Effect And Regulatory Mechanism Of Brassinosteroid On Stomatal Movement In Tomato

Stomata, formed by a pair of guard cells, plays an important role in gas exchange, water transpiration and stress response during the plant growth and development. The opening of stomata promotes plant growth by enhancing CO2 uptake and transpirational water loss, which are essential for photosynthesis and the uptake of nutrients from soil to plant. However, stomatal opening also causes undesirable and excess water loss from plants under drought stress and allows microorganisms to invade plants through stomatal pores. As a result, the stomatal movement is regulated by both extra-and intra cellular signals under a sophisticated and refined mechanism. Brassinosteroid （BR）, a growth-promoting steroidal plant hormone, has been reported to enhance the tolerance of plant against cold stress, salt stress, drought stress and diseases. Brassinazole resistant 1 （BZR1）, a transcription factor that plays a central role in BR signalling, regulates numerous BR-responsive genes. Therefore, it is significant to clarify the function and mediated proteins of BR in regulating stomatal movement.Here, we investigated the effects of exogenous EBR treatment and overexpressicn of BZR1-1D gene on stomatal movement in tomato（Solanum lycopersicum）. Isobaric tags for relative and absolute quantitation （iTRAQ） approach was used fcr protein identification and quantification to reveal the responsive protein during stomatal closure. In addition, we used ABA and JA mutants to study the regulation mechanism of BR in stomatal movement. The qRT-PCR method was also used to verify the transcriptional change of ABA and JA biosynthetic genes, demostrating the crosstalk between BR and ABA/JA in regulating stomatal movement. Following are the main results:1. BR can induce stomatal closure in tomato. The effect of EBR treatment on stomata] closure is in a dose- and time-dependent manner. The stomata from plants of transgenic line 35S::BZR1-1D::CFP#23 （BZR1-1D#23） showed a smaller aperture than those from the WT. The above results indicate that BZR1 plays a positive role in BR induced stomatal closure.2. iTRAQ-based quantitative proteomics technology was used to identify the BR responsive protein. A total of 214 differentially expressed （DE） proteins in response to EBR were identified in Ailsa Craig （AC） guard cells, while 98 DE proteins were identified in BZR1-1DU23 guard cells. Some identified proteins related to stomatal movement were also verified by qRT-PCR at transcriptional level. Gene Ontology, AgriGo and Kegg analysis of the DE proteins suggested that BR enhanced the expression of AA03 and LOX2/L0X1₅, enzymes involved in ABA and JA biosynthesis, respectively. In addition, the expression of PLCD, an activator of Ca2lin channel, Ca2+ binding protein CALR and Ca2+ channel TPC1 were all increased by BR. Ten enzymes related to the process of cytoskeleton and cell wall modification, were also increased by BR. Furthermore, many proteins involved in energy metabolism, photosynthesis, rene expression and protein metabolism were also identified.3. EBR application test in phytohormone mutants （sil, fail） and their WT, indicated that BR induced stomatal closure by enhancing AAO3-dependent biosynthesis of ABA and JAIl-mediated JA signalling. In the guard cells of transgenic BZR1-1D#23 plants, expression of genes related to biosynthesis of ABA and JA were also increased in comparison with those of the WT. Taken together, BR enhances the biosynthesis of ABA and JA via transcription factor BZR1, and fianlly regulates slomata closure.