Evidence For Apoplasmic Phloem Unloading Pathway In Apple Fruit

The pathway of phloem unloading was studied in developing apple fruit (Malus domestica Borhk cv. Golden Delicious) using a combination of carboxyfiuorescein transport, immunogold electron microscopy, and immunoblotting assay. The results showed that phloem-translocated carboxyfiuorescein remained confined to the phloem of apple 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 main and minor veins of 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.The hexose transporter was localized at subcellular level via immunogold electron microscopy in the phloem unloading zone of developing apple fruit. The gold particles representing hexose transporter were found to reside predominantly in the plasma membrane of sieve element / companion cell (SE/CC) complex, phloem parenchyma cells and other parenchyma cells. There was almost no gold particles at other subcellular compartments ( such as protoplasm and vacuole). The distribution pattern remained unchanged throughout the growing season, but the protein numbers varied. The density of immunogold particles located in the plasma membrane of sieve element was low at the early developmental stage, but obviously high at the middle and late developmental stage. There was no distinct difference on gold particles density in both the middle and late developmental stage, in which the gold particles were also located at the edge of sieve plate especially sieve pore. Furthermore, there were gold particles located on the plasmadesmata between parenchyma cells or two sieve elements. These results suggested that the hexrose transporter took part in the process of sugar unloading from the sieve element in apple fruit, and facilitated the assimilates transport into flesh cells. The immunoblotting of hexose transporter detected a 52-kD polypeptide for hexose transporter, and moreover, the apparent amount of hexose transporter also increased with fruit development. This provided a supporting proof for assays of immunolocalization. Furthermore, The protein amount in treated apple fruit with 500 u mol/L ABA is higher than in the control experiment. So, ABA promoted expression of the 52-kD hexose transporter at the translational level.The immunogold electron-microscopy technique was also used to determine the subcellularlocalization of vacuole acid invertase and cell wall acid invertase in developing apple fruit. The antibody against apple fruit acid invertase was used. The results showed that the vacuole acid invertase were mainly located in the vacuole and cell wall invertase mainly at the cell wall. The two antibodies can recognize each other.These results above provide strong evidence for the apoplasmic unloading pathway of sugar in apple fruit, and that the unloaded sucrose can be hydrolysed by the functional acid invertase localized at the cell wall before it is taken up into the sink cells by the hexose transporter or via plasmodesmata.