RESUMEN
A cytoplasm male sterile pepper (Capsicum annum L.) was examined using cytochemical method to study its pollen abortion. Thick sections of both anthers of male sterility line 8214A and its maintainer 8214B at different stages were stained using Periodic Acid-Schiff's (PAS) reaction to detect starch distribution. Anther structure and starch distribution in both anthers of male sterility and maintainer line were similar before the meiosis of microspore mother cells. After meiosis, the size of tapetal cells of fertile anthers of maintainer line increased and became high vacuolation. Abundant small starches appeared in the connective cells from tetrad stage to early stage of microspore development. At the late stage of microspore, the tapetal cells began to degenerate and the starches in the connective cells became large. Bi-cellular pollen synthesized starches after the large vacuole of vegetative cell disappeared, and abundant starches were stored in the mature pollen. In the anthers of male sterile line, meiosis of microspore mother could occurred and the tetrads could be formed in the locule, but the tetrads were extruded together because the locule could not enlarge its space. Finally, the tetrad microspores degenerated. The development of vascular tissue of the sterile anthers was normal and abundant starches were stored in the connective cells, which suggested that the function of plant transporting polysaccharide into anther was normal but tapetum could not transport the polysaccharide into locule. According to our result, the pollen abortion occurred in the tetrad stage and the abnormal development of tapetal cells might be the reason which induced tetrad microspore abortion in this male sterile pepper.
Asunto(s)
Capsicum/metabolismo , Citoplasma/metabolismo , Infertilidad Vegetal/fisiología , Polen/metabolismo , Almidón/metabolismo , Capsicum/ultraestructura , Flores/metabolismo , Flores/ultraestructura , Microscopía Electrónica de Transmisión , Polen/ultraestructura , Almidón/ultraestructuraRESUMEN
Potassium antimonite precipitation was used to locate calcium in the central cell of lettuce (Lactuca sativa L.) before and after pollination. At 3d before anthesis, two polar nuclei of central cell separately located at two polarity of the cell, and few calcium precipitates (ppts) appeared in the polar nuclei and cytoplasm, but some ppts in its small vacuoles. At 2d before anthesis, two polar nuclei moved toward the middle of the cell and fused to form a secondary nucleus, and the ppts evidently increased in the nucleus and cytoplasm. At 1d before anthesis, secondary nucleus again moved toward micropylar end and located near the egg to prepare for fertilization. Calcium precipitates were mainly accumulated in the secondary nucleus. After pollination and before fertilization, the distribution of calcium ppts was similar to that before pollination. At 4h after pollination, the central cell was fertilized, and calcium ppts evidently increased in the cell and numerous were accumulated in its nucleus and cytoplasm. At 6h after pollination, the primary endosperm nucleus completed its first division and formed two dissociate endosperm nuclei, and still many calcium precipitates appeared in the nucleus and cytoplasm. With endosperm development, calcium ppts decreased in the endosperm cell. At 1d after emasculated and without pollination, the secondary nucleus of the cell still bordered on the egg and some calcium ppts appeared in the secondary nucleus. The results indicated that the temporal and spatial changes of calcium in the central cell may play an important physiological role during the development of the central cell and endosperm.
Asunto(s)
Calcio/metabolismo , Lactuca/embriología , Lactuca/fisiología , Polinización , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Lactuca/crecimiento & desarrolloRESUMEN
Rho GTPases, including Rac1, Cdc42, play a critical role in the regulation of a variety of cellular processes such as cell morphology, cell migration, transcriptional activation and gene expression. We constructed several FRET-based single-molecule probes containing red fluorescent protein dsRed1, cyan fluorescent protein ECFP, the GTPase binding domain of the effector, Pak1 or N-WASP, and Rac1 or Cdc42. Rac1 and Cdc42 signaling pathways were activated in transfected cells by the inducer, insulin or bradykinin respectively. In vitro fluorescent spectroscopy assays showed that FRET phenomena were observed in transfected NIH3T3 and Hela cells. For all 3 signaling pathways in NIH3T3 cells, the values of FRET efficiency reached the highest after induction for 5 min, but the increasing extents of the values of FRET efficiency varied in 3 signaling pathways. The values of FRET efficiency decreased with the extention of the induction time, but differed significantly in the decreasing speed for the signaling pathways. Rac1 and Cdc42 activation assays indicated that Rac1 and Cdc42 were in the activated state (GTP-bound) in the induced cells. Their relative activated activities in the cells induced for different time were consistent with the values of FRET efficiency. The activated Rac1, Cdc42 signaling pathways led to the formation of lamelliopodia and filopodia in the transfected cells respectively. The results showed that these single-molecule probes could be used to directly monitor the spatial and temporal imaging of the induced activation of the signaling pathways in living cells. With these single-molecule probes, the GEF or GAP activities of putative regulatory proteins for Rac1 and Cdc42 were analyzed and judged, thus greatly simplifying the currently-used methods for identifying the regulatory proteins for Rho GTPases.
Asunto(s)
ADN Complementario/genética , Transferencia Resonante de Energía de Fluorescencia/métodos , Transducción de Señal/genética , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/fisiología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/fisiología , Animales , Humanos , Ratones , Células 3T3 NIHRESUMEN
Potassium antimonite was used to locate calcium in the synergids of lettuce (Lactuca sativa L) during their development. The two synergids on 3d before anthesis formed evident polarity with most cytoplasm located in the micropylar end and nucleus in the middle and a big vacuole in the chalazal end. At this time, calcium precipitates were a few in both cells. Calcium precipitates in the two synergids began to increase on 2d before anthesis. Synergid wall in the micropylar end thickened on 1d before anthesis, in which many calcium precipitates located. Near anthesis, synergids formed filiform apparatus in which abundant calcium precipitates accumulated to prepare for attracting pollen tubes entering. At anthesis, the distribution of calcium precipitates between two synergids was the same. At 1h after pollination, calcium precipitates evidently increased in one synergid that seemed to degenerate, the other one was persistent and the distribution of calcium granules did not change. Two synergids kept intact at 1d after emasculated, and the distribution of calcium precipitates did not display difference, suggesting that the degeneration of one synergid was caused by approaching pollen tubes which might give some signal to induce calcium increase of the synergid. Before fusion of sperm cell with egg cell, the cytoplasm of degenerated synergid embraced the egg and formed a thin layer between the egg and the central cell. Calcium precipitates in the different parts of degenerated synergid were closely connected with the fertilization: calcium precipitates accumulated in the near chalazal end of degenerated synergid at 1h after pollination. At 2.5h after pollination, the calcium precipitates increased at the chalazal end, especially abundant in the thin layer between the egg and the central cell. However, at 4h after pollination, the fertilization had finished at this time, the distribution of calcium precipitates in degenerated synergid changed again: the precipitates decreased at the chalazal end and increased at the micropylar end. The above-mentioned results suggested that calcium in the degenerated synergid played an important role during lettuce fertilization.
Asunto(s)
Calcio/metabolismo , Flores/metabolismo , Lactuca/metabolismo , Flores/crecimiento & desarrollo , Flores/ultraestructura , Lactuca/crecimiento & desarrollo , Lactuca/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
Potassium antimonite precipitation was used to located calcium in the egg cells (before and after anthesis), zygotes and proembryos of lettuce (Lactuca sativa L.). A few calcium precipitates (ppts) were located in the small vacuoles of cytoplasm of egg cell at 3 d before anthesis, when egg cells just formed. Then the small vacuoles fused to form some bigger vacuoles in egg cell at 2d before anthesis. Calcium ppts increased evidently in the cytoplasm and nucleus of egg cells at this time. At 1d before anthesis, a biggest vacuole located at the micropyle end of the cell and its nucleus was pushed toward the chalazal end of the cell, which made an evident cellular polarity. The number of calcium ppts in the egg cell markedly decreased, suggesting that change of calcium distribution may be related to the development of egg cell. After anthesis and before fertilization, calcium ppts were still few in the egg cells, and most of them were accumulated in the nucleus, especially in the vacuoles of nucleolus. At 4h after anthesis, egg cell was fertilized and the wall at the chalazal end of egg cell was formed completely. Calcium ppts evidently increased again in egg cell, and some big ppts appeared in the karyoplasm of nucleus and abundant small ppts in the large vacuole. At 9h after anthesis, zygote completed its first division. Calcium ppts in the nucleus and cytoplasm of two-celled proembryo began to decrease, and only some ones accumulated in the vacuoles of nucleolus. At 18h after anthesis, zygote divided several times and became a multi-celled proembryo. Calcium ppts in the cells of proembryo ulteriorly diminished but there were many ppts on the surface of proembryo. The result indicates that calcium in egg cell, zygote and the cells of proembryo orderly changes its temporal and spatial position, which suggests that calcium may play a role during the development of egg cell and zygote.
Asunto(s)
Calcio/metabolismo , Flores/metabolismo , Lactuca/metabolismo , Semillas/metabolismo , Flores/citología , Flores/ultraestructura , Lactuca/citología , Lactuca/ultraestructura , Microscopía Electrónica de Transmisión , Semillas/citología , Semillas/ultraestructuraRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Flores/metabolismo , Lactuca/metabolismo , Flores/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Lactuca/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
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.
Asunto(s)
Brassica/metabolismo , Calcio/metabolismo , Flores/metabolismo , Polen/metabolismo , Brassica/ultraestructura , Fertilidad , Flores/ultraestructura , Microscopía Electrónica de Transmisión , Infertilidad Vegetal , Polen/ultraestructuraRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Flores/metabolismo , Lactuca/metabolismo , Polinización/fisiología , Flores/ultraestructura , Lactuca/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.
Asunto(s)
Proteínas de la Nucleocápside/biosíntesis , Pichia/metabolismo , Proteínas Recombinantes/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Clonación Molecular , Proteínas de la Nucleocápside de Coronavirus , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/inmunología , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Flores/metabolismo , Lactuca/metabolismo , Flores/citología , Flores/ultraestructura , Lactuca/fisiología , Lactuca/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
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.