Role Of DPW2?DEFECTIVE POLLEN WALL 2?, An Acetyl Transferase In Rice Pollen Wall Development

As the male reproductive organ in plants,anther,is covered with cuticle on the surface,protecting the development of microspores.And the microspores within anther locules form a hydrophobic protective layer on the surface,which is called pollen wall with a major composition of sporopollenin.Anther cuticle is mainly constituted by cutin and wax,which contain the compositions of aliphatic and aromatic lipids.Similar to cuticle,the main components of sporopollenin are aliphatic and aromatic lipids.The outer pollen wall(also called exine)can protect pollen from environmental stresses,and also plays a significant role in male female interaction during pollination.Because sporopollenin,a hydrophobicity structure,is difficult to be degradaed,so far the mechanisms underlying the biosynthesis,transport and ploymerization of the monomers remain poorly understood,especially about the aromatic components and their functions.Rice male reproductive development is closely related to grain yield,thus,it is crucial to understand the molecular mechanism of anther and pollen development.This study charaterized a rice male sterile mutant,which is named dpw2(defective pollen wall 2)and revealed the important roles of DPW2,a HXXXD type acetyl transferase of the BAHD family in the formation of anther cuticle and pollen exine.We isolated two dpw2 allelic mutants.Genetic analysis suggests that these two mutants are controlled by a single recessive gene.Phenotypic analysis indicated that there are no obvious morphological differences between wild type and dpw2 during vegetative stage.But during reproductive stage,the mutant anther appeard paler and smaller than that of wild type.Pollen grains were sterile as revealed by iodine potassium iodide staining.Consistent of this,the DAPI stainning shows that microspores cann't undergo the second-round mitosis in dpw2.The semi-thin section observations indicate that tapetum degeneration was delayed in the mutant and most pollens are shrunken with irregular shape.Electron microscope observation results show that dpw2 mutant anthers had a dense cuticle layer,the degeneration of middle layer and tapetum was delayed,and,pollen exine and intine formation had defects,resulting in aborted pollen grains and male sterility.These observations suggest that DPW2 is required rice anther and pollen development.Map-based cloning and complementation experiment confirmed that male sterility of these mutants are caused by the disruption of a HXXXD type acetyl transferase encoding gene DPW2.DPW2 is ubiquitously expressed in different tissues but highly expressed in anthers at stage 7 to stage 11.DPW2 promoter activity analysis also shows that DPW2 is highly expressed in the tapetum and microspores.Transiently expression system using rice protoplast and tobacco epidermis cells shows that DPW2 is localized mainly in cytoplasm.Meanwhile,when pDPW2:DPW2-GFP is introduced into dpw2 mutants,the introduced construct fully rescues the male sterility of dpw2.Sudan red 7B staining analysis reveals that dpw2 anthers had more cutinized lipids in the anther and less lipids in pollen wall,indicating that mutation in DPW2 has great impact on lipid compound accumulation in pollen wall.Furthermore,the autofluorescence emitted by pollens in dpw2 pollen is less than that of wild type,indicating the reduction of phenolic compounds in the pollen wall of the mutant.Results of cuticular lipids analysis shows that the contents of the cutin monomer and wax components in the mutant are increased to different extents,suggesting that dpw2 had accumulated precursors of anther cuticle and pollen wall.Consistly in vitro enzyme activity assays demonstrated that recombinant DPW2 specifically transfers hydroxycinnamic acid moieties,using ?-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs as acyl donors,prefering to catalyze the feruloyl-CoA and 16-hydroxy-palmitic acid to form 16-feruloyl-palmitic acid.In conclusion,DPW2 catalyzes the synthesis of specific aromatic lipids,and these lipids take part in the formation of anther cuticle,pollen exine and intine.The study provides new insights into understanding the role of aromatic lipids during anther and microspore development.