The Regulatory Mechanisms Of Arabidopsis SYP32 On Pollen Wall Development And Pollen Tube Wall Integrity

Sexual reproduction of flowering plants is a reproductive mode in which parents produce female and male gametophytes through meiosis,which combine with each other to form fertilized eggs,and then gradually develop into a new individual.These processes include a series of important processes,such as gametophyte development,pollen tube elongation,double fertilization and embryo development.Each step is accurately regulated by a series of genes.Pollen is a male germ cell.The pollen wall forms a network structure,which is the most complex plant cell wall.The materials and regulatory factors required for cell wall biosynthesis are secreted to the plasma membrane and cell wall through vesicle trafficking.Vesicle transport in eukaryotic cells mainly includes budding,transport,tethering,anchoring and membrane fusion.The membrane vesicle transport process relies on the synergistic action of multiple conserved regulatory factors,such as coat protein,tether protein,SM protein,and SNARE(soluble Nethylmaleimide-sensitive factor attachment protein receptor)protein.Among them,SNARE proteins play a crucial role in anchoring and membrane fusion.In Arabidopsis,the cis-Golgi localized Qa-SNAREs are AtSYP3 family proteins,including AtSYP31 and AtSYP32,which regulate the anterograde transport from the ER to the Golgi.It is reported that AtSYP31 and AtSYP32 jointly regulate male gamete development with partially functional redundancy,but the molecular mechanism is still unclear.To investigate AtSYP32 functions on plant reproductive development,atsyp32 mutants,AtSYP32 RNAi lines,AtSYP31 and AtSYP32 consensus RNAi(AtSYP31/32 RNAi)lines,and AtSYP32 overexpression lines(AtSYP32 OE)were used to conduct in-depth research by means of developmental biology,molecular biology,cell biology and biochemistry.The results are as follows:1.The AtSYP32 depletion mutant is homozygously lethal.The atsyp32+/-,AtSYP32 RNAi,AtSYP31/32 RNAi,and AtSYP32 OE seedlings showed significant changes in root length,plant height,and fertility compared to wild-type,while the phenotypes of the atsyp32-2 complementary lines were completely restored,indicating that AtSYP32 regulates plant nutritional development and reproductive development.2.The pollen activity of the atsyp32+/-,AtSYP32 RNAi,and AtSYP31/32 RNAi lines decreased.In vitro germination showed that some atsyp32+/-pollen and pollen tubes exploded;In vivo germination revealed that some pollen grains could not complete hydration on stigma,some pollen tube elongation was hindered,and some of the germinated pullen tubes could not elongate straightly.All these defects lead to the failure of double fertilization in many ovules due to loss of pollen tube entering.Scanning electron microscopy observation revealed partial shriveled pollen grains and incomplete pollen walls.Transmission electron microscope observation showed that some pollen grains of atsyp32+/-,AtSYP32 RNAi and AtSYP31/32 RNAi lines could not form complete pollen walls,the intine thickness became thinner,and the organization of exine was abnormal.There was adhesion between pollen grains,or between pollen grains and the residual tapetum.The AtSYP31/32 RNAi lines exhibited more severe phenotypes.However,the atsyp31 mutant plants and pollen grains did not exhibit any phenotype.These phenomena indicate that AtSYP32 regulates pollen wall integrity and pollen tube elongation,while AtSYP31 enhances AtSYP32 function.3.atsyp32+/-mutant,AtSYP32 RNAi AtSYP31/32 RNAi lines had abnormal cellular structures in some pollen grains,the ER expanded,and a large number of different types of transport vesicles detained in the cytoplasm.These phenomena suggest that AtSYP32 regulates vesicle transport and pollen wall development during microspore development.Moreover,in AtSYP31/32 RNAi lines,a large number of pollen grains had no cellular structure,and some of them had plasmolysis.This phenomenon indicates that AtSYP31 enhanced the regulatory role of AtSYP32.4.Chemical staining and immunofluorescence detection revealed that in atsyp32+/-mutant,the distribution and contents of pollen wall components e.g.callose,and pollen tube cell wall components e.g.pectin,galactose uronan(HG),deesterified galactose uronan(HGs)and xyloglucan,changed significantly.These results suggest that AtSYP32 regulates the polar transport and secretion of these materials.5.Significant changes in the expression levels of genes in RALF4/19-LRX-AUN1 and RALF4/19-ANX/BUPS-MARIS pathways that regulate the integrity of pollen tube cell wall,as well as in the vesicle transport pathway related to cell wall biosynthesis,suggest that AtSYP32 regulates pollen wall development and the integrity of pollen tube cell wall by regulating the secretion of cell wall biosynthetic materials and regulatory proteins.6.Yeast two hybrid,luciferase complementation and BiFC proved that AtSYP32 interacted with the xyloglucan synthetase XXT5,which may accelerate synthesis and secretion of xyloglucan.AtSYP32 interacted with the receptor protein LRX11 and signal polypeptide RALF19,which may promote their secretion efficiency to pollen tube cell wall.Meanwhile,AtSYP32 interacted with SNARE proteins BET12 and AtSEC22 which is essential for male gamete development,and COPII coat protein SEC31B which is related to pollen fertility,suggesting that AtSYP32 regulates pollen wall development and the integrity of pollen tube cell wall via modulating secretion of cell wall synthetic materials and regulators.In summary,AtSYP32 plays a crucial role in plant reproductive development by controling vesicle transport to regulate pollen wall development and pollen tube cell wall integrity.