Mechanism Of A Secretory Lipid Transfer Protein,OsLTPL94,in Regulating Rice Pollen Development

The pollen of higher plants carries the genetic material of male parents and is essential for plant reproduction and the creation of genetic diversity.The pollen wall is a multilayer structure wrapped around the pollen surface.It protects the male gametophyte of plants from various biotic and abiotic stresses,and plays an important role in distinguishing other pollen and germination.Forming a unique pollen wall structure and elaborate exine pattern is a well-organized process that needs high coordination between reproductive cells and the neighboring somatic cells.However,molecular mechanisms underlying this process remain largely unknown.Lipid transfer proteins(LTPs)are a class of small and basic proteins with secretory signal peptides.LTPs are involved in all the key processes of plant biology and play a key role in pollen development.Therefore,an in-depth study on the function and regulatory pathway of LTP is of great significance in improving and clarifying the regulatory network of plant pollen development.In this study,we identified a rice male-sterile mutant,namely lipid transfer protein-like94(l94),and confirmed that OsLTPL94 encoding a secretory LTP plays a key role in rice pollen development by using genetical,cytological and biochemical assays.In addition,we found that ETERNAL TAPETUM 1(EAT1),a basic helix-loop-helix(b HLH)transcription factor,activated OsLTPL94 expression through binding to the OsLTPL94 promoter,suggesting that the function of OsLTPL94 in rice pollen development may be directly regulated by EAT1.The main results are as follows:1.l94 mutant was obtained from an ethyl methane sulfonate(EMS)-induced mutagenesis.Genetic analyses indicate that a single recessive mutation caused the malesterile phenotype of l94.Further cytological observation showed that the primexine of l94 microspores was irregular,and failed to form a normal exine pattern.The l94 microspores were shrunken and collapsed after stage 10.Meanwhile,an abnormal degradation with smaller and disorderly Ubisch bodies also occurred in the l94 tapetum.In addition,the expressions of several tapetum or pollen exine development-related genes were significantly altered in the l94 mutant.These results indicate that the pollen abortion of l94 may be due to the abnormal primexine pattern,which affects the correct deposition and assembly of sporopollenin.2.Two candidate genes located on chromosome 3 were unraveled by Mutmap analysis.Further CRISPR/Cas9 analysis confirmed that only the knockout lines of the LOC＿Os03g46110 gene mimicked the cytological abnormalities and male-sterile phenotypes of l94.A single single-nucleotide polymorphism(SNP)in the first exon of the LOC＿Os03g46110 gene in l94 led to a missense mutation of amino acid,and genetic analysis confirmed the co-segregation between the mutation site and the male-sterile phenotype.These results suggest that the LOC＿Os03g46110 gene is OsLTPL94.3.q RT-PCR analysis showed that the expression of OsLTPL94 peaked in stages 6–8 of rice anther development.Further β-glucuronidase(GUS)staining and RNA in situ hybridization assays showed that OsLTPL94 was mainly expressed in tapetum and microspores during meiosis,which also supported its roles in pollen and tapetum development.4.Phylogenetic analysis showed that OsLTPL94 encodes a type-G LTP and may have a conserved function in Poaceae plants.Subcellular localization showed that OsLTPL94 protein is specifically localized in the cell membrane;however,the mutation of l94 was located in its Nterminal signal peptide(SP)region,and the mutation weakened the plasma membrane specific localization.Further tobacco transient expression and yeast secretion assays confirmed the secretory ability of the SP of OsLTPL94,and the mutation may impair its secretory capacity.In addition,the protein truncation analysis showed that the plasma membrane localization of OsLTPL94 also needs its C-terminal glycosylphosphatidylinositol-anchor(GPI-anchor).5.Lipid-related genes are often directly regulated by many transcription factors.Our in vivo and in vitro investigations confirmed that the b HLH transcription factor EAT1 could activate OsLTPL94 expression through direct binding to the E-box motif of the OsLTPL94 promoter,suggesting that OsLTPL94 may act downstream of EAT1 in rice pollen development.Subsequently,we identified two independent allelic mutants of eat1,which showed abnormal pollen wall and tapetum development related to l94.The EAT1 in eat1-5 exhibited a deficiency in its transcription ability,and the expression of OsLTPL94 was also significantly downregulated.The EAT1 protein in eat1-6 still showed transcriptional activation ability,and the expression of EAT1 and OsLTPL94 were also positively correlated.These results support the hypothesis that OsLTPL94 may be a direct downstream target of EAT1.In conclusion,this study demonstrated that OsLTPL94 encoding a secretory LTP is required for rice pollen development,and proposed that the EAT1-OsLTPL94 module may play an important role in the coordinated development of tapetum and microspore,which provides new and valuable information for the regulation mechanism of rice pollen development.