Arabidopsis SNARE Protein SYP32 Participates In Vacuole Formation And Female Gametophyte Development

Vesicle transport plays an important role of intracellular transport and plant development.SNAREs(Soluble NSF Attachment Protein Receptor)are the key factor to regulate vesicle transport.The membrane fusion between target membrane and vesicle need three Q-SNAREs and one R-SNARE to form a SNARE complex.In Arabidopsis thaliana,SYP3(Syntaxin of plant 3)family has two members,SYP31 and SYP32.Our unpublished study showed that SYP31 and SYP32 are Functional redundancy during pollen development,and play a important role in Golgi morphology maintenance and secretion pathway.However,it is not clear whether there is a functional difference between SYP31 and SYP32,and the specific mechanism involved.In this study,through phenotype analysis and genetic complementation experiment we found that SYP32 play an important role in the development of female gametophyte.SYP32 knockdown mutant under the control of Dex inducible promoter exhibited abnormal vacuole of hypocotyl and cotyledon.Furthermore,the subcellular localization and protein interaction analysis suggested that SYP32 has functions different from that of SYP31,and SYP32 likely participates in the regulation of vacuolar transport pathway.1.SYP32 is essential for female development.syp32/+mutant(the GK₁09A09 insertion site is on the second exon,and the GK₉20F05 insertion site is on the second intron)offsprings segregate at a ratio of 1:1,suggesting a gametophytic transmission defect.The reciprocal crosses with wild type plants further showed that the transmission efficiency of syp32 female gametophytes was zero,implies that mutation in SYP32 led to the abortion of female gametophyte.It was found that about 50%of the embryo sacs were aborted in the pistil of the syp32/+mutant.Further analysis revealed that the development of embryo sacs in the mutant FG1 and FG2 stages was normal.However,female gametophyte at FG3 and FG4 stages began to show the loss of large central vacuole,while at FG5 stage obviously began to abort.In the same pistil of the syp32/+,the development of female gametophyte is obviously asynchronous.These results indicate that SYP32 plays an important role in the development of female gametophytes.2.In Dex::RNAi-SYP32 seedling,hypocotyl growth is inhibited and vacuoles are fragmented.The abortion of syp32/+female gametophyte leads to no homozygote.In order to study the function of SYP32 in the growth and development of diploid sporophytes,and to further study the mechanism of SYP32 in the formation of vacuoles,we generated transgenic plants expressing hairpin RNAi for SYP32 under the control of the inducible Dex promoter.We found that the expression of SYP32 decreased by half,and displayed small cotyledon and short hypocotyl.Hypocotyl growth defects in RNAi-SYP32 seedlings were more obvious upon dark treatment.SEM analysis indicated that the cells were much smaller and had abnormal cell wall.FM4-64 staining showed fragmented vacuoles in the cotyledon and hypocotyl cells.3.SYP32,but not SYP31,may be involved in regulating the vacuolar transport pathway.We showed that SYP31 was located in the cis-Golgi,consistent with previous research results.While SYP32 was located in the cis-Golgi,the trans-Golgi and the TGN(trans-Golgi network).Phenotype analysis found that syp31 homozygous mutation did not affect plant growth and development,suggesting that its function can be complemented by SYP32.The SYP32 T-DNA insertion caused the abortion of female gametophytes,and pSYP32::SYP31 transgene did not complement the defect of syp32/+.In addition,in Dex::RNAi-syp32 seedlings,the expression of SYP31 was up-regulated,but the plants showed a severe growth and development phenotype.These results indicated that SYP3 2 has its unique functions.We use TAP-MS(Tandem affinity purification-Mass Spectrometry)to identify proteins that interact with SYP31 and SYP32,respectively.Within proteins that co-purified with SYP32,but not SYP31,important proteins in vacuolar transport pathway,such as VTI11 and VAMP727 were identified.In short,in addition to overlapping functions of SYP31/32,SYP32 may have a specific role in regulating vacuolar transport.