Omic Analysis And Key Genes Research Of Self-incompatibility In Camellia Oleifera

Oil-tea tree(Camellia oleifera)is the most valuable forest species native to China.It is important in the forestry industry and ecological construction,and the seeds produce a useful cooking oil.The flowering rate of C.oleifera is high but the fruit set rate is low(< 5%)due to self-incompatibility(SI).The self-pollinated pollen tube in C.oleifera is inhibited in the upper ovary,at the base of the style,with the characteristics of programmed cell death(PCD).This confirms that C.oleifera is a late-acting self-incompatibility(LSI)plant type.The molecular mechanism of SI in C.oleifera is unclear.We conducted different pollination combinations with cultivars‘Hua Xin' and ‘Hua Jin' as the female and male parents,respectively.And we conducted physiological factor analysis of the pistils after self-pollination(SP)and cross-pollination(CP),screening of SI-related genes and metabolic pathways,and cloning and functional analysis of key genes.The resulting data help to resolve the physiological and molecular regulation of SI in C.oleifera.The main findings were as follows.1.Physiological and biochemical characteristics of self-and cross-pollinated pistils in Camellia oleifera.We used paraffin sectioning and squashing to observe that,after CP,the pollen tubes growed in the style,entered the ovary from the style after 48 h,reached the o vule after 72 h,and then entered the embryo sac through the micropyle.The growth rate of the self-pollinated pollen tube slowed after 36 h and self-incompatible reactions,such as swelling and folding of the pollen tube,appeared at 48 h.Between 48 and 72 h in SP,a small number of pollen tubes entered the upper ovary at the base of the style,but there were no pollen tubes in the ovule.This indicated that PCD of the self-pollinated pollen tube in C.oleifera occurred from 36 h to 72 h.Several physiological and biochemical indexes of the self-and cross-pollinated pistils were measured.Compared to CP,proline,plant reactive oxygen species(ROS),calmodulin(Ca M)and three protective enzymes that initially increased and then fell,strongly responded to the pollen tube PCD reaction from 36 to 60 h in SP.Mitogen-activated protein kinase(MAPK)and E3 ubiquitin ligase showed similar trends in SP and CP,but the activity of these two enzymes in SP was significantly higher than in CP at 36 h after pollination.This indicated that they were also involved in the SI response of C.oleifera.After pollination,ABA content was considerably higher in SP compared with CP from 2 to 96 h.ABA may inhibit the growth of pollen tubes.Additionally,there was a low(IAA + GA3 + ZR)/ ABA ratio during SP in C.oleifera.The content of ACCS and JA in SP was significantly higher than in CP,with nonobvious change trend in CP from 2 to 48 h.These physiological and biochemical indexes changed regularly during SI and they may be involved in the PCD of the self-pollinated pollen tube or involved in the self-defense responses caused by SI.2.Multi-omics and related metabolic pathway analysis of Camellia oleifera self-incompatibility.Transcriptome,proteome,and metabolome techniques were used to study the differences in the expression of genes,proteins,and metabolites in the pistil after SP and CP in C.oleifera.A total of 166,591 transcripts were identified in the database by third generation sequencing technology,with a 94.16% annotation rate.Using the transcripts as the reference sequence for second generation Illumina-seq,the average mapping rate was 81.82%,and 6851 proteins and 6455 metabolites were identified from the proteome and metabolome data.A total of 1197 differentially expressed transcripts(DETs),229 differentially abundant proteins(DAPs),and 44 differentially abundant metabolites(DAMs)were identified in the self-and cross-pollinated pistils at 48 and 72 h in SP and CP.KEGG functional enrichment analysis divided the DETs into 19 categories,including carbohydrate metabolism,amino acid metabolism,membrane transport,signal transduction,and other metabolic pathways.The DETs involved in the plant hormone signal transduction were mainly concentrated in the signal transduction pathway using JA and ethylene(ETH)as signal molecules.Analysis of the expression patterns of 18 reported PCD-related genes showed that most positively regulated genes showed relatively high expression levels in SP,and negatively regulated genes showed low expression levels in SP.We performed trend analysis and Pearson correlation analysis on DETs,DAPs,and DAMs and obtained190 transcripts,226 proteins,and 44 metabolites that responded strongly to SP and CP.These were involved in the MAPK signaling pathway,plant hormone signal transduction,ABC transport,and ubiquitin-mediated proteolysis.Multi-omics technology data provided support for analyzing the molecular mechanism of SI in C.oleifera,and the screened transcripts,proteins,and metabolites provided basic data for determining the regulatory network of the PCD-mediated SI molecular mechanism.3.Identification and functional study of key genes involved in the self-incompatibility of Camellia oleifera We identified and verified the function of Co PRF5,a gene related to the polar growth of the oil-tea pollen tube.Profilin(PRF)protein is involved in the regulation of the actin cytoskeleton.Proteome analysis showed that,at 72 h,PRF had higher levels in CP than that in SP.Five members in the PRF gene family were identified in the full-length transcriptome of C.oleifera.The full length of CDS was 396 bp,encoding132 amino acids.The members were all located in the cytoplasm.Phylogenetic tree analysis showed that Co PRF5 was clustered on the same branch with At PRF4 and At PRF5,which are involved in the polar growth of pollen tubes in Arabidopsis.Co PRF5 had considerably higher expression in pollen than other tissues.The in vitro pollen growth experiment showed that the length of the pollen tube was positively correlated with the expression of Co PRF5.Co PRF5 may participate in the polar growth of the oil-tea pollen tube.The S gene of C.oleifera was identified and its function was verified.A candidate S-RNase gene was selected from the transcriptome of C.oleifera.The CDS sequence of Co S-RNase was 717 bp and it encoded 239 amino acids.Sequence analysis showed that Co S-RNase had a histidine(His)site for degrading RNA and a proline(Pro)site for depolymerizing actin.This was consistent with the S-RNase structure in apples and pears.Co S-RNase was located in the cytoplasm,with the highest expression in the style,but no expression in pollen.The expression level of Co S-RNase in SP reached a peak at 24 h in the style and at 36 h in the ovary.It was significantly higher than in CP,and was strongly responding to the SI reaction of C.oleifera.The RNase activity determination experiment showed that the recombinant protein Co S-RNase had RNase activity.When added to the pollen culture the recombinant protein Co S-RNase significantly inhibited the growth of pollen tubes and the expression of the polar growth-related gene Co PRF5,and caused depolymerization of the actin cytoskeleton of the pollen tube.These data indicate that Co S-RNase is a signaling molecule involved in the death of self-pollinated pollen tubes in C.oleifera.Identification and functional verification of Co POD,the SI defense gene of C.oleifera,was also completed.Co POD was identified in the pistil of C.oleifera,with a full CDS of 1,218 bp encoding 406 amino acids.Co POD was located in chloroplasts.It had its highest expression in pollen,but no expression in the unpollinated style and ovary.Experiments on yeast two-hybrid,bimolecular fluorescence complementation and Pull-down revealed an interaction between Co POD and Co S-RNase.The expression of Co POD in the style and ovary after SP strongly responded to Co S-RNase,indicating that the expression of Co POD may be caused by a pollen tube self-protection system triggered by S-RNase attack.The recombinant protein Co POD promoted the elongation of pollen tubes and the expression of the polar growth-related gene Co PRF5 and reduced the degree of depolymerization of the actin cytoskeleton in the pollen tube by Co S-RNase.The expression of Co POD is a self-defense response mechanism of self-pollination pollen tubes in C.oleifera.This may be the reason that the self-pollinated pollen tubes in C.oleifera stopped growing at the base of the style,unlike GSI-type plants that stopped growing at the at one-third the length of the style.