Physiological And Molecular Mechanisms Of Brassinosteroids And Light Signal Regulating Photosynthesis In Tomato

In recent years,the area of facility vegetable cultivation in China has increased rapidly,ranking first in the world.In the process of facility production,there are a series of problems,such as insufficient light and poor environmental regulation capacity,which have a serious impact on the output of vegetables in facilities.As the main source of plant biomass accumulation,photosynthesis is directly related to vegetable yield and plant growth.Exploring effective ways to improve photosynthesis is of great significance to increase vegetable yield and enhance the adaptability of vegetables to adverse environment.Brassinosteroids（BRs）,as a kind of sterol hormone,are crucial to plant growth and development.BRs regulate many processes of plant growth and development and photosynthesis is one of them.However,previous studies about BRs regulating photosynthesis mostly stayed at physiological level,and molecular evidence is lacking.In addition to hormones,light can also be signal to regulate plant growth and development.After light signal received by photoreceptors,there will be a series of downstream responses.But the relationship between light signal and photosynthesis is still unclear.In this paper,we used the model plant tomato（Solanum lycopersicum L）as the research object,and studied the physiological and molecular mechanisms of brassinosteroids and light signal regulating photosynthesis though the methods of genetics,physiology,and molecular biology.We explained the function and mechanism of BRs level and BR signal pathway core transcription factor BZR1 in tomato photosynthesis;We found that light signal regulating tomato photosynthesis;We explored the function of BRs in the process of light signal regulating photosynthesis.The main research achievements are as follows:1.BRs regulate photosynthesis in tomato.Compared with wild type（WT）plants,dwf,the tomato BR synthesis impaired mutant,showed impaired plant height and a significant inhibition on photosynthesis.The net photosynthetic rate（Pn）,stomatal conductance（Gs）and transpiration rate（Tr）as well as the actual quantum efficiency of PSII(Ф_PSII)and the photochemical quenching coefficient（q P）were decreased in dwf mutant.In contrast to dwf mutant,overexpression of DWF（DWF:OE）improved plant growth and photosynthesis.The Pn,Gs and Tr were increased in DWF:OE plants,as well asФ_PSIIand q P.These results showed that the increase of endogenous BR levels would enhance the photosynthesis of tomato.Then,we evaluated the state of Calvin cycle of different BR levels tomato plants.The results showed that defects in BR biosynthesis inhibited the maximum Rubisco carboxylation rate（Vc,max）and the maximum Ru BP regeneration rate（Jmax）,whereas overexpression of DWARF significantly increased Vc,max and Jmax compared with WT.Taken together,all these results showed that BRs could enhance photosynthesis in tomato.2.BZR1 is involved in BRs regulating photosynthesis in tomato.The level of BZR1 protein in three different BR levels tomato plants was measured.It was found that the increase of BR levels would promote the accumulation of dephosphorylated BZR1（d BZR1）protein.Compared with WT,the growth of bzr1 mutant was weaker.Besides,the Pn as well asФ_PSIIand q P were lower in bzr1 plants as compared with WT plants,indicating that the loss of function of BZR1 impaired photosynthetic capacity in tomato.24-Epibrassinolide（EBL）application can improve Pn,Ф_PSIIand q P in WT plants.However,the decline of Pn,Ф_PSIIand q P in bzr1 mutant can’t be recovered by EBL application.Moreover,we found that EBL application can activate the state of Calvin cycle and increase Vc,max and Jmax in WT plants but had no promotion effect on bzr1 mutant.Taken together,all these results show that BRs can enhance photosynthesis in tomato through BZR1.3.BZR1 regulates the photosynthesis of tomato through transcriptional regulation of the Calvin cycle genes FBA1,RCA1,FBP5 and PGK1.Detection of the expression of 23 Calvin cycle genes in three different BR level tomato plants showed that more than half of the genes were up-regulated in DWF:OE plants,and the expression of FBA1,PGK1,RCA1,RCA3,FBP1,FBP2 and FBP5 were down-regulated in dwf mutant.We also evaluated the expression of Calvin cycle genes in WT and bzr1 mutant with or without the application of EBL,and screened seven genes FBA1,FBA7,FBA8,FBA9,PGK1,RCA1 and FBP5,which were affected by both EBL application and BZR1 mutation.Compared with the genes whose expressions were regulated by different BR levels,it was found that the expression of FBA1,RCA1,FBP5 and PGK1 was simultaneously regulated by BR levels and BZR1mutation and FBA1,RCA1,FBP5 and PGK1 were chosen for further study.We analyzed the regions（～2 kb）located upstream of the transcriptional start sites of FBA1,RCA1,FBP5 and PGK1 and found 4～11 d BZR1 binding sites.Subsequently,the directly binding of BZR1 to the promoters of FBA1,RCA1,FBP5 and PGK1 was identified by Ch IP and yeast one-hybrid assay.Furthermore,dual-luciferase assays verified that BZR1 could directly bind to the promoters of these genes and activated their expressions.For further study,we suppressed the transcription of FBA1,RCA1,FBP5 and PGK1 by virus-induced gene silencing（VIGS）.The results showed that the suppression of transcription of FBA1,RCA1,FBP5 and PGK1 led to stunted growth compared with TRV.Besides,the Pn,Φ_PSII and q P were significantly decreased by gene silencing.Unlike TRV plants,EBL application had no effect on silencing plants.Meanwhile,EBL application could improve Vc,max and Jmax in TRV plants,while silencing of FBA1,RCA1,FBP5 and PGK1 compromised increase in both Vc,max and Jmax by EBL.Taken together,all these results show that BZR1 enhances the Calvin cycle and improves tomato photosynthesis through transcriptional activation the expressions of FBA1,RCA1,FBP5 and PGK1.4.BRs are involved in photoreceptors phy A,phy B and CRY1a regulating tomato photosynthesis.Compared with WT plants,the Pn,Ф_PSIIand q P of tomato phy A mutant were significantly improved,while the Pn,Ф_PSIIand q P of phy B1B2,phy AB1B2 and cry1a mutants were significantly decreased.The examination of Vc,max and Jmax of these photoreceptor mutants showed the same pattern.All these results showed that far-red light photoreceptor phy A negatively regulated photosynthesis,red light photoreceptor phy B and blue light photoreceptor CRY1a positively regulated photosynthesis in tomato plants.We compared the BRs content,the expression of BR biosynthesis genes DET2 and DWARF4 and BZR1 levels among these plant materials.We found that the content of brassinolide（BL）was significantly decreased in phy A,phy B1B2 and phy AB1B2 mutants as compared with WT.The content of castasterone（CS）in phy A and phy B1B2 mutants showed no difference compared with WT.Besides,the content of CS in phy AB1B2 and cry1a mutants was higher than in WT.This indicated that the BRs content of phy A,phy B1B2,phy AB1B2 and cry1a mutants was down-regulated.Further comparison of the expressions of BR synthesis genes DET2and DWARF4 in these plant materials showed that the expression of DET2 and DWARF4 was up-regulated in phy A mutant.In addition,the expression of DET2 in phy B1B2,phy AB1B2 and cry1a mutants was down-regulated compared with WT.The expression of DWARF4 in phy B1B2 mutant was significantly decreased compared with WT,but the expression of DWARF4 in phy AB1B2 and cry1a mutants showed no significant difference compared with WT.Examination the levels of BZR1 protein in several experimental materials showed that d BZR1 protein was accumulated in phy A mutants but reduced in phy B1B2,phy AB1B2 and cry1a mutants.To verify that photoreceptors can affect tomato photosynthesis through BRs,we compared the photosynthetic capability of WT and phy B1B2 mutants with or without HBL（28-Homobrassinolide）treatment.It turned out that HBL treatment could restore the photosynthetic capacity of phy B1B2 mutant to the level WT plants under water treatment.Taken together,all these results show that photoreceptors phy A,phy B and CRY1a can regulate tomato photosynthesis.Besides,BRs are involved in photoreceptor phy B regulating tomato photosynthesis.