Mechenism Researches On The Formation Of Stone Cell, Russet Skin And Citric Acid Of Pear (Pyrus Pyrifolia Nakai) Fruit

The present work focuses on fruit quality of pear(Pyrus Pyrifolia Nakai) which is cultivated at the drainage of Yangtze River and the southern part of China. Aiming on revealing the mechanisms of stone cell formation, russet skin formation and citric acid accumulation of pear fruit, much work was performed in physiological, molecular and transgenic levels. Overall, this research provides the opportunity for further understanding of pear fruit quality and contributes to the breeding of pear. The main results in the present work are listed below:1. Research on stone cells of pear fruit(1) Two pear cultivars,’Guihua’and’Cuilu’which were with large difference in stone cell content, were chosen in this study. Throughout fruit development, stone cell and lignin contents increased firstly, reached the peak at 56 days after full bloom (DAFB) and then decreased in’Guihua’and’Cuilu’fruits. From 98 DAFB to fruit ripening, stone cell and lignin contents kept the relative low level in both cultivars. At the main stage of stone cells formation (from 42 to 70 DAFB), stone cell and lignin contents were much higher in’Guihua’than in’Cuilu’fruits and the gaps reached more than two times at 56 DAFB. Therefore, the ability of stone cells formation and lignin biosynthesis must be stronger in’Guihua’than in’Cuilu’fruits.(2) For further study on stone cells formation, PpCCR gene which was involved in lignin biosynthesis was isolated from pear fruit. Sequence analysis indicated it contained an open reading frame (ORF) of 1020 bp and encoded 339 amino acids with a molecular weight of 37.2kDa. Alignment of CCR proteins suggested that PpCCR shared the highly identity with other plants’CCR proteins. In addition, PpCCR contained the conserved NADP binding site and the CCR enzyme catalytic site. Phylogenetic tree analysis revealed that the plant CCR proteins could be divided into dicotyledonous plant group and monocotyledonous plant group. PpCCR belonged to dicotyledonous plant group and showed closed relationship with woody pants, such as poplar, eucalyptus and birch. Expression of PpCCR showed the similar profile with stone cell and lignin contents in ’Guihua’and’Cuilu’fruit. In addition, PpCCR exhibited the higher expression level in ’Guihua’than in’Cuilu’during stone cells formation, suggesting that there were obvious relationship between PpCCR expression, lignin biosynthesis and stone cells formation.In addition, PpCCRx and PpCCRx2 genes were found when PpCCR was identified. Comparing to PpCCR, a 282bp fragment, which was inserted into PpCCR sequence and named x fragment, was the most significant difference in these two novel genes. Due to the insertion of x fragment, both PpCCRx and PpCCRx2 genes were with 156bp ORF and this ORF could not encode a complete CCR gene. Sequence analysis for PpCCR, PpCCRx and PpCCRx2 in DNA and RNA level indicated that they were from different transcript.(3) For analyzing the functions of PpCCR and PpCCRx genes, they were introduced into wild-type Arabidopsis respectively via agrobacterium-mediated transformation. The result showed that overexpression of PpCCR effected the growth of wild-type Arabidopsis:dwarfed plant, increase of stem diameter and aggravated lignification of xylem. These results indicated that PpCCR was with the normal function in tissue lignification. However, overexpression of PpCCRx did not effect the lignification of wild-type Arabidopsis, indicating PpCCRx performed the abolished CCR function in tissue lignification. This result also suggested that x fragment disturbed the normal CCR function in PpCCRx and PpCCRx2 genes.(4) In this study, promoter sequence of PpCCR was isolated from pear genomic DNA. Through aligning the sequence in NCBI database, there was no plant nucleotide sequence showing the homology with PpCCR promoter, suggesting it was a novel plant promoter with unknown characteristics. In the present work, characteristic of PpCCR promoter was analyzed through PpCCR promoter driving GUS gene in Arabidopsis. GUS histochemical staining showed that the blue signals were mainly detected in the vessels, lignified tissues and the xylem of stem, while the signal could not be detected in wild-type Arabidopsis. These results indicated that PpCCR promoter was with stronger activity in lignified tissues.Due to the regulation of transcript factor onto promoter, the potential WRKY transcript factors which regulated PpCCR expression were examined. Through the expression analysis on eight WRKY members, WRKY2 showed the closed relationship with PpCCR in transcript level. In addition, both WRKY2 and PpCCR expression profiles were in accordance with the change of stone cell content. Therefore, WRKY2 transcript factor might regulate PpCCR expression directly or indirectly, and then regulate cells lignification and stone cells formation.(5) To understand the roles of Ca and B in stone cells formation, they were studied by spraying on’Syounan’pear respectively. The results indicated that both Ca and B did not affect the stone cell content in’Syounan’fruit. Further analysis revealed that Ca spraying did not affect its content in skin, flesh and stone cells of’Syounan’fruit. However, relative high Ca content in stone cells suggested that it played important role in stone cells formation. On the other hand, B spraying increased its content in skin and flesh but not in stone cells, suggesting that B was not necessary for stone cells formation.2. Research on russet skin of pear fruit(1) Russet skin was studied in the present work by using’GreenSyounan’which was a green skin mutant of’Syounan’pear.’GreenSyounan’showed the stable green skin trait in continuous observation. Quantified analysis for skin color of fruit indicated that ’GreenSyounan’exhibited the significant difference comparing to’Syounan’. Furthermore,’GreenSyounan’fruit accumulated much more sugar and acid than ’Syounan’in ripe fruit.(2) Transcriptome analysis in both’Syounan’and’GreenSyounan’skins was performed using RNA sequencing (RNA-Seq). This analysis revealed some different expression genes containing 154 up-regulated expression genes and 85 down-regulated expression genes. Pathway cluster analysis for these different expression genes suggested that flavonoid, phenylpropanoid and fatty acid biosynthesis pathways were up-regulated dramatically in’GreenSyounan’skin. Up-regulation of fatty acid biosynthesis implied that the wax on fruit surface should be paid more attention. Scanning electric microscopy (SEM) observation confirmed the wax layer exist on’GreenSyounan’fruit surface but not on’Syounan’. Therefore, the surface wax protected the epidermal cells of ’GreenSyounan’fruit, so that the fruit appeared the green color of chlorophyll. Up-regulation of flavonoid and phenylpropanoid biosynthesis pathways might result from the green trait of’GreenSyounan’because it had the stronger abilities in photosynthesis and carbohydrate flux comparing to’Syounan’.3. Research on citric acid metabolism in pear fruit(1)’Yandangxueli’was a pear cultivar with more citric acid than malic acid in ripe fruit, unlike’Gengtouqing’which represented most of the pear cultivars in organic acid composition. Acidity analysis indicated titrable acid in both cultivars increased at early development stage and then decreased until fruit ripening. However, titrable acid content in’Yandangxueli’was always higher than that in’Gengtouqing’from 42 DAFB to fruit ripening. Organic acid composition analysis indicated that citric acid accumulated at 70-154 DAFB in both cultivars, while malic acid accumulated at 42-112 DAFB. Because of the dramatic citric acid accumulation in’Yandangxueli’at later fruit development stage but not in’Gengtouqing’, the former was with the predominant citric acid while the later was with malic acid in ripening fruit.(2) To clarify the roles of organic acid metabolism-related enzymes in citric acid accumulation in’Yandangxueli’, five genes, Pp:mtCs, Pp:cyAco, Pp:cyIdh, Pp:mtMdh and Pp:cyMe encoding CS, Cytoplasmic ACO, NADP-IDH, NAD-MDH and NADP-ME respectively, were identified. Their expression levels and corresponding enzyme activities were examined in both’Yandangxueli’and’Gengtouqing’. The results suggested that there were no necessary relationship between the five enzymes and citric acid content. Expression profiles of Pp:vVAtpl, Pp:vVAtp2 (encoding V-ATPase) and Pp:vVpp (encoding V-VPPase) were also studied in both cultivars. Up-regulated expression of these three genes at latter fruit development stage in’Yandangxueli’but down-regulated expression in’Gengtouqing’might be inbolved in the predominant citric acid in’Yandangxueli’fruit.