CP24 Mediates Transcription Factor St904 To Regulate Chlorophyll Accumulation

Photoperiod is an important environmental factor that affects the growth and development of plants.Previous studies have identified and characterized some photoperiod response genes related to flower formation.Photosynthesis is also affected by light.Although many studies have identified PSI and PSII component proteins,such as CP24 and CSS,it remains to be determined which upstream regulatory genes regulates accumulation of these proteins and how they are in response to photoperiod to regulate photosynthesis.Previous study in our laboratory identified an light response gene St904(DMG400033904)in Solanum tuberosum L.In potato genome database,St904 is annotated to be a single MYB transcription factor family in terms of its amino acid sequence.Literature retrival finds there is no function report on St904 and its homologs.Beginning with St904 cDNA,we used design and synthesis of recombinant genes,gene in vivo transformation,phynotype assay of the recombinant genes,gene expressission assay and EMSA(Electrophoretic Mobility Shift Assay)to disclose phynotype of change of St904 expression and reason for phynotype formation.The main results are as follows:1.Synthesis of 35S-St904 and His-St904 gene cDNA,subcloning of 35S-St904 to obtain a recombinant vector,transform Agrobacterium GV3101,and select and obtain a positive recombinant Agrobacterium tumefaciens strain GV3101-pC-35S-St904+/-and GV3101-pC-35S-His-St904,transform the recombinant gene into Solanum tuberosum L and Nicotiana tabacum,and screen to obtain positive transformed plants;induce expression of His-St904 gene in prokaryotic cells,isolate and purify to obtain His-St904.2.Inhibition of St904 expression in transgenic Nicotiana tabacum and Solanum tuberosum led to a darrk green and thicker leaf,14%and 23%higher chlorophyll content than WT,respectively,as well as in advance flowering time.Overexpressing of St904 expression in vivo resulted in reversely phynotypy.The result indicated that St904 involved in the metabolism of chlorophyll accumulation and flowering time.3.We assayed expression of eight genes related to chlorophyll accumulation.The result showed that light expression of CP24(chlorophyll-binding protein 24)and CSS(Chlorophyll synthase)as well as St904 were promoted by light and inhibited by darkness.Inhibition in darkness no long existed when St904 were overexpressed.CP24 and CSS expression increased obviously in St904 overexpression transgenic lines.The result indicated that St904 expression was opened by light and closed by darkness.4.The results of EMSA experiments conducted with His-St904 showed that His-St904 protein can bind to CSS and CP24 promoter in various extent.Lagging bands were obviously visible when CP24 promoter was incubated with verious concentrations of His-St904 protein.The result indicated that His-St904 protein can bind to CP24 promoter.Light-responsive transcription factor St904 positively regulates CP24 expression,which in turn changes chlorophyll accumulation.Light induces St904 up-regulated expression.In turn CP24 accumulation increases and is equipped by more photoprotection pigment,which result in a decreased chlorophyll accumulation.Light attenuation allows less St904 expression and CP24 accumulation is down along less St904.More chlorophylls fills in CP24 instead of the other pigments.A higher chlorophyll contents is displayed.The result imply that St904,in response to light regulates CP24 accumulation and balance binding of chlorophyll and CP24.