The Role Of Aspartic Protease A39 In Cell Death Of Arabidopsis Anther Tapetum

Programmed cell death(PCD)is a genetically controlled and actively ordered way of death in order to maintain a stable internal environment and better adapt to the living environment under certain physiological or pathological conditions.PCD has important biological significance.The timely degradation of the tapetum of flowering plants plays a decisive role in the formation of male gametes(pollen).The degradation process of tapetum cells has a typical PCD characteristic.At present,understanding of protease and other molecules involved in and the mechanism behind the PCD of the anther tapetum are still lacking.Aspartic proteases(ASPs)are the second largest class of proteases in Arabidopsis.Studies have shown that this family of proteins may be one of the main function performers in the process of plant PCD.Therefore,ASP gene A39 specifically expressed in anthers were selected for genetic analysis to explore the function of ASPs in the anther tapetum PCD.A T-DNA insertion mutant of gene A39(Salk?134919,abbreviated as T-A39)was obtained for the functional research and the insertion of the TDNA mutant into the promoter region of the A39 was confirmed by threeprimer method.Real-Time quantitative reverse transcription PCR(q RTPCR)demonstrated that the expression of A39 gene was about 5 times higher in T-A39 than in wild type.It was speculated that the T-DNA fragment contains a 35 s enhancer leading to increased transcription.The genetic observation of homozygous T-A39 showed that there was no significant difference between the vegetative growth period of T-A39 and WT,but the seed setting rate in the reproductive period was about 36% lower than that of WT.Scanning electron microscopy(SEM)confirmed the abortion of TA39 pollen.To further confirm that the genetic phenotype of T-A39 is associated with overexpression of A39,an overexpression vector(Ubiquitin+A39CDS)were constructed and a new overexpressing plant Over-Eexpression-A39(referred to as OE-A39)was obtained.q RT-PCR demonstrated that the expression of A39 gene in OE-A39 was about 8 times higher than that of WT.The genetic observations of OE-A39 also have traits similar to T-A39.Based on the genetic analysis of T-A39 and OE-A39 overexpressed plants,we confirmed that the overexpression of A39 gene did affect pollen development and lead to a decline in seed setting rate in Arabidopsis thaliana.In order to further study the genetic function of A39,a homozygous mutant Knockout-A39(referred to as KO-A39)was obtained by CRISPR-Cas9 technique.Sequencing results indicated that KO-A39 had a single base A insertion at exon + 111 bp,which resulted in the early termination of amino acid coding and transcription.q RT-PCR showed that the expression of A39 gene in KO-A39 decreased by about 50% compared with WT.Considering that this mutation led to premature termination of translation,we believed that KO-A39 strain could be used as a genetic material for complete deletion of A39 gene for related genetics and functional analysis.Analysis of seed setting rate showed that KO-A39 decreased by about 44% compared with WT.SEM also showed abnormal development of pollen wall of KO-A39.SEM results showed that the pollen wall of KO-A39 was abnormal,and it had the genetic traits similar to T-A39 and OE-A39 in pollen abortion and seed setting reduction.Cytological characteristics of anther development in detail in T-A39,OE-A39 and KO-A39 were compared.The results of semi-thin section showed that both overexpression(T-A39 and OE-A39)and deletion(KOA39)of A39 resulted in abnormal morphological and structural development of anther microspore in the 9th stage.At the 12 th stage,the microspore of the three mutants underwent concentrated degradation,which eventually led to the reduction of the number of mature pollen and fertility.In order to further study the expression profile of A39 gene,we also constructed,transformed and screened pro A39:GUS strains which has expression of GUS driven by A39 promoter.The results of GUS staining showed that A39 was highly expressed in tapetum cells of anther development stage 9,which indicated that the expression pattern of A39 gene was consistent with the period of cytological changes in the mutants concerned.In addition,we also carried out evolutionary analysis of A39 and its homologous genes A51 and A52.Phenotypic observation of A51 and A52 related genetic materials(KO-A51,KO-A52,OE-A51,OE-A52)showed that the seed setting rate did not change.The seed setting rate of a39/a51 double mutant obtained by hybridization technology was lower than that of WT,but there was no significant change compared with KO-A39.The above results demonstrated that A39 plays an important role in the middle and late stages of anther development.Overexpression and deletion of A39 function can lead to abnormal pollen development.A39 may be involved in the process of pollen wall formation and tapetum PCD.Further investigations of A39 will provide new ideas and understanding for exploration the function of ASPs gene in plant PCD and reproductive development.