Molecular Mechanism Of Brassinosteroids And Boron Deficiency Response Genes TCH4 And STOP1 Involved In Low Boron Response In Arabidopsis

Boron(B)is an essential micronutrient for plants.B deficiency leads to a wide range of morphological and physiological symptoms.One of the earliest responses to B deficiency in plants is the inhibition of primary root length.In our previous studies,the content of various phytohormones changed under B deficiency,but the molecular mechanism why B deficiency inhibits plant root growth is still unclear.Brassinosteroid(BR)are a class of plant hormones that play an important role in plant growth and development,biotic and abiotic stresses.TCH4 is a member of the xyloglucan glycosyltransferase/hydrolase(XTHs)family.It is very sensitive to the external environment,and various external stimuli can increase the expression level of TCH4.STOP1 is a zinc finger transcription factor that plays an important role in the regulation of aluminum toxicity.At present,there are few reports on whether BR is involved in the response to B deficiency,whether cell wall modifying enzymes are involved in the regulation of B homeostasis,and whether transcription factors participate in the response to B deficiency.Therefore,it can provide theoretical support for understanding why B deficiency inhibits the root growth and enrich the regulatory network of B deficiency response by studying the role that BR and other B deficiency-responsive genes play in the inhibition of primary root growth under B deficiency.This study explored that B deficiency-induced root growth inhibition is mediated by BR in Arabidopsis,the role of TCH4 in boron homeostasis under Bdeficiency conditions and the role of STOP1 in response to B deficiency,by using Arabidopsis as the research object.The main results obtained are as follows:(1)Boron deficiency-induced root growth inhibition is mediated by BR signalling regulation in ArabidopsisWe identified BR-related processes underlying B deficiency at the physiological,genetic,molecular,cell biological and transcriptomic levels and found strong evidence that B deficiency can affect BR biosynthesis and signalling,thereby altering root growth.RNA sequencing analysis revealed that 4,441 genes were significantly differentially expressed between the B-deficiency and B-sufficiency conditions,and the expression of 45.9% of the genes regulated by B deficiency was also regulated by BRs,indicating a high degree of co-regulation between them.Furthermore,we found that more than 60% of the co-regulated genes responded oppositely to B deficiency and BRs.The BR signal enhanced mutant bes1-D and transgenic line p BZR1-bzr1-D exhibited tolerance to low boron stress,while the BR receptor mutants bri1-119 and bri1-301 showed insensitivity to low boron stress.Under B-deficiency conditions,exogenous 24-epibrassinolide(e BL)rescued the inhibition of root growth,and application of the BR biosynthesis inhibitor BRZ exacerbated this inhibitory effect.Under B deficiency,the dephosphorylated BES1 decreased,while the phosphorylated BES1 increased.By analyzing the endogenous BR content and the expression of BR synthesis genes,we further found that B deficiency hindered the accumulation of brassinolide(BL),which may occur through a reduction in BR6ox1 and BR6ox2 m RNA levels.When the BL concentration is low under B deficiency,BL no longer binds to the BRI1 receptor at the cell surface,which weakened the BR signalling,and further inhibited the expression of BSU1,so that BIN2 kinase was activated.Transcription factors BES1 and BZR1 were phosphorylated by BIN2,which made them stay in the cytoplasm and could not enter the nucleus to start BR response,resulting in the inhibition of root growth.(2)TCH4 involves in the regulation of cell wall under B-deficiency conditionsThe results of pTCH4:GUS showed that TCH4 was expressed in cotyledons,hypocotyls,epidermal hairs,lateral root node at seedling stage,was expressed in the young tissues of rosette leaves,stem leaves,flowers and pods,and was up-regulated by B deficiency.The results of pTCH4:TCH4:GFP confirmed that TCH4 is located in cell wall.The results showed that the overexpression lines of TCH4 was more sensitive to B deficiency than wild type,while the knockout lines of TCH4 was more resistant to B deficiency than wild type during the seedling,flowering,and mature stages.TCH4 has little effect on B absorption,transport and B homeostasis.The results showed that TCH4 affected the ratio of different forms of pectin,and the structure of cell wall under B deficiency by detecting the content of cellulose,hemicellulose,pectin,KDO and the transmission electron microscope observation of the cell wall.The fluorescence immunocytochemistry of JIM5 and JIM7 and the determination of PME enzyme activity indicate that TCH4 reduced the degree of esterification of pectin under B-deficiency conditions.The up-regulated expression of TCH4 under B-deficiency conditions increases the modification of hemicellulose,which may change the spatial structure of hemicellulose,affect the ratio of pectin in different forms and reduce the degree of esterification of pectin,thereby making the cell wall more thicker and more deformed,affecting plant growth.(3)Mechanism of STOP1 increased the adaptability to low boron stress by upregulating the expression of NIP5;1In this study,we found that stop1 mutants showed more sensitive to low boron,and the overexpression lines of STOP1 showed more resistance to low boron through the phenotypic screening of mutants,indicating that STOP1 participated in the response to B deficiency.We found that STOP1 affects the absorption of B by influencing the expression of B transporter NIP5;1 under B deficiency by analysing the B concentration and the expression of B transporter between wild-type,mutants and overexpression lines of STOP1.Our results showed that the expression levels of NIP5;1 changed between mutants and overexpression lines of STOP1 under B deficiency by the hybrid materials of stop1 × p NIP5:NIP5:GFP and OE-STOP1 ×p NIP5:NIP5:GFP.The expression level of NIP5;1 was lower in the stop1 mutant and higher in the overexpression lines of STOP1 under B deficiency.Our data proved that STOP1 is located upstream of NIP5;1 in response to B deficiency by constructing the hybrid material with nip5;1 mutant and overexpression of STOP1.Furthermore,we proved that STOP1 can interact with NIP5;1 by yeast one-hybrid and tobacco coinjection.It showed that STOP1 can positively regulate the expression of NIP5;1under B deficiency,affect the absorption of B under B deficiency,and respond to B deficiency stress.