Primary Studies On Physiological And Molecular Mechanisms Underlying Root Border-like Cell Development In Stylosanthes Guianensis

Border-like cells?BLCs?present in the outermost layer of plant root cap,which not only can secret compounds?e.g.,mucilage?into rhizosphere,and thus influence rhizosphere environment,but also protect root tip development and growth.In the study,using a Stylosanthes guianensis?stylo?genotype,RY2 as a material,we investigated dynamic formation and structure of BLCs.Subsequently,RNA-seq technique was conducted to identify differential expression genes in stylo primary root tips and BLCs,which might be involved in BLC formation,and partially elucidate physiological and molecular mechanisms underlying BLC development.The main results are shown as follows:1)Border-like cells were obviously observed in stylo tap root tips and lateral root tips in both solid MS growth medium and nutrient solution culture.On solid MS growth medium,fresh weight,length,and number of cell layers of BLCs around the primary root tip were significantly increased within 1-3 days after seed germination.On the 3^rd day after seed germination,BLCs formed a yellow-brown sheath,which fresh weight,morphology and structure remained relatively stable after that.2)During the development of BCLs,structure and activity of cells at different sites of BLCs were significantly different.From 2^nd to 5^th day of BLCs formation,cells on both top and bottom of BLCs were loosely connected.Furthermore,cells with fluorescein diacetate?FDA?staining were significantly decreased,suggesting that cell activity was significantly reduced.However,cells in the middle of BCLs exhibited tight connection,and high rates of FDA staining,indicating that the cell activity remained relatively high.3)On solid MS growth medium,application of sucrose could significantly affect BLC formation in stylo.Compared to plants with sucrose application,fresh weight,length,and average width of BLCs around the tap root tip without sucrose application were reduced by 50%,14%and 32%,respectively,indicating that sucrose might regulate BLC development in stylo.4)With the aid of RNA-seq technique,differential gene expression profiles were investigated during BLC development around the tap root tips in stylo.The results showed that a total of 5,667 genes were identified with differential expression during the 1^st,2^nd and 3^rd day after seed germination in stylo tap root tips and BLCs.Among them,differential expression genes were 3,383 between the 1^st and 3^rd day after seed germination in tap root tips and BLCs.According to the KEGG metabolic pathway analysis,these genes were mainly enriched in pathways such as fatty acid synthesis,cutin/suberine/wax biosynthesis,pentose and glucuronic acid conversion,plant hormone signal transduction,and starch and sucrose metabolism.Furthermore,15genes with differential expression patterns were selected for qRT-PCR analysis.The results showed that expression patterns of the selected 15 genes were consistent,and thus confirmed the results from RNA-seq analysis.5)Among the differential genes,several up-regulated genes were predicted to be involved in pectin metabolisms,including pectin methylesterase?PME?,pectate lyase?PL?and polygalacturonase?PG?.Furthermore,it was observed that pectin acid staining was relatively high between epidemic cells and BLCs,indicating that pectic acid was accumulated in BLCs,which might be regulated by PME,PL and PG.