Application Of Low Temperature Electron Microscopy Of Arabidopsis Inflorescence, The Flower Morphology And Flower Nectary Ultrastructure Of The Study

The cryoelectron microscopy was used for the investigation of development of shoot apex, initiation and development of inflorescence and flower, and ultrastructure of floral nectaries of Arabidopsis thaliana. In the development of shoot apex, the relationships between thechanges of morphology, structure and changes of ultrastrcture was probed. The point of view about morphogenesis sequence and characteristics of different parts of flower was brought up. The relationships between the changes of nectary ultrastructure and the accumulation, transportation and secretion of nectar were discussed. We found that there was no fusion at all between the vesicle and plasmalemma in the process of vesicle secretion. It is quite different from the modle of vesicle secretion (i.e. exocytosis ) and is first reported by us.The initiation and development of Arabidopsis thaliana apex was studied with high pressure freezing and freeze substitution. The results indicated that the process of the apex development was divided into vegetative and reproductive shoot apex. The vegetative shoot apex was also divided into two stages. During the early stage of vegetative apex, the apical meristem was flat and the tunica—copus was not apparent. The apical meristem at the late stage was dome- shaped and tunica-corpus and cytohistological zones were clearly observed. When vegetative apex changed to reproductive one, thetunica-corpus and cytohistological zonalization disappeared. However, the mantle—core structure was not apparent in the inflorescence apex. The ultrastructure of the shoot apex in development illustrated that the organelles in the apical meristematic cells appeared immature in the early stage of vegetative shoot apex. They were small and inner membrane structure of mitochontria and plastids was simple. During the late stage of vegetative shoot apex, the matural organelles was shown. They were larger, abundant and the membrane structure was clear. The organelles in tunica and rib zone were more differentiated than that of the other zones. However, organelles of the meristematic cells of reproductive apex became immature again. The results reflect that the changes of morphology, structure and ultrastructure during the development of shoot apex are associated with differentiation of cells and organelles, and can show different characters in morphology, structure and physiology at various stages.Scanning electron microscopic studies of development of the inflorescence and flower were performed. Several floral primordia were produced spirally from the florescence apex which became dome-shaped during a pause. Floral primordia differentiated upwards from the lower position of the inflorescence primordium. Occassionally the branch which arised in axils of cauline leaves, developed into a new inflorescence. Occurring sequence of the floral organs was four sepals, six stamens, two carpels, four petals and four or six nectaries in five whorls. The nectaries initiated later and appeared breast-shaped. Two medial nectaries were positioned between the bases of a pair of medial stamens (two pairs medial stamens in all) or four near the bases of medial stamens, while two lateral nectaries were situated individually near the two lateral stamens. The matural nectary consisted of epidermis with thick cuticle on the surface, nectariferous tissue and vascular bundle.The morphology, development and ultrastructure of the nectary of Arabidopsis thaliana were studied by high pressure freezing and freeze substitution. The relationships between the changes of ultrastructure and nectar secretory mechanism were discussed. During the early stage of the nectary development, the nectary primordium was a group of meristem which had larger nucleus, dense cytoplasm and small vacuoles. The organelles appeared in immatural state. Epidermis differentiated first and the cell lengthened in tangential direction, and starch grains accumulated in the plastids. Nectariferous tissue differentiated afterwards. The cells divided in all directions and organelles differentiated gradually and increased in number. The vascular tissue appeared last and consisted of sieve elements and companion cells only. Abundant plasmodesmata were between the sieve elements and companion cells, and sieve—pored structures and multivesicular structures were found between the companion cells and their neighbouring cells. Before nectar secretion, epidermis with small vacuoles, more Golgi body and vesicles were observed. The vesicles with different substances inside secreted out and no any fussion occurring between the vesicles and plasmalemma was observed. The ER appeared short cisternae and distributed in the cytoplasm or near the plasmalemma. The number of mitochondria increased by their own division. A few dense staining cells in the nectariferous tissue appeared with irregular nucleus, vacuoles and Golgi bodies, plastids and mitochontria distributed abnormally. During the se-cretion, the number of dense staining cells increased and gradually connected with the modified stamata, but only a few Golgi bodies were observed. ER appeared active and cisterae dilated. A large number of rough ER situated along the plasmalemma or connected with the plasmodesmata. Afew smooth ER surrounded small vacuoles and formed the sheath. The starch grains reduced gradually and mitochondria with delicated inner meberane structure was observed clearly. In the heavy nectar secretion, the dense staining cells increased fast and became the main tissue of the nectary and only a few normal cells were among them.From the changes of ultrastructure of nectary development, several suggestions were given as following: 1. The regular changes of the vacuoles volume during the development of nectary might be considered with the changes of other organelles, accummulation and degradation of substances in the cells at different developmental stage. The vacuole volume changes from small to larger because of the differentiation of the cells and it is possible that the vacuoles from larger to small is associated with the accummulation of substances in the nectary cells before nectar secretion. With the nectar secreting out, the vacuole volume changes again from small to larger during the secretion. 2. The change amount of starch grains before or during the secretion might be related with their storage in plastids as a part of nectar and then decreases as nectar secretes out. 3. The number of Golgi body and its vesicles increases before secretion might supply the nutritional substance for the growth of cell wall and cuticle. ER increase in number during the secretion might be for modification and transportation of the nectar. Nectar secretion might require energe supplying from the mitochontria increasing in number. 4.1t is defferent from the typical model of the process of vesicle secretion that the vesicles, in our research, do not fuse with plasmalemma as they secrete out from the cell.In short words, the pathway of nectar secreting in the nectary of Arabidopsis thaliana is that: nectar from the phloem sap is transported from sieve tubes to the nectariferous cells by the plasmodesmata, sieve—pore—shaped structures and multivesicles structures; it is stored in the plastids of nectariferous cells and then degrades there; after that it is modified in ER or Golgi bodies; finally the nectar is accummulated in the dense staining cells and secretes out through the modified stomata.The cryoelectron microscopy is a new techninics for the sample preparation. It includes: freeze fixation, freeze substitution, freeze drying, cryosection, cryoscanning electron microscopy and cryotransmission electron microscopy.The sample can be fixed very fast (0.1ms—Is) and preserve the original structure in natural state. In our studies, the phenomena of non-fusion between the vesicle and plasmalemma, as the vesicle secreting out, can be observed clearly. However, it was suggested as a nodule—like structure on the cell wall by Mansfield and Briarty (1990) when the sample was prepared by conventional method. In addition, the nectar covers the top of nectary by using the cryoscanning electron microscopy and it is difficult to be observed when the sample is prepared by the chemical fixation and dehydration, because the nectar is easily lost in the fixed and dehydration solution during the sample preparation. Technological innovation is very important for the electron mcroscopic analysis.