Study On Characteristic Of Transport And Accumulation Of Sugar In Developing Pyrus Ssp. Fruit

Distribution and accumulation of assimilates in the fruit is the most important factor to determine the fruit quality. The accumulation of sugar is not just the result of interaction of the enzyme, but also have a very close relationship with the beginning of sugar accumulation, cellular pathways of phloem transport, sugar transporter interactions and the main regulator of sugar transport and its regulation mechanism, and many other factors. Photosynthate assimilates from source leaves transport through the phloem by sorbitol and/or sucrose form into the fruit to be stored and used, and the unloading of assimilates from phloem sieve elements to sink cells is an important part of the throughout process of assimilation transport. In comparison, the knowledge about the mechanism of sugar unloading and accumulation during the development of pear fruit is very limited at home and abroad.Chinese traditional late-maturing, green pear varieties ’Yali’(Pyrus bretschneideri Rehd. cv.’Yali’) and precocious, beige sand pear cultivars ’Aikansui’ (Pyrus pyrifolia Nakai. cv.’Aikansui’) from Japan were used for the materials in this paper, and the changes of soluble sugar and metabolism enzyme and their relationship, the vascular bundles in fruit phloem cells were isolated or linked, the cloning and expression of fruit sugar transporter gene, the ultracytochemical localization of ATPase and other content during the development of pear fruit were studied from cellular, physiological and molecular level respectively, the results will help to lay a theoretical basis for promoting fruit sugar accumulation and transformation and for improving the quality of pear fruit. Main results as follows:1. Components and soluble sugar content was determined by high-performance liquid chromatography (HPLC) techniques during the fruit development of cv. Yali and cv.Aikansui. The results showed that:fructose and sucrose are the most main soluble sugar in the cv. Aikansui mature fruit, about 70% of total soluble sugars, glucose and sorbitol content minimum; fructose and sorbitol are the most main soluble sugar in the cv. Yali mature fruit, accounting for about 70% of total soluble sugar and more, followed by glucose and sucrose. Both in the fruit development process, the total soluble sugar content did not differ significantly, but sucrose content have significant differences, the sucrose content of cv. Aikansui mature fruit is about four times of the cv. Yali mature fruit. The analysis revealed that the taste value of soluble sugar in the matured fruit has correlation with the proportion of sugar in the total soluble sugars. Regardless of the type of high-sucrose or low-sucrose varieties, the taste value of fructose is the maximum and is the main sugar to determine the final quality; and the taste value of sucrose change in different types of fruit due to its content, mainly affecting the flavor of the fruit sugar.2. The activity of sucrose metabolizing enzymes were measured during the fruit development of cv. Yali and cv. Aikansui, the results showed that:whether Yali or Aikansui from the cell division until the fruit before rapid enlargement, the activity of cleavage enzymes higher than that of synthesis enzymes, so the cleavage enzymes are dominant; after fruit rapid enlargement, sucrose synthase (SS) and sucrose phosphate synthase (SPS) activity gradually began to sync up to the ripe fruit close to the sharp rise to maximum value; on the contrary, the activity of SS appeared as first and then decreased in pear fruit after the rapid enlargement, while the SPS activity was gradually increased to the highest until the mature fruit, the activity of enzymes VIN, NIN, SD and CIN in the fruit close to the mature sharply decreased. Overall, in the Aikansui fruit SPS activity trends are similar with the SS during the fruit development, however in the Yali fruit SS has been rising in the early, premature 30-40 d to reach the highest, and then with the fruit development decreased gradually to mature at a lower level, SPS activity was very low activity in early development, after cells begin to expand its activity has been rising sharply approaching maturity.3. The distribution of vascular bundles of pear fruit were observed by the using of red ink markers and biopsy technique, vascular tissue is the well developed and located in the reticular distribution within the parenchyma of fruit. Using transmission electron microscopy, the ultrastructural changes of the fruit phloem cells during the development is obvious. Large pre-cavity molecular sieve, sieve elements gamut and more around the residual protoplasm, cell cytoplasm with dense, rich in a variety of organelles, cell gap, tough between sieve elements and companion cell plasmodesmata density, and SE-CC complex and parenchyma cells between the phloem and parenchyma cells between the plasmodesmata density is very small, almost non-existent plasmodesmata, plasmodesmata in the sieve element side of the channel, In the companion cell side of the formation of multi-branch channel; with fruit development, phloem parenchyma cells have many vesicles, endoplasmic reticulum and Golgi apparatus, during the rapid cell enlargement and fruit maturity, sieve element happen to be black particulate matter clogging phenomenon, sieve elements embedded narrow, pearl wall thickening, the formation of pearl cells; sieve elements and companion cell and the surrounding parenchyma cells with thickened cell walls, plasmodesmata clearly is blocked. These characteristics mark the possibility of symplasm isolation between the sieve element-companion cell complex and phloem parenchyma cells, the role of assimilates transport are high impedance, and thus the pathway of transmembrane apoplastic unloading was formed.4. The pathway of phloem unloading was studied in developing pear fruit cv. Yali and cv. Aikansui using a combination of carboxyfluorescein (CF) transport. The result showed that phloem-tanslocated carboxyfluorescein remained confined to the phloem of pear fruit and not into the surrounding parenchyma tissues even 72 h after application on the treated petiole during the fruit developing. The results obtained from the different sample position and developmental stage were in agreement with the above point. This suggested the symplasmic isolation of SE-CC complex from the surrounding parenchyma cells and that the photoassimilate unloading from phloem must be facilitated via the sugar transporters.5. To obtain the full-length cDNA sequence of sucrose transporter protein and sorbitol transport protein genes, RT-PCR and RACE techniques were carried out using pairs of degenerate oligonucleotide primers designed on the basis of conservative amino acid sequence of different plant sucrose transporter protein and sorbitol transport protein genes.The specific primers based on the cloned sequences were designed and used to isolate the genes of sucrose transporter named PySUT1 and sorbitol tranporter named PySOT2. Through the structure and amino acid sequence analysis of the phylogenetic tree, indicating that these two genes is highly conserved with Apple MdSUT1 and MdSOT2 gene respectively in the structure and function, belong to the same subfamily. The expression analysis of the PySUTl genes and PySOT2 gene in pear fruit of cv. Yali and cv. Aikansui, found that PySUTl genes and PySOT2 genes are constitutively expressed during the fruit development, and the expression levels differences occur in the different periods of fruit development because of fruit soluble sugar and metabolic enzyme changes. Bioinformatics analysis shows that, PySUTl genes and PySOT2 gene encodes a protein with typical features of MFS members, in the middle of a hydrophilic molecule central cytoplasmic loop and the 12 transmembrane domains, are high-affinity transporter. Initially speculated that these two genes respectively encoding the normal proteins with sucrose transporter function and sorbitol transport function, and play an important role during the processes of sugar transmembrane transport in the sink cells. The specific function of the genes will require further experiments to prove.6. Subcellular localization of ATPase activity in the fresh of developing pear fruit was carried out using a lead phosphate precipitation techinique via electron-microscope. The ultrastructure of phloem and its surrounding cells was also observed. The results showed that ATPase activity was dominantly localized on the plasma membranes of sieve element-companion cell complex. The distribution of ATPase activity in the phloem parenchyma cells and flesh parenchyma cells changed during the different developmental phases of the fruit. It is particularly interestin to observe a band along the plasmalemma of flesh parenchyma cells adjacent to the phloem, the band highly enriched with the ATPase activity, forming a ring of encirclement that surrounds the phloem. The ATPase subcellular distribution pattern and ultrastructure of phloem provided a proof for the apoplasmic unloading pathway of photo-assimilates from SE-CC complex in the fresh of developing pear fruit.