Role Of Metacaspase In Formation Of Pear Stone Cell

Stone cell is a kind of cell unique in pear(Pyrus)fruit,which causes rough flesh and less juice,and is one of the important factors affecting the taste and processing quality of pear fruit.’Dangshan Su’ Pear is a characteristic traditional variety cultivated in China.Due to poor management and genetic characteristics in recent years,stone cell is particularly serious.Program cell death(PCD)and Secondery cell wall(SCW)thickening of parenchyma cells are accompanied by cellulose and lignin deposition to form stone cells.Metacaspase(MC)is a multifunctional protein that involved in PCD regulation,cell cycle and senescence,and oxidative stress.The bioinformatics analysis of the MC family in the Pyrus bretschneideri genome was carried out by protein sequence alignment and collinearity analysis.The key gene PbMC1a/1b was screened by combining physiological indicators,tissue-specific expression and real-time fluorescent quantitative PCR analysis.PbMC1a/1b was then stably transformed into Arabidopsis thaliana and transiently infested pear leaves to verify the relationship with lignin and stone cell formation.The interaction protein PbRD21 was screened by yeast two-hybrid and bimolecular fluorescence complementation experiments.Finally,bioinformatics analysis was carried out on the Rocaceae NAC gene family.Genes related to lignin synthesis and regulation of PCD were screened with fruit development transcriptome data.Sequence analyses of the PbMC1a/1b and PbRD21 promoters were used to screen the PbNAC gene that may regulate PbMC1a/1b and PbRD21.The main results of the study are as follows:1.A total of 11 PbMC genes were identified by the analysis and screening of the pear genome.Eight pear PbMC genes were mapped on five chromosomes,and three genes were mapped on the scaffold.According to the analyses of gene structure,protein motifs and phylogenetic development topology,the PbMC family was divided into two subfamilies.The PbMC genes within each subfamily had similar exon and intron structures.Each subfamily PbMC protein had conserved motifs.The pear and Arabidopsis MC protein structures contained the P10 and P20 subunits unique in the MC family.Genome-wide/segmental replication plays a key role in the expansion of the PbMC family,and purification selection was a major force to drive the evolution of the PbMC family of genes.PbMC1a/1b had a high expression level in early fruit development and was consistent with the trend of stone cells and lignin by analyses of lignin and stone cell contents,tissue-specific expression and qRT-PCR analysis of PbMC gene expression in pear fruit development.2.The full-length ORF of PbMC1a/1b was amplified by PCR and constructed into pCAMBIA1300 and p-TRV2 vectors.The PbMC 1a/1b protein was localized in the cytoplasm and nucleus by transient transfer into epidermal cells of tobacco leaves.Relative to wild-type Arabidopsis,overexpression of PbMC1a/1b resulted in plant dwarfing,the cell wall of vessels,xylary fibers and interfascicular fibers thickening,lignin content increasing,and the expression of lignin biosynthetic genes increasing.Yeast two-hybrid and X-α-Gal experiments indicated that the PbMC1a/1b protein physically interacts with PbRD21 and bimolecular fluorescence complementation showed their interactions were in the cytoplasm and nucleus.Simultaneously,transient expression of PbMC1a/1b and PbRD21 led to significant changes in lignin content in pear leaves and expression of lignin biosynthetic genes.Taken together,these results indicate that pear PbMC1a/1b plays an important role in cell lignification and lignin deposition,possibly by interacting with PbRD21 to enhance mRNA levels of some lignin synthesis-associated genes.3.A total of 171 NAC genes in apple(Malus domestica),114 in peach(Prunus persica),1 13 in plum(Prunus mume),127 in strawberry(Fragaria vesca)and 183 in pear(Pyrus bretschneideri)by analyzing and screening genomes.Based on gene structure,protein motif analysis,and topology of the phylogenetic tree,the NAC family was classified into 33 groups.By comparing and analyzing the unique NAC subgroups in Rosaceae,we identified 19 NAC subgroups specific to pear.Among them,146 PbNAC genes were located on 16 chromosomes,and 37 PbNAC genes were located on scaffold contigs.We also found that WGD/segmental duplication played critical roles in the expansion of the NAC family in pear,such as the 83 PbNAC duplicated gene pairs dated back to the two WGD events.Further,we found that purifying selection was the primary force driving the evolution of PbNAC family genes.Transcriptome data in fruit development combined with functional analysis of homologous genes revealed that four PbNAC genes were involved in phenylpropanoid biosynthesis and regulation of PCD.Analyses of promoter sequences of PbMC1a/1b and PbRD21 showed that these two genes had NAC specific recognition motifs.These indicated that these four PbNAC genes had the potential to regulate these two genes.