Phloem Unloading Pathway In Walnut Fruit

The pathway of phloem unloading was studied in developing walnut (Juglans regia L.) fruit using a combination of tissue slice, carboxyfluorescein transport, immunogold electron microscopy, and immunoblotting assay. The results showed that the tissue structure of the fruit pericarp may be divided into three parts: exocarp, mesocarp and endocarp. The developmental stage can be divided into the early stage and late stage. The mesocarp was the main part of pericarp, and the cell of endocarp was small and transparent in early stage, and it became into hard shell that was made of lignification stone cells. The tissue of vascular bundles was well developed and became ereticulate vein. There were no found vascular bundles in the endocarp, so it was a natural layer of isolation in walnut fruit.The ultrastructure of phloem and its surrounding parenchyma cells in developing walnut fruit was for the first systematically investigated via transmission electron microscope during whole developmental process of the fruit. The results showed that plasmodesmata exist at the thickened part of cell wall between sieve elements (SEs) and companion cells (CCs) both in pericarp of fruit and pericarp of seed, with the plasmodesmata furcating at the CC side and keeping a single oriface at the SE side .In pericarp of fruit the SEs are larger than the CCs, while in pericarp of seed the SEs are smaller than the CCs. The CCs contain an electron-dense protoplasm and nucleus, and are enriched with mitochondrion, endoplasmic reticulum and Golgi apparatus or dictyosomes in their cytoplasm. There was almost no plasmodesmata between the SE-CC complex and its surrounding phloem parenchyma cells in the pericarp of fruit, but it was opposite in the pericarp of seed, resulting in the symplasmic isolation between them in pericarp of fruit, which may be associated to apoplasmic transportation, of course there were symplasmic links between them in pericarp of seed. Plasmodesmata were found numerous both between phloem parenchyma cells and between flesh parenchyma cells (non-phloem parenchyma cells), as wells as between phloem parenchyma cell and flesh parenchyma cell. The relationships between the ultrastructure of the fruit phloem and its surrounding parenchyma cells and the photoassimilate unloading pathway in developing walnut fruit were discussed. It was considered that the apoplasmic unloading may be the main pathway of the photoassimilates unloaded from SE-CC complex of pericarp of fruit, and that the apoplasmic and symplasmic pathways may coexist during postphloem transport of photoassimilates in developing walnut fruit.The phloem-translocated carboxyfluorescein remained confined to the phloem in the pericarp of fruit and oppositional, it diffused into the surrounding parenchyma tissues in the pericarp of seed after application on the treated petiole during the fruit developing. This provided a supporting proof for assays of ultrastructure. The immunoblotting of acid invertase detected a 60-kD polypeptide for acidinvertase, and moreover, the apparent amount of acid invertase also increased at the early developmental stage (30DAFB) and decreased at the late developmental stage (80DAFB). The immunogold electron-microscopy technique was also used to determine the subcellular localization of cell wall invertase in developing walnut fruit. The antibody against apple fruit acid invertase was used. The results showed that the cell wall invertase mainly in the cell wall and the two antibodies can recognize each other. The gold particles representing acid invertase were found to reside predominantly in the cell walls of sieve element / companion cell (SE-CC) complex, phloem parenchyma cells and other parenchyma cells. There were almost no gold particles at other subcellular compartments (such as protoplasm and vacuole). The density of immunogold particles was low at the early developmental stage (10DAFB), but obviously high at the after developmental stage (30DAFB) and lately, was low at the late developmental stage (80DAFB). The distribution...