Development Of Endosperm Cells In Wheat And Rice

The endosperm, rich in starch and protein, is the largest part of the kernel and takes up nearly90percent of the kernel’s mass. Starch and protein are stored in the form of amyloplast and protein body respectively. Elucidating their development regularity may be a great help to both theoretical and practical guiding significance.In our study, developmental time of developing caryopsis from rice (Yangdao6) and wheat (YangMai16) is accurately tagged. Histochemical assays, light microscopy, electron microscope (SEM and TEM) were employed to investigate the structure features, developmental process, formation mechanism and physiological function of endosperm, of which the developmental formation of the amyloplast and protein body are mainly observed. Elucidation of above questions will somehow deepen the study of endosperm development, enrich the knowledge of cell biology, crop science, and provide theoretical basis for mass production and crop quality improvement.Main results were as follows:1. The development of rice and wheat caryopses.The fastest growth rate of rice caryopsis appears to be within first few days after anthesis, till10days after followring (DAF) the shape of rice caryopsis is no longer changed. In wheat, the growth rate is relatively slow, till30DAF maximum volume reached.Starch first accumulated in the rice ovary wall, and lasts long during the whole developmental process. I2-KI staining on the rice endosperm gets darker with DAF increases. In wheat caryopsis, starch begins to accumulate from6DAF in endosperm, thickness of the pericarp declines as DAF increases, with its starch degrading gradually. Vascular bundle region, embryo and peripheral endosperm cells have a relatively higher and long-lasting dehydrogenase activity, while the endosperm cells, shows little activity in10DAF, and no activity when grain-filling finished. In wheat, ventral vascular bundle region and pericarp both show a high dehydrogenase activity, the endosperm shows a declined activity in17DAF. 2. Freshweight and dryweight of rice and wheat caryopsis grows under a typical "S-shaped" behavior, following a first slow then fast and finally slow rate.3. Respiration rate of rice caryopsis maintained a high level at first, then declines rapidly till15DAF; while in wheat, the rate first drops sharply from5to20DAF, from20to30DAF slowed down, and30to35DAF, increases rapidly.4. Effects of varying fixative and staining methods on the structure display of wheat and rice endosperm cells.Conventional glutaraldehyde-osmium tetraoxide (GA-OSO4) fixation better in preserving cell ultrastructure, such as amyloplasts, protein bodies, Endoplasmic reticulum and vacuoles, while potassium permanganate fixation proves to be better fixative for endomembrane system. Semi-thin sections staining were carried out using toluidine blue (TBO), polychromatic dye, periodic acid Schiff (PAS), coomassie brilliant blue and PAS-TBO counterstaining. The results were as follows:(1) GA-OSO4fixation and TBO stain preserve cell structures well, amyloplasts, protein bodies, cell walls and nucleus all have a better display than amyloplast envelope and tonoplast. Samples fixed with Potassium permanganate show a good result in endomembrane system display, like nuclear membrane, tonoplast and amyloplast envelope. Besides, amyloplast stains easily, while other cellular tissues not.(2) GA-OSO4fixation and polychromatic dye provide the uniformly color, moderate contrast, with cell wall and protein body being stained blue, amyloplast envelope and inner septum stained rose red. Samples fixed in potassium permanganate present a slight different result, in that nulear being blue, cytoplasm and stroma being rose red, and amyloplast poorly stained.(3) Only amyloplasts from samples fixed with GA-OSO4and stained with PAS can be colored rose red and other organelles are unstainable. When fixed with potassium permanganate, results improved, in that endosperm cell wall, protein body, amyloplast and its envelope and septum are all well colored.(4) Samples fixed with above GA-OSO4and PAS, when counterstained with TBO show a improved results in color reactions, cell wall, protein body, stroma and nuclear etc all well displayed. But the amyloplast from samples fixed with potassium permanganate can be unevenly and over stained by PAS-TBO.In most cases, cells with GA-OSO4fixation have a better result than with potassium permanganate fixation, and of all the staining methods, TBO and polychromatic dye have the best effect. By comparing effects of2different fixation and5staining methods on wheat semisections, we found that GA-OSO4fixed samples, stained by TBO, polychromatic dye, periodic acid PAS, coomassie brilliant blue, are superior to those fixed with potassium permanganate, but in PAS-TBO counterstaining, potassium permanganate fixation made the best performance. Therefore, the selection of appropriate fixation and staining methods should be based on experimental destination.Through observation in the ultra-thin sections, we find that the GA-OsO4fixation was a better fixative in preserving endosperm cell intrinsic feature, but lacked clarity in membranous structure display. Potassium permanganate fixation, although was able to show the structure of the cell endomembrane better and its contrast was satisfactory, but it was poor on the polysaccharide and protein colour generation. We chose the latter method to study physiological activities of endoplasmic reticulum and results are optimistic.5. Initiation, development and proliferation of rice and wheat amyloplast.Rice endosperm amyloplast is commonly formed from proplastids. Utilizing GA-OSO4and Potassium permanganate fixation respectively, we find that:samples fixed with GA-OSO4although have better effection preserving the microstructure of endosperm cells, show a low contrast in amyloplast envelope; while samples fixed with potassium permanganate perform better in membrane structure display, but perform poor in cell stroma and organelle stroma fixation. As amyloplasts proliferation mainly manifested in their envelope changes, the latter fixation method are our main choice.Rice endosperm amyloplasts proliferate mainly by means of envelope protrusion, constriction, invagination, middle septum dividing, expansion of amyloplast envelop et al, among which the first two are the most common ones in earlier stage of endosperm cell development. Two middle septum formation mechanisms have been observed, first is the amyloplast envelop invaginate to amyloplast stroma, when it touched opposite envelop, two new amylopplast appeared, another is two amyloplast envelops move in opposite direction, two new amyloplasts generated when two envelops linked. In the amyloplast proliferation process, two or more ways of amyloplast proliferation could be observed in a amyloplast.Wheat endosperm amyloplast, differ from rice, has two types of amyloplast, starch begins accumulating in proplastids from6DAF, transforming into amyloplasts. Each amyloplast contains2or more starch granules. Large amyloplasts proliferate by means of envelope protrusion and constriction, of which are observed by both fixation methods.From8to14DAF, envelope of large amyloplasts either invaginate into stroma dense vacuoles, or protrude outwardly, forming a new type of amyloplast, called small amyloplast. From16DAF, small amyloplasts accumulate, not only by way of envelope protrusion, but by self-constriction or middle septum division as well.6. Formation and accumulation of rice and wheat protein body.Two types of protein bodies are found in rice endosperm, protein body I, Pbl and protein body Ⅱ, PbⅡ. PbⅠ, derives from the secretion of Rough Endoplasmic reticulum, is a spherical, concentric circle-like structure(on the section),which surface covered with ribosomes and polyribosomes, and is possibly a product accumulated through RER inward secretion layer-by layers. Matured PbⅠ usually coated by a single layer of discontinuous ribosome, and can be fused by vacuoles with protein matrix.PbⅡ, developed from vacuoles, mostly are irregular clots like. When vacuoles start accumulating proteins, they turn into PSV, in which crystal structures of storage proteins can be detected. Enriched PSVs are found in wheat endosperm cells from6DAF, in the form of spherical particles. Meanwhile, RER retains a high level of activity (of which matrix electron density higher than cell matrix), which stacked together, and thus forming either looped or reticulated structure. Some RER’s cavity or terminal may be protruding where storage protein is accumulated.Most rice and wheat protein bodies are stored in the sub-aleurone layer, seldom seen in endosperm cells.7. The surface of the geometric characteristics analysis for rice endosperm starch granules by using Image J.Based on the optical microscope and scanning electron microscope images of rice starch granules, the image analysis software image J was introduced in analysis of starch granules of surface features. Image analysis presented in two ways:outline and ellipse fitting. Analysis of indicators chosen area, perimeter, major, minor, circularity and Feret. Finally, the particle image acquisition and image pre-treatment were discussed.8. Discussion on the existence of amyloplast which contain single starch grain in rice endosperm.5DAF and mature rice caryopsis were made to consecutive semithin sections, GA-OSO4and potassium permanganate fixation were used for different days after followering rice caryopsis, Meanwhile, chalky caryopsis was took as contrast, both of them were observed ultrastructure by TEM and SEM. The results found that the vast majority of the rice endosperm amyloplast was compound grain amyloplast, a few was single grain amyloplast.