Functional Analysis Of HOTHEAD Family Member OsHTH1 In Rice Anther Development

The cuticle is a complex hydrophobic layer that covers the epidermis of all aerial parts of higher plants.It deposits on the epidermal cell wall as barriers to control the fluxes of gases,water and solutes,and plays roles in protecting plants from biotic and abiotic stresses.Cuticle deposits on the epidermal anther wall is one of the most important adaptations for protecting pollen from biotic and abiotic stress.Cutin polymer matrix and waxes are the main components of cuticle.However,research about the molecular mechanism underlying the biosynthesis of the monomers in anther cutin isn't quite as clear.In Arabidopsis,it has been reported that HOTHEAD family is involved in the synthesis of cutin monomers.In this study,we focus on a rice anther epidermis-specifically expressed gene OsHTHI,which is homologous genes with Arabidopsis HTH/ACE.We used RNA interference and CRISPR technology to down-regulate expression of OsHTHl gene.By analysis of cytology,molecular biology and biochemistry to confirm function of OsHTHl in anther and pollen development.The main results of this study were as follows:1.We constructed OsHTH1 CRISPR vectors and overexpression vectors,and obtained OsHTH1 T1 generation CRISPR transgenic plants and T0 generation Overexpression plants respectively.2.Expression pattern analysis of OsHTH1.qRT-PCR result showed that OsHTH1 was specifically expressed in spikelet development 7 stage,but barely expressed in the vegetative organs;in situ hybridization analysis of OsHTH1 revealed it was specifically expressed in the epidermis cell layer of the anther wall and the significant signal was detected at vacuolated microspore stage.3.Agronomic traits observation of HTH1-RNAi plant.HTH1-RNAi lines displayed a similar phenotype to that of the wild type in vegetative growth.However,a small and blanch anthers were observed in HTHl-RNAi plant after heading,we inspected fertility of pollen using I2-KI stain,the result showed the number of fertile pollen was significantly reduced in HTH1-RNAi plant.A large proportion of pollen isolated from these plants failed to stain with FDA and therefore was likely to be dead.The setting percentage of HTH1-RNAi seeds were notable decline that might be caused by lacked normal mature pollen grains.4.Phenotype analysis of hthl mutant T,generation.Compared with wild type,anthers of single nucleotide deletion mutant hth1 showed short and shrink appearance;I2-KI and FDA stain results showed that pollen sterility and pollen vitality obviously decreased compared with wild type.These results were consistent with OsHTHl-RNAi lines.5.Cytological analysis of HTH1-RNAi anther.Anther paraffin sections revealed that anther wall was collapsed and pollen degraded in vacuolated pollen stage.We noted the H7Hl1-RNAi anther outer surface was obvious shrink and collapsed at mature pollen stage,microspore looked like be a shrivelled spherical under the scanning electron microscopy.Transmission electron microscopy analyses showed that cuticle was thinner in the outer layer of the anther compared with wild type and the microspore development was finally disrupted.6.Aliphatic Composition analysis of the HTH1-RNAi Anther.GCMS analysis showed significant defect in the biosynthesis of cutin monomers in HTH1-RNAi anther.Most of cutin monomers in anther surface was obvious decreased,while total wax almost equivalent with wild type.For this gene is strongly expressed in the epidermal cells of anthers,our results suggest that OsHTH1 involves in biosynthesis of cutin monomers and plays an important role in anther cuticle development during rice male reproductive.