Characteristics Of Carbohydrate Unloading In Kiwifruit(Actinidia Deliciosa)

Photosynthates unloading in phloem have an important regulatory role in sugar distribution and accumulation. Sugar unloading, metabolism and accumulation, are great significant for sink-source regulation and quality improvement. To ascertain the mechanism of carbohydrates unloading in kiwifruit, we investigated the carbohydrates unloading pathway in kiwifruit(Actinidia deliciosa) and possible regulated genes involved in unloading at physiological, biochemical, cellular and molecular level. Besides, we transformed a hexose transporter(a key gene involved in the process of sucrose apoplastic unloading in apple fruit) into Actinidia deliciosavia the method of agrobacterium mediated transformation. The results are as follows:1. To know the sugar unloading pathway in kiwifruit, we detected components of phloem unloading exudations, observated patterns of vascular bundles plasmodesmata in fruit and the distribution of fluorescence trancer signal. We found that the dominant carbohydrate in the exudations of leaf stalk and fruit stalk phloem was sucrose, but no sorbitol, stachyose and other compounds for long distance transportation. It meaned sucrose was the carbohydrate involved in the long distance transportation in kiwi. By the way, vascular bundles ultrathin section technique and structural analysis by electron microscope showed that there was no plasmodesma around the interface between the sieve element-companion cell complex and surrounding parenchyma cells during the whole fruit development precess. Meanwhile, numerous plasmodesmata were found around the interface between parenchyma cells and pulp cells. Additionally, we used carboxyl fluorescein, which is a symplastic free diffusion tracer, to instruct the unloading path of sucrose in kiwifruit, and the result showed that the dye was always confined to vascular bundles throughout the fruit development without diffusion into surrounding parenchyma cells. These results suggest that phloem unloading pathway of sucrose in kiwifruit(Actinidia deliciosa) is apoplastic pathway rather than symplastic pathway.2. To explore the mechanism of sugar accumulation and catabolism during the post-phloem unloading transport in kiwifruit, we studied the key enzymes and key genes expression, which participated in the sugar transportation after phloem unloading and sugar metabolism.The results showed that sucrose concentration had a significant decline before the secondary rapid expansion of kiwifruit(Actinidia deliciosa). Along with the maturity of the fruit, sucrose concentration was increased trends. However, small variations of glucose and fructose concentrations were observed during the growth of fruit. In the process of postripeness after harvest, sucrose, glucose and fructose accumulated fast in the process of softening. After sucrose unloading in the phloem, key enzymes during the process of sucrose decomposition include sucrose synthase(SUSY)、acid cell wall invertase(CWINV)、neutral invertase(NINV)、vacuolar acid invertase(vAINV). Enzyme activity analysis showed that SUSY in kiwifruit behaved higher enzyme activities than CWINV、NINV and vAINV. Meanwhile, we detected gene expression of six sugar transporters(SUTs) and found that SUT1, SUT3 expressed highly in fruit. Besides, we detected only little expression of CWINV in fruit. However, SUSY1 had high expression in fruit. The results may suggest that CWINV is not the key gene in the process of sucrose unloading. Sucrose in the apoplatic space may be transported directly into the parenchyma cells and stroed or metabolized. Apoplastic sucrose from phloem unloading may be transported into parenchyma cells by SUTs, and hydrolysised by SUSY. It means that SUSY is the key enzyme in the process of sucrose post-phloem unloading transport.3. We constructed an over-expression vector of MdHT2.2, which is a hexose transporter involved insucrose apoplastic unloading in apple fruit, and then transformed it into Actinidia deliciosavia the method of agrobacterium mediated infection. After detecting the antagonistic strains with RT-PCR, we obtained the transformation strains withMdHT2.2 over-expression. We test the transgenic somaclone materials and found that there is no significant difference between kiwi of MdHT2.2 over-expressed transformed plants and wide type on the sugar concentration. However, transgenic plants could not take roots in the medium of 1/2 MS while the indolebutyric acid(IBA) concentration is 0.1 mg.L-1. When IBA concentration increased to 0.5 mg.L-1, transgenic somaclone kiwi can take roots. It suggests that sugar signals mediated by MdHT2.2 are associated with auxin signals.