Expression Pattern And Functions Of Small Heat Shock Proteins(SHSPs) In Nicotiana Benthamiana

The small heat shock proteins(SHSPs)are a kind of important stress-resistance proteins,which are widely present in a variety of plants with functional diversity.N.benthamiana is one of the important model plants for studying the interaction between plants and stresses,however,the knowledge on its SHSPs remains a little.Our previous studies have shown that one of its SHSPs may be involved in viral infection.To further investigate the function of SHSPs,43 members of SHSP family were firstly identified by bioinformatic methods from the genome database of N.benthamiana.Their basic features,classification,and evolutionary relationship were characterized by using multiple sequence alignment and other computer programs.Their expression patterns were analyzed using qRT-PCR in different tissues of N.benthamiana.The results showed that the expression levels of most SHSPs were higher in the leaves than those in the flowers.Quantitative results in the responses of N.benthamiana to various stresses showed that most SHSP genes were induced by high temperature stress;only CI-CV subtypes,which have been predicted to be located in the cytoplasm-nucleus,were induced by drought stress.Both ABA and SA treatments significantly altered the expression pattern of SHSPs gene family in N.benthamiana,in which ABA mainly affected the expression of cytoplasm-nuclear subtype genes but SA also altered the expression of other subtypes.The effects of four plant viruses[including Rice stripe virus(RSV),tobacco rattle virus(TRV),Tomato mosaic virus(ToMV),Turnip mosaic virus(TuMV)]infection on the expression of SHSPs genes were also analyzed in N.benthamiana.The results showed that 11 SHSPs genes(including HSP-01,-02,-06,-07,-25,-18,-20,-22,-34,-31 and-43)were induced by all tested viruses.The promoter activity assays further demonstrated that plant virus infection enhanced HSP-05,HSP-07 and HSP34 promoter activity.It was found that all 11 SHSPs genes affected by viral infection were also regulated by SA,indicating that these SHSPs may play roles in the viral infection by involving the SA pathway.In order to further analyze the function of SHSPs,two SHSPs(NbHSP-07 and NbHSP-34)were expressed in Escherichia coli and purified for the assays of activity in vitro.The results showed that both proteins possessed the excellent activity of molecular chaperone.In order to analyze the function of SHSPs,the HSP-07 protein was validated by using laser confocal microscope and immunocolloid gold labeling techniques to be localized into the cytoplasm of N.benthamiana leaf cells,indicating the SHSPs protein should function as molecular chaperone in the cytoplasm.Furthermore,transgenic seeds overexpressing HSP-07 displayed higher germination rate and healthier growth/development than those of wild-type seeds in assays treated with heat stress,suggesting that the SHSPs could protect the seeds from heat stress and contribute to seed germination and seedling growth/development under the stress.This study could be helpful for understanding the roles of SHSPs in stress resistence and normal growth/development.