduction, we analyzed the expression levels of the genes coded CaM which are protein combined with Ca2+. The results are as follows:1. The influence of the BRs act on the Ca2+messenger systemCa2+mainly distributed in cell walls, intercellular space, vacuole, and there are only a few Ca2+distributing in the chloroplast and cytoplasm in the control cells, after1000nM BR treatment for3hours, Ca2+gathers and distributs nearby vacuole membrane and cell membrane, it seemd to enter cytoplasm by the Ca2+channel in vacuole membrane and cell membrane, or the closed Ca2+channel blocked Ca2+entering, meanwhile, the distribution of Ca2+in the cytoplasm and chloroplast increased; after6hours’BR treating, the distribution of Ca2+in the cytoplasm and chloroplast continues to increase, the distribution of Ca2+in the cell walls reduced; after9hours’BR treating, the distribution of Ca2+in the cytoplasm and chloroplast decreased, and the distribution in the intercellular space and vacuole increased, while the distribution of Ca2+in the cell walls obviously reduced. It was suggested that BR had the function of transferring and releasing Ca2+of cell walls. The effect of BR on Maize leaf cells Ca2+distribution was similar to Arabidopsis.2. The mechanism research of BR’s effect on Ca2+distribution in plant cells(1) The effect of BR on the expression levels of the genes coded Ca2+channelCNGC2and CNGC12were genes which encoded Ca2+channel in the cell membrane, after1000nM BR treatement for3hours, CNGC2and CNGC12expression level all obviously decreased, which indicated that BR could block extracellular Ca2+into cytoplasm; after1000nM BR treatement for6hours, the CNGC2and CNGC12expression levels recover; after1000nM BR treatement for9hours, the CNGC2and CNGC12expression levels obviously increased. The TPCI and TPC2were genes respectively coded Ca2+channel in the vacuole and lysosome. The expression levels of TPC1and TPC2significantly decreased after1000nM BR treatment for3hours, but the TPC1expression level was obviously higher than the control without BR treatement; the expression level of TPC2also significantly increased after1000nM BR treatement for9hours.The results showed that BR could block the fast rising of the cytoplasm Ca2+concentrations, the expression levels of the genes coding vacuole Ca2+channel recovered earlier than the Ca2+channel genes in the cell membrane and lysosome, which indicated that lots of Ca2+in the vacuole entered cytoplasm earlier than extracellular calcium store and other intracellular organelles including lysosome, etc.(2) The effect of BR on the expression levels of genes coding Ca2+-ATPaseACA8and ACA10were genes which encoded Ca2+-ATPase located in the cell membrane, after1000nM BR treatement for3and6hours, the expression levels of ACA8and ACA10had no significant change; after BR treatement for9hours, the expression levels of ACA8and ACA10significantly increased; ACA4and ACA11were genes coding Ca+-ATPase located in the vacuole membrane, the change of ACA4and ACA11expression levels were similar to the change of ACA8and ACA10. ACA2was one gene coding Ca+-ATPase located in the endoplasmic reticulum, the highest peak of the expression level of ACA2also appears after1000nM BR treatement for9hours.The results indicated that the expression level of Ca2+-ATPase genes increased after BR treatement for9hours, and high concentrations cytoplasm Ca2+were infused into extracellular and intracellular calcium store including intercellular space, vacuole, endoplasmic reticulum, etc. that lead to the regulation of cytoplasm calcium homeostasis.3. The effect of BR on the expression level of the genes coded calcium-binding proteins CaMIt was found that the expression level of CAM1, CAM2, CAM4, CAM6, CAM7genes all decreased after1000nM BR treatement in Arabidopsis for3,6,9hours. It showed that BR could reduce the expression level of the genes coded CaM, and regulate the process of Ca2+signal transduction.4. The negative regulation of CAM7on BR regulation process of plant growth and developmentThe Arabidopsis plantsover expressing CAM7were short and delayed in development. The expression level of the genes regulated BR synthetic and signal transduction genes decreased in the plants over expressing CAM7. It showed1that CAM7had negative function in the regulation of BR regulation in the process of plant growth and development.These results indicated that BR treatment could inhibit Ca2+channel genes expression, restrain the fast rising of the cytoplasmic Ca2+, with prolonged BR treatment time, Ca2+distributed in vacuole, cell walls, intercellular space, endoplasmic reticulum, lysosome had successively entered cytoplasm by Ca2+channel; meanwhile, after BR treatment for9hours the expression level of the genes coding Ca2+-ATPase significantly increase to promote cytoplasmic Ca2+efflux, which made cells at stable calcium ions solutions. Ca2+binding protein CAM7had negative function in the regulation of BR on the process of plant growth and development.