| Dehiscence of mature fruits is achieved through a series of specialized cells differentiation that form a specialized dehiscence zone structure.The fruit dehiscence and the seeds release are an important way for plant to complete their life history and species reproduction.Arabidopsis thaliana(L.)Heynh.has the similar characteristics and regulatory mechanisms with traditional oil crops in fruit growth and development.Therefore,using Arabidopsis as a model plant to study the mechanism of fruit dehiscence can help us further analyze mechanism of fruit dehiscence and serve its future production and application.Cellulases,hemicellulases,and pectinases are directly involved in the modification and degradation of plant cell walls,and therefore they are considered to play an important role in the process of plant organ abscission and dehiscence.However,at present,none of the cellulase and hemicellulase genes involved in the dehiscence process of Arabidopsis silique have been reported.In our study,a cellulase gene CEL6 and a hemicellulase gene MAN7 were identified through the Arabidopsis gene database and co-expression database,and then functional studies were performed on these two genes.The main results and conclusions obtained were as follows:1.Through using RT-q PCR to detect the expression level of CEL6 and MAN7 at different stages of fruit development,we found that CEL6 and MAN7 maintained relatively high levels expression in the whole stage 17 of silique development,and MAN7 even reached its peak of the expression in stage 18.The results from comparing expression levels of CEL6 and MAN7 in stage 17 the Col-0 silique and dehiscence related transcription factors ind and alc mutants siliques showed that the expression levels of CEL6 and MAN7 in ind and alc mutants were significantly reduced.These results indicated that CEL6 and MAN7 express in stage 17 silique and their expression were partial regulated by transcription factors IND and ALC.2.By culturing the promoter GUS and fusion protein GUS transgenic plants and using GUS staining to investigate the expression patterns of CEL6 and MAN7,we found that CEL6 and MAN7 expressed in vegetative and reproductive organs.Among them,CEL6 and MAN7 expressed mainly in the region of replum,valve margin and funiculi,which associated with organ abscission and fruit dehiscence.In addition,the GUS signal of CEL6 and MAN7 in valve margin and replum reduced or disappeared in ind and alc mutant,which indicates CEL6 and MAN7 expression in silique dehiscence zone,and their expression in this place is regulated by IND and ALC.3.By investigating the t-DNA insertion mutants of CEL6 and MAN7,we found that cel6 mutants,man7 mutants,and cel6 man7 double mutant siliques generally spend 1-2 days longer to maturation compared with wild type.Their silique dehiscence zone differentiation and programmed cell death in the separated layer were delayed,and their cells in separated layer became smaller and more narrow compared with wild type.However,in previous study of gene expression patterns,there was no GUS signal at the corresponding period and place,which indicates that CEL6 and MAN7 mutation affect dehiscence zone differentiation indirectly.In addition,TEM images showed that most of the cells in separated layer of wild type stage 18 B silique were completely degraded,while the separated layer cells in mutants were remain intact during stage 18 B.Dehiscence rate statistics among wild type and mutants also showed that the dehiscence rates of all the mutants were lower than that of wild type,and dehiscence rates of the cel6 mutants and cel6 man7 double mutant were lower than that in man7 mutants.These results indicate that mutations of cel6 and man7 delay the ripening of the silique,affect dehiscence zone differentiation,prevent the degradation of the separated layer cells and reduce silique dehiscence rate,which proves that CEL6 and MAN7 play an important role in fruit development and dehiscence.4.Through culturing over-expressing transgene lines of CEL6 and MAN7,and counting their dehiscence rate,we found that over-expressing CEL6 and MAN7 only increased the dehiscence rate of near mature silique in less degree,and not significant difference was shown compared with wild type.5.By introducing pectinase mutants adpg1-1 and adpg2-1,we obtained all possible combination of cel6-1,man7-3,adpg1-1 and adpg2-1 of double,triple,and quadruple mutant lines through crossing.Phenotypic analysis of various double,triple and quadruple mutants combination from four mutants,including adpg1-1,adpg2-1,cel6-1,and man7-3,indicated that the indehiscence phenotypes of adpg1-1 single mutant and the adpg1-1 adpg2-1 double mutant were more severe than those of the cel6-1 single mutant and the man7-3 single mutant or the cel6-1 man7-3 double mutant.The indehiscence phenotype were even stronger by combining the cel6-1 and man7-3 single mutations or the cel6-1 man7-3 double mutation to adpg1-1 single mutant and the adpg1-1 adpg2-1 double mutant.On the other hand,the indehiscence phenotypes of the cel6-1 and man7-3 single mutants and the cel6-1 man7-3 double mutant were stronger than the phenotypes of the adpg2-1 single mutant.By combing the cel6-1 and man7-3 single mutations or the cel6-1 man7-3 double mutation to adpg2-1 single mutant,indehiscence phenotypes became more severe compared with adpg2-1 single mutant but similar to the cel6-1 and man7-3 single mutants and the cel6-1 man7-3 double mutant.These results indicated that the phenotypes of the cel6-1 and man7-3 single mutants and the cel6-1 man7-3 double mutant were epistatic to adpg2-1.In summary,our results revealed that CEL6 and MAN7 acted indirectly on the cell differentiation during silique development,and contributed to silique dehiscence by promoting cells lysis in separated layer.Our research also demonstrate that the degree of silique dehiscence can be manipulated by altering the activity of multiple types of cell walldegrading enzymes.This provided thetheoretical and technical support for culturing oil crops in different indehiscence level in the future. |
