The Role Of Lignin Metabolizing Enzymes In The Synthesis Of Pear Stone Cells

Stone cell is one of the important factors affecting the quality of pear fruit,and lignin is a major component of pear stone cells,both the genetic characteristics of varieties and external environment or cultivation of technical measures can affect the lignin formation.In pome fruit production,not only can fruit bagging avoid pollution from external harmful substances and improve quality,but also can affect the formation of stone cells.The previous studies on the effects of bagging focused on fruit quality,pest control,stone cell development and related enzyme activities changes of pear fruit,bagging affected the formation of stone cells still can not forme a clear a consensus.In this study,79 pear varieties were used as test materials,determinations of the stone cells content in the pulp and lignin content in stone cells were carried out firstly,then 'Dangshansuli' were used as test materials,we centred the study around stone cells and lignin,studied the regular pattern of stone cells and lignin content,related enzyme activities and gene expression under the bagging conditions during the development of pear fruit,PAL(phenylalanine ammonia lyase),C4H(cinnamate-4-hydroxylase),C3 'H(p-coumaroyl-shikimate/quinate 3'-hydroxylases),HCT(hydroxycinnamoyl transferase),CCoAOMT(caffeoyl CoA-O-methyl-transferase),POD(peroxidase)genes were cloned and nalaysed,and C4H,POD,CCoAOMT subcellular localization analysis were conducted.The results are as follows:1.By the combination of freeze separation and hydrochloric acid treatment method,the contents of stone cells in 78 pear cultivars were determinated separately.Results showed that most stone cell content ranged from 0.05%to 0.5%,only a few stone cell contents were extremely low or concentrated in more than 0.8%;lignin contents accounted for 26-38%of stone cell composition.2.15-year-old 'Dangshansuli' were used in this study,the effects of fruit bagging on fruit fresh weight,stone cell content,lignin content and lignin metabolism related enzyme activities were analyzed at different stages during fruit development.The results showed that:with the growth of fruit,fruit fresh weight was an "S" curve dynamic growth and fresh weight of bagging was slightly less than the control at mature.Stone cells contents and lignin contents in fruit showed a decreasing trend after the rising firstly during fruit development,and the bagging were higher than the control.The lignin contents in stone cells by bagging increased gradually with the growth of fruit,but always were higher than the control.PAL and CAD activities were subjected to different degrees of inhibition by bagging,but G-POD and S-POD activities appeared a rising phenomenon,there was a similar trend between POD ? diversification and lignin content in stone cells.Correlation analysis showed that the lignin contents in stone cells were significantly related with fruit fresh weight and POD ? activity,initially suggested that POD ? plays a very important role in the metabolism of lignin in 'Dangshansuli'.3.In order to study the expression patten of lignin metabolism-related genes,'Dangshansuli' were used as test materials,combined with changes of lignin content,the effects of fruit bagging on real-time quantitative expressions of lignin metabolism-related genes at different developmental stages of 'Dangshansuli' were analyzed.The results showed that:during the stone cell and lignin synthesis productive period(within 46 d after full bloom),gene expression patterns varied with different genes,different gene expression patterns of the same genge family were also different.PAL,C4H,4CL1,4CL2,CAD1,HCT3,,CCoAOMT,POD1 and POD2 had higher expression levels during the lignin synthesis productive period;CAD2,HCT2 and POD4 have higher expression levels when the fruit approaching maturity;and HCT1 and HCT4 appeared the lower expression levels during the peak period of lignin synthesis;POD3 had a higher expression in the early period of lignin synthesis and was almost no expression at 46 d after flowering.Bagging treatments can inhibite or promote the expression patterns of different gene at different stages.PAL,C4H,4CL2,C3'H,HCT3,CCoAOMT,POD1,POD3 can be better to reflect the variation of lignin in pulp,and CAD,HCT1,HCT4,POD2,POD4 are similar with the variation of lignin in stone cells.Thus,they may play different roles in lignin metabolism.4.PAL(KJ577540),C4H(KJ577541),C3'H(KJ577546),HCT(KJ577545),CCoAOMT(KJ577544),POD1(KJ577542)and POD2(KJ577543)genes were cloned and sequenced using RT-PCR from 'Dangshansuli'.In nucleotide sequence,PAL,C4H,HCT,CCoAOMT,POD1 and POD2 encoded respective amino acid sequence which comprised of 720,504,438,247,325 and 336 residues respectively,and they were closely related with the Rosaceae by phylogenetic analysis.C3'H encoded an amino acid sequence which comprised of 512 residues,phylogenetic analysis analysis revealed that there was a father genetic relationship between it and Populus tomentosa.Further,the rest of their stuuctures were predicted and analyzed through the software.5.C4H,POD,CCoAOMT genes functions in pear were further researched by using'Dangshansuli' as material.C4H ORF,POD ORF with restriction sites respectively were amplified by PCR,pCAMBIA1302-C4H-GFP and pCAMBIA1302-POD-GFP were constructed by fusing C4H,POD and GFP respectively;CCoAOMT ORF with restriction sites was amplified by PCR,pBI121-CCoAOMT-GFP was constructed.Subcellular location analysis using the expression vectors of C4H,POD and CCoAOMT constructed showed that C4H was located in the cell membrane and had membrane localization function,POD and CCoAOMT were both located in nucleus and had nuclear localization functions.