Molecular Mechanism Of Transcriptional Regulatory Protein AMD1 Involved In Rice Male Development

Pollen development includes a series of biological events that require precise gene regulation.Although several transcription factors(TFs)have been shown to play roles in maintaining pollen fertility,the major regulatory networks underlying tapetum development and pollen wall formation are largely unknown.Herein,we report that ABERRANT MICROSPORE DEVELOPMENT1(AMD1),a protein annotated previously as unknown protein,is required for tapetum development and pollen exine patterning in rice(Oryza sativa L.).The phenotypic,cytological and biochemical analyses suggested that AMD1 interacts with TAPETUM DEGENERATION RETARDATION(TDR),a key transcription factor for tapetum development,and binds the promoters of two pollen exine formation related genes DPW and OsPKS2 to activate their expression.These findings suggest that AMD1 may be involved in pollen development by interacting with TDR and acting as an upstream transcriptional regulator of DPW and OsPKS2.The main results are as follows:1.A male-sterile mutant amd1 was generated from 9311 by EMS-induced mutation.Compared with the wild type,the amd1 mutant showed no significant difference in the vegetative growth stage,but its anthers at heading stage were slightly smaller than that of the wild type and were pale yellow.The results of pollen staining by FDA and I2-KI showed that the pollen grains of amd1 were inactive.The results of semi-thin section,transmission electron microscope and scanning electron microscope showed that amd1 had defects in tapetum development and pollen wall formation,indicating that AMD1 is involved in tapetal PCD and exine formation.2.Using MutMap analysis,we found a SNP on chromosome 1 with highest SNP index.This SNP is located in the third exon of LOC＿Os01g55094 and induces a stop codon,leading to premature termination of protein translation.This SNP was co-segregated with malesterile phenotype.Furthermore,the null knockout mutant of AMD1,cr-1 and cr-2,in ZH11 background were obtained by CRISPR/Cas9 technique.Both null knockout mutants produced sterile pollen,similar to that of amd1 mutant.These results indicated that LOC＿Os01g55094 is AMD1.3.qPCR analysis showed that the expression of AMD1 peaked in the 5-6 mm florets.Further cross-section observations of GUS stained anthers and RNA in situ hybridization analysis indicated that AMD1 was mainly expressed in the tapetum and microspore during meiosis,and only expressed specifically in the tapetum during tapeta PCD and exine formation.These results support the role of AMD1 in pollen and tapetum development.Subcellular localization analysis showed that AMD1 protein is located in the nucleus,and this necleus-localization is dependent on the two nuclear localized signals in the protein sequence.Next transcriptional activity analysis indicated that AMD1 protein may function as a transcriptional activator.4.Transcriptomic analysis revealed that the genes related to cutin,wax and fatty acid metabolic pathways were down-regulated in the mutant.Further qPCR analysis verified the results of the transcriptomic analysis.We next used yeast one hybrid and D-LUC reporting system to analyze the down-regulated genes,and found that DPW and OsPKS2,two important genes in exine formation,might be directly regulated by AMD1.These results suggested that AMD1 may affect the development of rice pollen exine by directly regulating the expression of OsPKS2 and DPW.5.Given that tapetal PCD was defected in amd1,and that AMD1 might acts as transcriptional activator in tapetum,we speculated that AMD1 may also be involved in the transcriptional regulation network of tapetum.To this end,we used Y2H system to screen the yeast libraries of rice panicle c DNA.Through Y2H screening,we found that AMD1 could interact with TDR,a key transcriptional regulator of the tapetal PCD.Further BiFC,LCI and Co-IP analyses verified that AMD1 indeed interacts with TDR in plant cells.Additionally,the results of differentially expressed genes analysis suggest that AMD1 and TDR may have the common targets.These results imply that AMD1 may be involved in the development of rice tapetum by interacting with TDR.In summary,we demonstrate that AMD1 is a novel component in the regulatory network of rice male development by participating in tapetal PCD and exine formation.Our work provides new insights into the regulatory network of sexual reproduction in rice and a useful target for genetic engineering of new hybrid rice varieties.