The response of Pyropia haitanensis to inorganic arsenic under laboratory culture.

Up to now, complicated organoarsenicals were mainly identified in marine organisms, suggesting that these organisms play a critical role in arsenic biogeochemical cycling because of low phosphate and relatively high arsenic concentration in the marine environment. However, the response of marine macroalgae to inorganic arsenic remains unknown. In this study, Pyropia haitanensis were exposed to arsenate [As(V)] (0.1, 1, 10, 100 µM) or arsenite [As(III)] (0.1, 1, 10 µM) under laboratory conditions for 3 d. The species of water-soluble arsenic, the total concentration of lipid-soluble and cell residue arsenic of the algae cells was analyzed. As(V) was mainly transformed into oxo-arsenosugar-phosphate, with other arsenic compounds such as monomethylated, As(III), demethylated arsenic and oxo-arsenosugar-glycerol being likely the intermediates of arsenosugar synthesis. When high concentration of As(III) was toxic to P. haitanensis, As(III) entered into the cells and was transformed into less toxic organoarsenicals and As(V). Transcriptome results showed genes involved in DNA replication, mismatch repair, base excision repair, and nucleotide excision repair were up-regulated in the algae cells exposed to 10 µM As(V), and multiple genes involved in glutathione metabolism and photosynthetic were up-regulated by 1 µM As(III). A large number of ABC transporters were down-regulated by As(V) while ten genes related to ABC transporters were up-regulated by As(III), indicating that ABC transporters were involved in transporting As(III) to vacuoles in algae cells. These results indicated that P. haitanensis detoxifies inorganic arsenic via transforming them into organoarsenicals and enhancing the isolation of highly toxic As(III) in vacuoles.

Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation.

Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca(2+) precipitates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of calcium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca(2+)](cyt), and in pollen and style maturation during the progamic phase.

[ATPase distribution in fertile and sterile anther of a genic male sterile Chinese cabbage].

Lead precipitation technique was used to locate Adenosine Triphosphatase (ATPase) in the fertile and sterile anthers of a genic male sterile Chinese cabbage (Brassica campestris L. ssp. Chinensis Makino var. communis Tsen et Lee),which would help us to understand the relationship between ATPase and sterility of anthers of the cabbage. At megaspore mother cell (MMC) of fertile anther many ATPase reactive precipitates were located in nucleus but few of the precipitates in cytoplasm of the cell. Meantime, some ATPase reactive precipitates also specially appeared in mitochondria of the MMC. After meiosis of MMC, the precipitates in cytoplasm of early microspores increased evidently and then decreased step by step with development. The ATPase reactive precipitates in tapetal cell also increased ultimately in early microspore stage and then decreased with development of anther. When microspore formed a large vacuole, which is late stage of microspore, the ATPase reactive precipitates were located in its mitochondria. After microspores mitosis a few of the ATPase reactive precipitates appeared in pollen grains and tapetal cells. More ATPase reactive precipitates appear in MMC of sterile anther than in fertile anther but fewer of them in mitochondria. Although more ATPase granules appear in abnormal tetrad microspores which degenerate by cytoplasm shrinkage and plasmolysis. The relation between ATPase and male sterility of the cabbage was discussed.

[Advances in cell biological researches on male sterility of higher plants].

Male sterility of higher plants is multiform in pollen abortion and has varied and complicated mechanisms. It is an active field to probe the mechanisms. Recently, some new results in this field have been obtained by using the methods of cell biology, including the structure and function of tapetal cell, the changes in Ca(2+) distribution, ATPase activity distribution, cytoskeleton array and programmed cell death in anther cells. All of the results gave us some new understanding for the process of pollen abortion. These results will make a link between the researches of individual and molecular level in male sterility of higher plants, and help us understand the mechanisms of male sterility of higher plants. This paper summarizes the knowledge about aborting process of male sterile anther obtained by the methods of cell biology.

[The character of calcium distribution in developing anther of lettuce (Lactuca sativa L.)].

Potassium antimonite was used to locate calcium in the anther of lettuce (Lactuca sativa L) during its development. At the early stage of anther development there were few calcium granules in microspore mother cells and the cells of anther wall. After meiosis of microspore mother cells, calcium granules first appeared in the tapetal cells in which some small secretive vacuoles containing many calcium granules were formed and secreted into locule. Then, the tapetal cells began to degenerate. At the late stage of microspore, tapetal cells completely degenerated and its protoplast masses moved into anther locule with many calcium granules. Few calcium granules were precipitated in the microspores just being released from tetrad, but some on the surface of exine. Then calcium granules appeared in the nucleus and cytoplasm of early microspores, as wall as in the exine. When microspores formed some small vacuoles containing some calcium granules, and then the small vacuoles fused to form a large vacuole, the calcium granules in the nucleus and cytoplasm evidently decreased, microspore developed to the late stage. The result suggested that calcium is related to the formation of large vacuole in microspores. The wall of microspore also is a main location of calcium granules during its developing. At early microspore some calcium granules began to accumulate in exine, which suggested calcium related with exine formation. At late stage of microspore, most of calcium granules were mainly deposited on the surface of exine. After the first mitosis of microspores, the large vacuole of bicellular pollen disappeared and calcium granules in the large vacuole went back to cytoplasm again. When bicellular pollen synthesized starches some calcium granules appeared on the surface of starches, which suggested calcium may regulate starch synthesis. With amount of starches increasing, calcium granules disappeared from pollen cytoplasm and only some of them located on the surface of pollen.

[Calcium distribution in fertile and sterile anthers of a genic male sterile Chinese cabbage].

Potassium antimonite was used to locate calcium in the fertile and sterile anthers of a genic male sterile Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) to probe the relation between Ca(2+) and fertility and sterility of anthers of the cabbage. During fertile anther development, calcium granules increase in number in anther wall cells after meiosis, and then appeared also in locule, suggesting a calcium influx into locule from anther wall cells (Plate I-4). Then the number of calcium granules in microspore cytoplasm also increased at early stage (Plate II-1), accumulated mainly on the membrane of small vacuoles which were fusing to form big ones to make a polarity in the cell and to prepare asymmetric division of microspore (Plate II-3,4). After microspore division and the big vacuole decomposition, many calcium granules accumulated again on the membrane of the vacuoles (Plate III-1,2), displaying calcium regulates vacuole formation and decomposition during pollen development. In sterile anthers, abnormal distribution of calcium granules first appeared in callus wall of microspore mother cell (Plate IV-1). However, only a few calcium granules appeared in early microspores, which then could not form small vacuoles and finally a big vacuole (Plate IV-2,3). The aborting microspores degenerate by cytoplasm shrinking (Plate IV-5,6). The difference pattern of distribution of calcium granules between the fertile and sterile anthers indicates that anomalies in the distribution of calcium accumulation are correlated with the failure of pollen development and pollen abortion.

[The dynamics of calcium distribution in stigma and style of lettuce (Lactuca sativa L.) before and after pollination].

Potassium antimonite was used to deposit calcium in the stigma and style of lettuce (Lactuca sativa L.) before and after pollination. The stigma of lettuce is two splits. Abundant calcium granules are displayed in the wall of papillae on the receptive surface of stigma before and after pollination, which may facilitate pollen germination. However, a few calcium granules in the wall of epidermis cell on no-receptive surface. Calcium distribution in style presents a gradient in transmitting tissue and parenchyma cells from the top to the base of the style before pollination. After pollination, calcium in transmitting tissue distinctly increased and its gradient distribution became more evident. Pollen tubes grow in the intercellular gaps of transmitting tissue. When pollen tubes grew into transmitting tissue, calcium granules in parenchyma around transmitting tissue decreased, suggesting a calcium movement was controlled by pollen tubes. The calcium gradient distribution also appeared in the trachea of vascular bundle of style. In general, calcium in style displays a feature of time-special distribution: transmitting tissue doesn't need much more calcium that is only stored in the parenchyma before pollination. However, calcium in parenchyma cells may be transported to transmitting tissue and make the latter contain more calcium to form an evident calcium gradient and meet the requirement of pollen tubes directionally growing after pollination. This is the second sample of calcium gradient existing in style, which was found by using potassium antimonite method.

[The dynamic of calcium distribution during megasporegenesis of lettuce (Lactuca sativa L.)].

Potassium antimonite was used to deposit calcium in the young ovule of lettuce (Lactuca sativa L.) at megasporogenesis stage to study the relationship between calcium and megaspore degeneration. At the megaspore mother cell stage, few calcium granules were formed in the cell (Plate I-1, 2). After meiosis of megaspore mother cell and forming an arrayed tetrad in a line (Plate I-3), three megaspores degenerated one by one from the micropyle end. In the process of degeneration, the numbers of calcium granules decreased in the three megaspores. After the first megaspore degenerated, the number of calcium granules decreased in the second megaspore, which began to degenerate (Plate II-7, 8). The third megaspore also had its number of calcium granules diminishing before it degenerated (Plate III-13, 14). The fourth megaspore always accumulated many calcium granules in the cytoplasm during its development (Plate IV-17, 18) and finally becomes functional one that will develop into an embryo sac (Plate IV-20). Megaspore degeneration is a process of programmed cell death which may be closely related with change in calcium content: when a megaspore of tetrad decreases calcium content the cell begins to degenerate, and when calcium increases in the cell, it will continue to develop into a functional megaspore. This is the first report about calcium distribution in megaspores of a tetrad during megasporogenesis in higher plants and will open a door to study the physiological function of calcium in megasporogenesis.

[The distribution of calcium in the stigma and style of tobacco during pollen germination and tube growth].

After pollen grains of tobacco landed on stigma they begin to hydrate and form many small vesicles containing some calcium grains in cytoplasm. The calcium stored in pollen wall is released into tectum of stigma to make a calcium-rich environment. When a pollen tube penetrates the tectum and grows between stigma cells, numerous calcium precipitates appear in the tip tube wall. The length of style of tobacco is 4 cm, and the pollen tube need take 44 h to reach the ovary. The style was artificially divided into 4 stages and each 1 cm respectively. There were only a few of calcium precipitates in the transmitting tissue of style from anthesis to 11 h after pollination. A calcium gradient in the transmitting tissue of style was formed at 22 h after pollination: only a few calcium precipitates found in the transmitting tissue of the style under stigma and at stage 1, 2 and 3, and many of them were located in the transmitting tissue of style near ovary (stage 4). When the flowers were emasculated and unpollinated at 1 d after anthesis, no calcium gradient in the transmitting tissue of style could be identified because some precipitates were also accumulated in the transmitting tissue at stage 1. When a flower without pollination was kept for 3 d, some calcium precipitates were formed in the cells of stigma, and the cells of the whole transmitting tissue contained the same quantity of calcium precipitates. To check the ability of pollen to germinate and grow in a low calcium environment, pollen grains were cultured in a medium containing 0-0.1% CaCl(2).2H(2)O. The result of in vitro assay confirmed that tobacco pollen can germinate and the pollen tube can grow in an environment with a very low concentration of calcium, which may be similar to the environment in the stigma. A few calcium precipitates were accumulated in stigma and upper transmitting tissue of tobacco to make a calcium gradient in the style. If the calcium in the style at 1 cm increases it will be increased more at 4 cm, and more in ovules, and more in synergid cells to keep the calcium gradient. When the emasculated flowers were not pollinated for 3 days the calcium in upper transmitting tissue evidently increases. The calcium in style is abundant in all plants, but the distribution of calcium in style is different between different plant species. For this difference, it may differ from types of style, and in the plants with short style the calcium gradient in the style is too small to be detected. But for tobacco with style 4 cm long, the gradient can be identified using antimonate method.

[The ultrastructural observation of anthers of Chinese cabbage's mail-sterility].

The fertile and sterile anthers of a Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) were observed using electron microscope to find the ultrastructural feature of sterile anthers. The earliest abnormal phenomenon in sterile anther was nucleolus of sporogenous cells locating in the edge of nucleus. During microspore mother cell development, callus wall surrounding the cell displayed uneven in the thick ness and was discontinuous,and the some cytoplasm leaked out of the cell from some rifts in the wall. After meiosis of microspore mother cells, the cells of tetrad were irregular and some of them contained several nuclei. The exine of pollen began to be formed in tetrad in this cabbage. The evident disorder during exine formation in the sterile pollen occurred during its primexine formation and then the sporopollenin was irregularly deposited to form a layer of uneven and discontinuous pollen exine. Cytoplasm of aborting microspores contracted and finally degenerated after them released from tetrad. The tapetal cells of fertile anther began to synthesis abundant lipid material during microspore development. However, the tapetal cells of sterile anther did not synthesis lipid material during microspore aborting. The microspore abortion was first and tapetal degeneration second. Therefore, aborting microspore induced the functional default of tapetal cells synthesizing lipid material. The ultrastructural results on this study further complete and correct our previous results obtained by light microscope.

[The cytochemical observation of anthers of Chinese cabbage's male-sterile].

A Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) produces 1/4 male sterile and 3/4 fertility in offspring. The sterile plant can be identified from the color of corolla that is some white when it grows out. The fertile and sterile anthers were researched using cytological and cytochemical methods. Thick sections of both anthers of different developmental stages were stained with Toluidine blue for general cytological observation and stained with the periodic-acid-Schiff's (PAS) technique to detect polysaccharides (red), with Sudan black B (SBB) to detect lipids (black). Before meiosis of microspore mother cells, connective tissue of both fertile and sterile anthers stored a lot of starch grains. Neither starches nor lipid drops were in tapetal and microspore mother cells. The only difference of both anthers was that the tapetal cells of sterile anthers contained more vacuoles than those of fertile anthers. After meiosis of microspore mother cells, the starch grains in connective tissue of fertile anthers disappeared, the tapetal cells synthesized abundant lipid drops, and the microspores also began to accumulate lipid drops. In sterile anthers, the starch grains in connective tissue also disappeared, but only a few lipid drops appeared in tapetal cells. The tapetal cells, however, became red, suggesting the cell contained some polysaccharide material. Pollen abortion in sterile anthers occurred in this stage. The aborting microspores accumulated very less lipid drops in its cytoplasm than those in fertile the starch This result suggested that in the cabbage, the starch grains in connective tissue were transformed into polysaccharide and transported to tapetal cells, then these cells transformed polysaccharide into lipid material that was absorbed by developing microspore. In sterile anthers, however, polysaccharide in the tapetal cells could not be transformed to lipid. The functional default of tapetal cells during lipid metabolism led to microspore abortion. This is new sample in which the functional default of tapetal cells will make pollen abort, and will enhance research field in male sterile in higher plants.

[The electron microscopic in situ hybridization and its application].

The technique of electron microsco-pic in situ hybridization is applying in situ hybridization at the electron microscopic level. It is mainly used in the ultrastructural localization of the lablled DNA, RNA and RHA in a cell and/or a tissue. In this paper I mainly elaborated its establishment and classification, and the operation procedure of nonradioactive electron microscopic in situ hybridization and some points for attention. In the end I also discussed its application for research.