Identification,Characterization And Functional Analysis Of Apa1331(Apical Panicle Abortion1331) And Apa1304 In Rice

Panicle degeneration is a serious physiological defect and causes significant reduction in the yield.Panicle degeneration is often seen due to extreme climate conditions and its molecular basis remained largely unexplored.Although,several mutants have been reported to cause the panicle degeneration due to micro-mutations in the genome but their mechanism remains a dilemma.In the present study,we have identified and characterized the panicle degeneration related mutants named as“apa1331（apical panicle abortion）and apa1304”that were derived from an EMS mutagenized population of a maintainer line（Yixiang 1B）.Both apa1331 and apa1304 have revealed significant differences in the fertile length of panicle.The important results of present study are given below.1.Phenotypic analysis of apa1331 revealed significant reduction in grain yield and exhibited up to 54%degeneration in the apical spikelets.Histochemical analysis revealed the triggered level of reactive oxygen species（ROS）and TUNEL（terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling）assay has demonstrated that DNA fragmentation has occurred in the apical spikelets of apa1331.Genetic mapping coupled with Mutmap revealed that the degeneration phenotype in apa1331 was mapped on the 4th chromosome and harbored an SNP（A>G）in the fourth exon of LOC＿Os04g40720.This was a non-synonymous mutation that changed the genetic codon for AAC（asparagine）>GAC（aspartic acid）.APA1331 is a novel gene and codes for an uncharacterized protein that only contains a conserved coiled coil domain（CCD）.Genetic segregation analysis presented that trait of panicle degeneration in apa1331 is controlled by single recessive gene,and knock out lines of APA1331 have displayed the panicle abortion phenotypes.Comparative transcriptomic and q RT-PCR analysis of panicles of WT and apa1331,revealed the down regulation LTP’s（LIPID TRANSFER PROTEINS）genes and altered fatty acid metabolism in apa1331.Likewise,the apical spikelets displayed the dissimilar load of phytohormones especially the cytokinin and abscisic acid.Fatty acid contents analysis has also revealed the significant reduction in the contents of palmitic and octadecanoic acid in the apical panicles of apa1331.Our results support that disruption in the APA1331 causes the degeneration of panicle and transcriptomic profiling reveals the panicle degeneration phenotype can be associated with fatty acid metabolism.2.The phenotypic analysis revealed that apa1304 displayed severe degree of degeneration（up to 49%）.Histological and TUNEL assays have demonstrated that boosted level of ROS caused damages to the DNA and internal entities of glume cells.Genetic mapping coupled with mutmap revealed the candidate gene of apa1304 harbored a SNP（T>C）,which altered the triplet codon from GTC（valine）into GCC（Alanine）in the second exon of LOC＿Os04g37950.It is a novel gene and encodes for UBIQUITIN CARBOXYL-TERMINAL HYDROLASE DOMAIN CONTAINING PROTEIN（UCHDP）.CRISPR cas9 lines that were knocked out against APA1304 have displayed the apical degenerated spikelets that verified function of UCHDP for normal panicle development.Subcellular localization has discovered that APA1304 was localized into both nucleus and cytoplasm.Apa1304 spikelets were subjected to further phytohormones analysis and revealed a highly significant contents of cytokinin and gibberellins in upper than middle and lower panicle.Our results present the novel role of UCHDP for the normal panicle development in rice.3.To examine the relevance of protein ubiquitination with panicle development in rice,we present a comparative ubiquitome profiling in an apical panicle abortion 1304 mutant（MT）,which exhibited apical spikelets degeneration,and its originated wild type rice variety,Yixiang 1B（WT）.Our proteomic analysis using high-throughput mass spectrometry and highly sensitive di-gly-lys antibody enrichment method allowed in-depth monitoring of ubiquitinated peptide tags:204 of up-regulated modified sites and 56 of down-regulated modified sites in MT.A motif analysis of ubiquitinated sites revealed that 1^stmost enriched motif（XXXXXAXK）contained an alanine at-2 position in both MT and WT but some other motifs were also found.Gene Ontology classification revealed significantly enriched ubiquitinated proteins for transport and integral membrane components in MT.Conserved ortholog set classification and KEGG metabolic annotation assisted to find prevalence of ubiquitinated peptide tags for post-translational modifications and endocytosis.Integration of protein domain annotations revealed‘protein kinase domain’and‘leucine rich repeat’were preferential targets of ubiquitination in MT.These results suggest the novel role of ubiquitin in endocytosis for panicle development together with some other diverse functions of ubiquitome in panicle development.The current study provides the novel role APA1331 and APA1304 for normal panicle development.Although,the possible regulation of mechanism is supported by transcriptomic and ubiquitomic studies but physical evidences are needed yet in further experimentation.What are the down and upstream transcription factors or genes of APA1331 and APA1304?How exactly the APA1331 and APA1304 regulate production of ROS in their candidate pathway would be the topic of further studies.