Molecular Mechanism Of Cellulase In Anther Development

Microspore mother cells are encased by callose after meiosis in the anther of Arabidopsis,and microspores are wrapped by the tetrad wall to form tetrads.In the stage of tetrad,as the innermost layer of the anther wall,tapetum synthesizes a large amount of pollen wall precursors,and transports these raw materials to tetrads through the polar degradation of tapetal cell wall,thereby ensuring the normal deposition of the pollen wall onto the microspores.After then,the tetrad wall is degraded,and the microspores covered with pollen wall are released from tetrads.Eventually,the pollen wall is fully developed;microspores form mature pollen grains.During this developmental process,the primary cell wall of microspore mother cells undergoes an orderly transition from the tetrad wall to the pollen wall.In addition,the degradation of tapetal cell wall is also a prerequisite for ensuring the successfully transportation of pollen wall raw materials.Cellulose is an important component of cell wall.Studies have shown that cellulose in the cell wall of the tapetum and the primary wall of microspores mother cell in Arabidopsis are degraded during meiosis.However,it is still unknown which cellulases are involved in the process;the effects of this process on the secretion of tapetum and the formation of pollen walls of microspores is unknown.In this paper,6cellulase genes(CELA,CELB,CELO,CELP,CELR,CELS),localized in the early stage of anther development,were identified from 75 candidate genes by gene annotation and expression analysis.Among which,CELA and CELB are the members of the glycosyl hydrolase 9(GH9)family.Protein localization showed that they are specifically located in the cell wall of tapetum and the microspores during meiosis.CELR and CELS are hydrolases;CELO and CELP are the member of endo-β-1,4-MANNAN(MAN)family,which are mainly located in the cell wall of the middle layer and/or the tapetum..Subsequently,we obtained homozygous mutants of those cellulases genes mentioned above by molecular identification.Alexander staining showed that the pollen viability of these mutants were not affected,suggesting that there is the genetic redundancy.Presently,the related double and multiple mutants are being hybridized.In addition,according to the 14 developmental stages of anther in Arabidopsis,we used Safranin O-fast green dye to specifically stain the cellulose in developing anthers.The results showed that in normal anthers,the degradation of cellulose in the cell wall of the tapetum began at stage 5;and basically end at the stage 7,the degradation of the cellulose of primary wall began at stage 5;and end at stage 6.On the other hand,the transcription factors specifically expressed in the tapetum form a transcriptional regulatory pathway(DYT1-TDF1-AMS-MS188),in which,DYT1,TDF1 and AMS are responsible for the early development of the tapetum and play an important role in pollen wall formation and tetrad release.Therefore,combining with the expression patterns of CELA and CELB,we analyzed their genetic pathway.The results showed that both CELA and CELB share the similar expression trends,and they were slightly downregulated in dyt1,and significantly downregulated in tdf1.Combining with their protein localizations,we speculate that CELA and CELB may be expressed in both tapetum and microspores,and their expression in tapetum are mainly downstream of TDF1.We will perform cytological observations on cellulose degradation in each mutant to determine their degradation.We will also analyze the transcriptional pathways that regulate those candidate genes foe cellulose degradation.Therefore,the further analysis on the cell wall degradation process of tapetum and microspores will provide a theoretical basis for in-depth understanding the synergistic regulation of tapetum development and pollen wall formation.