Arabidopsis Sar1 GTPases Of The COP? Complex Regulate Pollen Development

Pollen development is a pre-requisite for double fertilization in angiosperms.COP? mediates anterograde transport of vesicles from the endoplastic reticulum to Golgi and components of the COP? complex have been reported to regulate either sporophytic or gametophytic control of pollen development.The Arabidopsis genome encodes five Sar1isoforms,the small GTPases essential for COP? formation.By using a dominant negative?DN?approach,Sar1 isoforms were proposed to have distinct cargo specificity despite their sequence similarity.In this study,we examined the function of Sar1 isoforms by taking a reverse genetic approach.The main results and conclusions presented in this thesis are as follows:?1?Functional loss of Sar1b results in male sterility.To examine the function of three Sar1 isoforms in Arabidopsis,we took a reverse genetic approach.We characterized T-DNA insertion mutants of Sar1a and Sar1c.Functional loss of both genes did not affect plant growth as compared to wild type.We generated sar1b mutants by CRISPR/Cas9.Two mutant alleles were identified,sar1b-1 and sar1b-2,both of which expressed mutated Sar1b transcripts with pre-stop codons resulted either from an insertion or from a deletion in the Sar1b coding region.Vegetative growth of both sar1b mutants was comparable to that of wild type.However,siliques of the homozygous sar1b mutants did not elongate in contrast to those of wild type.Siliques of sar1b or sar1b pollinated with wild-type pollen contained a full seed set whereas mature wild-type siliques did not set seeds after pollination with sar1b or sar1b pollen,suggesting that sar1b is male sterile.Indeed,by alexander staining and scanning electron micrographs?SEMs?of dehiscing anthers,we determined that mature sar1b anthers did not produce viable pollen as did wild type,demonstrating that functional loss of Sar1b causes male sterility.?2?Tapetal defects of sar1b start at the tetrad stageTo determine what have caused the male sterility of sar1b mutants,we performed plastic embedding and transverse section of developing anthers.At anther developmental stage 6-7,sar1b started to differ from wild type.The tapetal cells in sar1b were hypotrophic containing larger central vacuoles than those of wild type.At the unicellular stage and bicellular stage,a large amount of sporopollenins was deposited to the developing microspores that gradually formed their reticular pollen coat structure in wild type.By contrast,dense materials,likely sporopollenins,were deposited to the intercellular space between the tapetum and the middle layer rather than the surface of developing microspores in sar1b.There were aggregates of dense materials deposited in pollen sacs without attaching to the surface of microspores.At the tricellular stage,the tapetal layer degenerated completely while the microspores developed well-pattern coat structure in wild type.By contrast,a hypotrophic tapetum was still visible in sar1b.Microspores in sar1b pollen sacs were degenerated and all cellular organelles disintegrated.These results supported a key role of Sar1b in sporophytic male fertility.?3?Sar1b loss-of-function interferes with the timing of tapetal PCDTo determine whether the timing of tapetal PCD was affected by Sar1b loss-of-function,we performed terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling?TUNEL?assays.At stage 10,TUNEL-positive signals appeared in the tapetal layer of wild-type anthers.Anthers of sar1b at the same stage did not show TUNEL-positive signals in the tapetal layer.At stage 11,TUNEL-positive signals were not only in wild type but also in sar1b.At stage 12,the tapetal layer degenerated completely in wild type.By contrast,a hypotrophic tapetum was still visible in sar1b.These results suggested that Sar1b participates in tapetal PCD.?4?Tapetum-specific expression of Sar1b partially rescues anther defects of sar1bBecause the function of sar1b tapetum seem to be affected,we tempted to hypothesize that the role of Sar1b in sporophytic control of pollen development was played through its expression in tapetum.To test this hypothesis,we expressed Sar1b under Pro_A9,a tapetal specific promoter.Examination of dehiscing anthers by SEM showed the production of pollen grains at the dehiscing anthers of the Pro_A9:GFP-Sar1b;sar1b plants.These results suggested that tapetal expression of Sar1b at least partially rescued pollen defects of sar1b.?5?Sar1c can replace Sar1b in sporophytic control of pollen developmentBased on histochemical GUS staining of reporter lines,both Sar1b and Sar1c were expressed in all cells during anther development.The fact that the sar1b single mutants were completely male sterile while sar1c was normal suggested a distinct function of Sar1b and Sar1c despite the same spatiotemporal expression.To test this hypothesis,we examined the ability of exogenous Sar1c versus Sar1b to complement the anther developmental defects of sar1b by generating the Pro_UBQ10:Sar1b;sar1b and Pro_UBQ10:Sar1c;sar1b transgenic plants.Either transgene was able to restore male fertility of sar1b based on alexander staining and SEM analyses of dehiscing anthers.These results suggested that although Sar1b and Sar1c do not have redundancy in sporophytic control of pollen development,they are exchangeable.?6?Sar1b and Sar1c redundantly regulate male gametophytic developmentExcept for their expression in sporophytic cells in anthers,both Sar1b and Sar1c were substantially expressed in microspores.To explore whether Sar1b and Sar1c play roles in male gametophytic development,we crossed sar1b and sar1c by using sar1c as the pollen donor to pollinate sar1b pistils.The sar1b;sar1c male gametophytes could not be transmitted based on reciprocal crosses and segregation analysis.These results suggested that Sar1b and Sar1c redundantly mediate male gametophytic function.To determine which processes were affected by Sar1b and Sar1c loss-of-function that caused male gametophytic lethality,we examined pollen development of the sar1b/+;sar1c plants.By alexander staining,DAPI staining,and SEMs,we found that around 50%pollen produced by the sar1b-1/+;sar1c plants were aborted while wild type or either single mutant contained almost 100%normal pollen grains at maturation.These results suggested that Sar1b and Sar1c play redundant roles in gametophytic control of pollen development.?7?sar1b;sar1c caused the arrest of pollen development at the stage 10To determine the exact defects occurred in sar1b;sar1c pollen,we performed plastic embedding and transverse section of developing sar1b/+;sar1c anthers.There was no tapetal hypotrophy in sar1b/+;sar1c anthers,which differs from that in sar1b and consistent with the fact that gametophytic but not sporophytic defect resulted in pollen abortion in sar1b/+;sar1c.Defective microspore development was started at anther developmental stage 10 a portion of microspores in sar1b/+;sar1c pollen sacs showed the detachment of PM from pollen coat.Later on,wild-type microspores went through bicellular to tricellular mature pollen.By contrast,a portion of microspores in sar1b/+;sar1c pollen sacs showed degenerating cytoplasm and finally collapsed.In this study,we demonstrated that Sar1 isoforms perform distinct yet inter-changeable roles in pollen development.Sar1a does not play a role in pollen development.Sporophytically expressed Sar1b is critical for pollen development,whose absence can be compenstated by exogenous Sar1c.On the other hand,gametophytically expressed Sar1b and Sar1c redundantly regulate pollen development.