Histology And Cytology Of Resin Ducts At Different Development Stages In Pinus Massoniana

The structure, distribution, initiation, development and the changes of histochemistry and ultrastructure of resin ducts were studied with the methods of paraffine section, thin section and ultra-thin section during development of resin ducts in Pinus massoniana. Cytochemical localization of pectinase and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase with transmission electron microscope (TEM) were employed to show the changes of these enzymes activity during development of resin ducts. The results are as follews:1 Structure, distribution and development of resin ductsThe resin ducts were relatively large intercellular spaces surrounded by a ring of parenchyma cells, called the epithelium. Surrounding the epithelium there were usually one or two layered sheath cells, which were parenchyma cells in some primary resin ducts and dead cells with thick walls in other primary resin ducts and secondary resin ducts. The resin ducts were present in all organs but absent in cytoledon in Pinus massoniana.The resin ducts were formed by schizogeny according to characteristics of cell morphology during development of resin ducts. During the resin duct formation a series of changes in structure take place and three different stages can be divided, namely initial stage, formative stage and mature stage, respectively. To further reveal essence of resin duct formation, the electron microscopic cytochemistry of enzyme was firstly employed to study changes of pectinase during development of cortical resin ducts. At the initial stage, the activity of pectinase was first appeared in the swollen cell wall corners among the future epithelial cells and later done along the walls. At the formative stage, the intercellular space, which formed by pectinase degrading middle lamella, first appeared in the corner among the forthcoming epithelial cells and then gradually enlarged. The activity of pectinase was gradually decreased with intercellular space expansion. At the mature stage, there was no activity of pectinase outside the walls of epithelial cells. These results indicated that pectinase degraded the middle lamella region of the future epithelial cell walls during the resin duct development. The cytochemical evidence supports that the resin ducts are formed by schizogeny and the cytochemical method can be used for a number of developmental processes, which were involved in cell wall breakdown.2 Histochemical changes during resin duct developmentAt the initial stage, a few resin droplets and starch grains were observed in the cytoplasm of initial cells. At the formative stage, the quantity of starch grains gradually increased while their volume enlarged. Resin droplets had a similar charge to starch grains. At the mature stage, the volume of starch grains obviously decreased and the number of starch grains decreased slowly. Whereas the number of resin droplets increased and no great changes took place in volume of droplets. These results suggested the changes of starch grains have relationship to the synthesis of resin and the precursor of resin might come from starch grains.3 Ultrastructure changes during resin duct developmentAt the initial stage, the cytoplasm of the initial cells was dense and the plastids were abundant. Plastids are large and elliptical, which contain one or two starch grains, without internal structure visible apart from a few short membranous profiles. A small number of osmiophilic droplets were present in the plastids. At the formative stage, the number of plastids, mitochondria and Golgi bodies in the cytoplasm increased. The plastids were commonly sheathed with endoplasmic reticulum (ER). The larger osmiophilic droplets in cytoplasm and the smaller osmiophilic droplets on the plastids envelope, mitochondrion envelope and in Golgi vesicles obviously increased in number. At the mature stage, the cytoplasm of epithelial cells became thin with a small nucleus. The number of mitochondria and Golgi body decreased, but numerous plastids still existed. Most of the plastids did not contain starch grains or only contained small ones. All of these showed that the structures of plastids in epithelial cells greadually became well developed and the synthetical activity of resin greadually enhanced during resin ducts formation.4 Sites of resin synthesis and the modes of its eliminationWith the conventional TEM, the osmiophilic droplets were seen in plastids and ER at the initial stage. At the formative stage, the number of osmiophilic droplets in plastids increased, osmiophilic droplets were obseved in mitochondria, ER and Golgi body. At mature stage, numerous osmiophilic droplets appeared on plastids envelope and both sides of the plasmalemma. At the same time, the ER had close relationship to the plasmalemma. Therefore we came to the conclusion that the synthesis of resin take places separately in various organelles, but mainly in plastids.3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase is a key enzyme in the mevalonic acid pathway leading to terpenoid production. To further reveal accurate site of resin synthesis, cytochemical localization of HMG-CoA synthase during resin duct development was used to investigate resin synthesis. At the initial stage, the activity of HMG-CoA synthase was first observed in plastids and mitochondria. At the formative stage, the activity of HMG-CoA synthase was found in ER and Golgi body besides plastids and mitochondria. The activity of HMG-CoA synthase gradually increased and came to maximum at the mature stage in above noted organelles. So we suggested the plastids were the most important organelle for resin synthesis and the mitochondria follows. The Golgi body and the ER were not important organelles for resin synthesis. The ER plays a role in transport of oleoresin from the plastid and the mitochondria to plasmalemma. These results are similar to that from conventional TEM.Analysizing based on above observations, there are three modes of resin elimination from the protoplast. 1) by fusing with plasmalemma, ER eliminates oleoresin to the outside of plasmalemma. 2) plasmalemma invaginations surround resin droplets and detach them. 3) the resin fuses with plasmalemma and discharged from protoplast. In processes of elimination, the resin may be degraded to pass wall, then aggregated again outside wall.