Guard Cells Of Stomata Research Articles

Epigenetic and epiplastome variability in apozygotic progenies of sugar beet with 5-azacytidine

Soaking seeds in 5-azacytidine induced epigenetic and epiplastome variation in sugar beet. It hastens the early flowering of sugar beet, decreased the amount of not-flowering plants (flowerlcss, stemlcss), increased the number of second order branches in plants (epigenetic variability). 5-azacytidine influenced on the number of sub-cellular organelles in cytoplasm. It was also shown that 5-azacytidine reduces the chloroplasts number in stomata guard cells by 15–20% (epiplastome variability). It is possible that correlation exist between epiplastome and epigenetic variability in plants treated by 5-azacytidine.

Variability of Chloroplast Number in Populations of Stomata Guard Cells in Sugar Beet Beta vulgaris L.

Diploid sugar beet plants demonstrate a broad variability of the number of chloroplasts in stomata guard cells, which is related to mixoploidy of cell populations in leaf apical meristems (epigenomic variability). In addition to random organelle segregation between daughter cells, this variability is affected by factors disrupting the mitotic cycle: (1) plant treatment with a mitotic poison, such as colchicine; (2) duration of the life cycle of a plant; the variability in second-year plants is greater than in first-year ones; (3) the mode of plant reproduction; the variability in inbred plants is greater than in the initial population. Treatment of germinating seeds with a diluted colchicine solution increases the number of organelles in cells in the myxoploid generation (generation C0) and the variance of the distributions in the first vegetation year. The variability in the organelle number in stomata cells correlates with that in maternal meristem cells. It is concluded that the epidermal cell monolayer (including stomata guard cells) keeps record of the epigenomic and epiplastome variability in meristem cells. The variability of the number of chloroplasts in stomata guard cells is approximated by binomials with negative powers.

Inducibility by pathogen attack and developmental regulation of the rice Ltp1 gene.

Using a genomic clone encoding a rice lipid transfer protein, LTP1, we analysed the activity of the 5' region of the Ltp1 gene in transgenic rice (Oryza sativa L.) during plant development and under pathogen attack. The -1176/+13, -556/+13 and -284/+13 regions of the promoter were fused upstream from the uidA reporter gene and nos 3' polyadenylation signal, resulting in the pdelta1176Gus, pdelta556Gus and pdelta284Gus constructs which were transferred to rice by microprojectile bombardment. Histochemical and fluorometric GUS assays and in situ detection of uidA transcripts in transgenic homozygous lines harbouring the pdelta1176Gus construct demonstrated that the Ltp1 promoter is preferentially active in aerial vegetative and reproductive organs and that both specificity and level of expression are regulated during organ development. In leaf sheath, GUS activity which is initially strictly localized in the epidermis of growing tissue, becomes restricted to the vascular system in mature tissues. In expanded leaf blade, expression of the uidA gene was restricted to the cutting level suggesting inducibility by wounding. Strong activity was detected in lemma and palea, sterile glumes, and immature anther walls and microspores but not in female reproductive organs. No GUS activity was detected during seed embryo maturation whereas the uidA gene was strongly expressed at early stages of somatic embryogenesis in scutellum tissue. The Ltp1 transcripts were found to strongly accumulate in response to inoculation with the fungal agent of the blast disease, Magnaporthe grisea, in two rice cultivars exhibiting compatible or incompatible host-pathogen interactions. Analysis of pdelta1176Gus leaf samples inoculated with the blast fungus demonstrated that the Ltp1 promoter is induced in all cell types of tissues surrounding the lesion and notably in stomata guard cells. In plants harbouring the Ltp1 promoter deletion construct pdelta556Gus, activity was solely detected in the vascular system of mature leaves whereas no uidA gene expression was observed in pdelta284Gus plants. These observations are consistent with the proposed role of LTP1 in strenghtening of structural barriers and organ protection against mechanical disruption and pathogen attack.

Modelling of variability in chloroplast number of stomatal guard cells of sugar beet

Variability in chloroplast number of stomatal guard cells of inbred line of sugar beet was studied in correlation with the level of cell populations mixoploidy. Control plants (seeds were soaked in distilled water before sowing) were compared with plants under the study (seeds were soaked in a weak colchicin solution for 24 hours before sowing). The variability of chloroplast number in stomata guard cells for both control and experimental plants can be described by either binomials with integer positive indexes or integer negative indexes (negative binomials), or Neyman “contagious” distribution. It was demonstrated that both cell population mixoploidy level and variability of chloroplast number in cells rise when plants get older. A comparison in plants of the first and second years of vegetation was also studied.

Transfer of PLRV resistance fromSolanum verrucosum Schlechdt to potato (S. tuberosum L.) by protoplast electrofusion

Somatic fusions between an accession of the diploid wild speciesSolanum verrucosum and a dihaploid S.tuberosum genotype were produced in order to incorporate resistance to potato leafroll virus (PLRV). In total 15 somatic hybrids out of 16 regenerants were obtained. Identification of hybrids was based on additive RAPD patterns, general morphological characteristics, chromosome numbers and chloroplast counts in stomata guard cells. A field trial was performed with the hybrids, their two parents and the control cultivar Kennebec to assess field performance and phenotypic variability. Yield parameters varied considerably among somatic hybrids. Some of the hybrids gave significantly higher yields, tuber numbers and tuber weights than both parents. Pollen fertility of hybrids ranged from 19 to 77%. Twelve hybrids were found to be resistant to PLRV.

Action of Photosynthetic Diuron Herbicide on Cell Organelles and Biochemical Constituents of the Leaves of Two Soybean Cultivars

Two varieties of soybean (Glycine max L. cv. Clark and Crawford) showed a high sensitivity to low concentrations of diuron herbicide [3-(3,4-dichlorophenyl)-1,1-dimethyl urea]. Depending on the application dose of diuron, chlorosis, necrosis, and wilting of leaves ending in death after 7 to 10 days were observed. These symptoms were more obvious in the Clark variety even at the lowest concentration of diuron (1 ppm, 4.29 μM). Electron microscopic observation of treated plants revealed disorganization in the ultrastructure of mesophyll cell organelles. Disappearance of starch grains, decrease of plastoglobuli, and swelling and disarrangement of grana thylakoid of chloroplasts were observed in comparison to untreated plants. The guard cells of stomata of the Clark variety were seriously affected. The chlorophyll content of diuron-treated plants was sharply lowered. On the other hand, proline, soluble protein, and carbohydrate contents of leaves were increased in diuron-treated plants. The protein pattern of treated leaves of the two cultivars was altered, and the most profound change is the appearance of a new ∼207-kDa polypeptide.

Induction of novel organelle DNA variation and transfer of resistance to frost and Verticillium wilt in Solanum tuberosum through somatic hybridization with 1EBN S. commersonii

Somatic hybridization can be used to induce genetic variability in plastidial and mitochondrial genomes, and transfer multiple uncloned genes across sexual barriers. Somatic hybrids were produced between a dihaploid clone of the common potato, S. tuberosum subsp. tuberosum, and the wild sexually incongruent diploid species S. commersonii. Fusion products were selected on the basis of callus growth and regeneration in vitro. Genome composition of putative somatic hybrids was determined by flow cytometric analysis of nuclear DNA content, RAPD analysis, and Southern analysis with probes specific to organellar DNA. All regenerated fusion products proved to be hybrids based on RAPD analysis. Seventy per cent of somatic hybrids were (near) tetraploids, 22% (near) hexaploids and 8%(near) octoploids. A high correlation was found between the nuclear DNA content determined by flow cytometry and the number of chloroplasts in stomata guard cell pairs. Somatic hybrids inherited the parental plastids in a random manner. On the contrary, they preferentially inherited the mitochondrial DNA fragments of S. tuberosum. The majority of them had a rearranged mitochondrial genome with fragments from both parents. Hybrids were highly vigorous and morphologically more similar to the cultivated than to the wild parent, produced tubers on long stolons under long photoperiod conditions, showed a high degree of flowering, but did not produce pollen. In addition, somatic hybrids were generally more resistant to frost and Verticillium wilt than the cultivated parent, indicating the introgression of relevant resistance genes from the wild species into the genetic background of S. tuberosum.

Heterologous and homologous transgenic expression directed by a 2S seed storage promoter of Brassica napus

Few plant genes have been analysed in both homologous and heterologous transgenic systems. In this study, deletion mutants of the storage protein promoter napA fused to the receptor gene uidA (GUS) were analysed for their ability to direct tissue-specific expres sion in transgenic tobacco as well as transgenic Brassica napus. In seeds, qualitatively similar results have previously been obtained, demonstrating that transcription factors in the heterologous tobacco system recognized the napA promoter cis elements, more or less in the same way as in B. napus (Ellerstrom et al., 1996; Stalberg et al., 1996). However, in anthers of the transgenic plants, clear differences were noted. The napA promoter constructs were inactive in transgenic B. napus anthers. In contrast, tobacco anthers displayed activities of similar magnitudes to those previously found in the seed for the respective promoter constructs. Interestingly, in seven constructs the activity in the anthers was retained dow nstream from an imperfect ABRE element, whereas no activity could be detected in the seed. Another clear difference was that a region from −211 to −152 silenced the expression in anthers whereas this region had no effect on the activity in the seed. Likewise, in tobacco the napA promoter showed a low activity in leaves. Histochemical staining of young tobacco leaves showed that this activity was considerably higher in stomata guard cells than in the mesophyll cells while the leaves of the B. napus plants had a diffuse and barely detectable staining in the mesophyll cells. The high level of napA transcription in tobacco anthers indicates that the set of transcription factors and corresponding cis-sequences that direct tissue-specific transcription in this organ are similar to those responsible for seed-specific expression. However, comparison of the levels of expression in anthers and seeds in individual plants revealed that there was no correlation between the activities in the two organs, which suggests that positional effects influence the transcription complexes differently in seeds and anthers. Further, this study shows that careful analysis of expression directed by promoter mutants in a heterologous transformation system might reveal important cis-elements, not discernible in the tighter homologous situation

Actin filaments of guard cells are reorganized in response to light and abscisic acid.

We recently showed that treatment with actin antagonists perturbed stomatal behavior in Commelina communis L. leaf epidermis and therefore suggested that dynamic changes in actin are necessary for signal responses in guard cells (M. Kim, P.K. Hepler, S.O. Eun, K.-S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Here we show that actin filaments of guard cells, visualized by immunofluorescence microscopy, change their distribution in response to physiological stimuli. When stomata were open under white-light illumination, actin filaments were localized in the cortex of guard cells, arranged in a pattern that radiates from the stomatal pore. In marked contrast, for guard cells of stomata closed by darkness or by abscisic acid, the actin organization was characterized by short fragments randomly oriented and diffusely labeled along the pore site. Upon abscisic acid treatment, the radial pattern of actin arrays in the illuminated guard cells began to disintegrate within a few minutes and was completely disintegrated in the majority of labeled guard cells by 60 min. Unlike actin filaments, microtubules of guard cells retained an unaltered organization under all conditions tested. These results further support the involvement of actin filaments in signal transduction pathways of guard cells.

Ultrastructural detection of sucrose synthase distribution in developing maize leaves

A developing maize leaf grows by the activity of a basal meristematic region and an adjacent elongating zone, resulting in a morphological and functional gradient along the leaf. We have used this system to detect the spatial and temporal expression of an enzyme, sucrose synthase, which plays a pivotal role in the sucrose import-export transition which occurs along a monocotyledon leaf. Immunogold labeling was used to detect the cellular and sub-cellular distribution of sucrose synthase (SS) at the electron microscopical level; the protein was visualized using a polyclonal antiserum on embedded tissue sections. Immunolabel was observed in the cytosol of dividing meristematic cells, expanding cells of the elongation zone, and in differentiating cells of young photosynthetic tissue. In fully differentiated leaf tissue, however, the protein was no longer immuno-detectable in photosynthetic cells, but was present in the guard and subsidiary cells of stomata and in companion cells within the phloem tissue of vascular bundles. The tissue- and cell-specific localization of sucrose synthase changes along the growing leaf as a function of the developmental state and the associated need for sucrose import or export.

A new species of Eoginkgoites from the Upper Triassic of North Carolina, USA

The foliage genus Eoginkgoites represents a superficially ginkgoalean bennettitalean leaf restricted to the Late Triassic of North America. A new species, Eoginkgoites sydneyi is described from the Pekin Formation in North Carolina. The number of lateral pinna pairs is typically four, but varies from three to five. Previously described species have a maximum of three lateral pairs. The new form is most similar in morphology to E. gigantea from the upper Stockton Formation of Pennsylvania. Eoginkgoites sydneyi and the Chinle Formation species E. davidsonii share several cuticular features unusual to the Bennettitales including straight anticlinal epidermal cell walls and radiating striations on the stomata guard cell thickenings. The additional pinna pairs of the new species suggests support for the hypothesis that the Eoginkgoites leaf morphology was derived from a typical pinnate form by loss of lower pinnae and shortening of the rachis. Recognition of this unusual leaf morphology in the Triassic provides further evidence for an emerging picture of greater morphological diversity in the early Bennettitales.

Anther wall layers control pollen sugar nutrition inLilium

Anthers ofLilium were for the first time investigated at the ultrastructural level in order to appreciate the possible ways of sugar transport in the microsporangium. Our results have shown that the cells of the outer anther wall layers and the cell of the connective were interconnected by plasmodesmata, thus allowing assimilates to travel through the symplasmic pathway from the vascular bundle to the most internal middle layer (ML 1). ML 1 was devoid of cell communication throughout pollen development. Tapetal cells were also lacking plasmodesmata on their external face towards ML 1, but adjacent tapetal cells developed lateral junctions: the tapetum could represent a syncytium. Sugars destinated to pollen in the loculus have then to cross the ML 1 and the tapetal layers by the apoplasmic pathway; it is suggested that these two envelopes could be involved in the control of sugar transport from the outer anther wall layers to the locular fluid. Before microspore mitosis, the tapetum degenerated but ML 1 remained structurally unchanged. During pollen development, the guard cells of stomata were lacking cell communication, and preserved their starch content, which could be the sign of photosynthesis within the anther wall. In order to check whether these structural disconnections in anther tissues corresponded to physiological barriers, isolated pollen and stamens were cultivated during the anther maturation phase, on a medium containing increasing concentrations of sucrose (0 M, 1/6 M, 1/2 M, 1 M). After 7 days of culture, isolated pollen was engorged with starch grains and was unable to germinate, whereas in cultivated stamens, pollen did not contain any starch grain: sporophytic tissues, however, accumulated abnormal amylaceous reserves. These results strongly suggest that the anther wall layers, in particular ML 1, starve pollen with sugars during its maturation. They are acting as a physiological buffer storing nutriment surplus in starch grains.

The blue light‐responsive AthH2 gene of Arabidopsis thaliana is primarily expressed in expanding as well as in differentiating cells and encodes a putative channel protein of the plasmalemma

According to our previous studies the Arabidopsis gene AthH2 which is inducible by blue light and phytohormones codes for an intrinsic membrane protein. It bears a resemblance to several distinct channel proteins of plant and animal species classified as the MIP/NOD-26/GlpF family. In the present study biochemical analyses and electron microscopic immunochemistry were used to elucidate the subcellular location of the AthH2 protein. The results clearly demonstrate that it is an exclusive constituent of the plasmalemma. Furthermore, the expression of the AthH2 gene in transgenic Arabidopsis plants containing the promoter region of AthH2 fused to the beta-glucuronidase (gus) reporter gene was studied. The in situ localization of gus activity revealed that the specific promoter is temporally activated by light in expanding and/or differentiating cells comprising newly formed tissues and organs: root elongation zone, guard cells of stomata, vascular bundle sheaths, filaments of stamen and young siliques. Several sites of gus expression coincide spatially with those of in situ hybridization and the immunocytochemical reaction, respectively, suggesting that the AthH2 promoter had correctly responded to light as an important exogenous factor with relevance to the complex pattern of differentiation. Studies with protoplasts from plants transformed with an antisense construct revealed a water transport capacity of the AthH2 protein.

Reproduction and continuity of chloroplasts in spermatophytes

Without the continuity of chloroplasts there would be no higher life. Chloroplast reproduction and continuity can be studied in the guard cells of stomata, utilizing the result of reproduction, viz., the number of chloroplasts. An annotated list of such numbers from 261 families of spermatophytes is provided. For each family and many subfamilies or tribes are given the numbers of genera and species investigated as well as the ranges and medians of 1909 generic and 6161 specific chloroplast numbers, predominantly from my own countings. The driving forces behind the reproduction and continuity of chloroplasts are reviewed and discussed in the light of some figures of the list and other evidence. The results support the hypothesis that nuclear DNA replication paves the way for chloroplast reproduction and that the nuclear DNA amount is instrumental in ensuring the continuity of chloroplasts throughout meristematic cell successions.

Rha1, a gene encoding a small GTP binding protein from Arabidopsis, is expressed primarily in developing guard cells.

The rha1 gene from Arabidopsis encodes a small GTP binding protein belonging to the Ypt/Rab family. Transgenic Arabidopsis plants containing the promoter region of the rha1 gene fused to the beta-glucuronidase (gus) reporter gene revealed gus expression limited mainly to the guard cells of stomata, the stipules, and the root tip of young plants. In flowering plants, expression was found predominantly in the receptacle and in guard cells of the different flower organs. High GUS activity could also be seen in callus tissue and developing seeds. No detectable activity was present in other plant tissues; activity could not be induced by various treatments. GUS activity was visualized histochemically using both 5-bromo-4-chloro-3-indolyl beta-D-glucuronide and a newly developed GUS substrate: Sudan II-beta-glucuronide. The latter precipitates as red crystals at the site of GUS activity. Results obtained by the gus analysis were confirmed by whole-mount mRNA in situ hybridization. A hypothesis for the function of the Rha1 protein is discussed.

Heterogeneous growth of plant tissues

Heterogeneous growth is defined as different rates or patterns of growth in adjacent tissue regions, in contrast to homogeneous growth where a region expresses a uniform rate or pattern of growth. Heterogeneous growth is inspected in a variety of plant tissues and the pattern of expansion is characterized for each. In the case of epidermal cell proliferation, different growth rates for cell plates and old walls lead to the feature of coordinated growth in which slow growth of the former is compensated for by a faster rate of the latter. Examples include leaf epidermal cells above veins growing differently from those above areole regions, and pairs of guard cells of stomata ceasing to expand before other epidermal cells. In the alga Coleochaete only marginal walls grow, and at different rates around the colony, to generate a fractal, stochastic type of coordinated growth. In the fern gametophyte there are complex gradients of differential growth rates. Epidermal cells of apices are often of mixed growth, as cells at the summit undergo two dimensional expansion while cells along the flanks express one dimensional expansion. Coordinated growth requires matched rates where the constraining effect of the slower growing region is compensated for by a faster rate in an enacircling region compared to the average rate of the overall tissue. Mixed and differential growth patterns do not necessarily create constraints and so lead to smooth tissue expansion. Emergence of some constraints leads to breaking of symmetry and disruptive growth as in the appearance of new axes found in organs and epidermal derivatives. In planar development heterogeneous growth appears to be the rule, and homogeneous growth the exception.

Heterogeneous growth of plant tissues

Heterogeneous growth is defined as different rates or patterns of growth in adjacent tissue regions, in contrast to homogeneous growth where a region expresses a uniform rate or pattern of growth. Heterogeneous growth is inspected in a variety of plant tissues and the pattern of expansion is characterized for each. In the case of epidermal cell proliferation, different growth rates for cell plates and old walls lead to the feature of coordinated growth in which slow growth of the former is compensated for by a faster rate of the latter. Examples include leaf epidermal cells above veins growing differently from those above areole regions, and pairs of guard cells of stomata ceasing to expand before other epidermal cells. In the alga Coleochaete only marginal walls grow, and at different rates around the colony, to generate a fractal, stochastic type of coordinated growth. In the fern gametophyte there are complex gradients of differential growth rates. Epidermal cells of apices are often of mixed growth, as cells at the summit undergo two dimensional expansion while cells along the flanks express one dimensional expansion. Coordinated growth requires matched rates where the constraining effect of the slower growing region is compensated for by a faster rate in an enacircling region compared to the average rate of the overall tissue. Mixed and differential growth patterns do not necessarily create constraints and so lead to smooth tissue expansion. Emergence of some constraints leads to breaking of symmetry and disruptive growth as in the appearance of new axes found in organs and epidermal derivatives. In planar development heterogeneous growth appears to be the rule, and homogeneous growth the exception.

Visualizing Changes in Cytosolic-Free Ca2+ during the Response of Stomatal Guard Cells to Abscisic Acid.

In this paper, we report the results of a detailed investigation into abscisic acid (ABA)[mdash]stimulated elevations of guard cell cytosolic-free Ca2+ ([Ca2+]cyt). Fluorescence ratio photometry and ratio imaging techniques were used to investigate this phenomenon. Guard cells of open and closed (opened to 10 to 12 [mu]m before treatment with ABA) stomata were microinjected with the fluorescent Ca2+ indicator Indo-1. Resting [Ca2+]cyt ranged from 50 to 350 nM. ABA (100 nM) stimulated an increase in [Ca2+]cyt in 68 and 81% of guard cells microinjected in the open and closed configuration, respectively. All stomata were observed to close in response to ABA. Increases ranged from 100 to 750 nM above the resting concentration and were arbitrarily grouped into five "classes." ABA-stimulated increases in [Ca2+]cyt were not uniformly distributed across the cytosol of guard cells. Rapid transient increases in [Ca2+]cyt were also observed in the guard cells of stomata microinjected in the closed configuration. We concluded that the ABA-induced turgor loss in guard cells is a Ca2+-dependent process.

Nitrogen Supply and Chloroplast Reproduction.

Dicotyledonous plants usually found in habitats with more nitrogen were showing fewer, not more, chloroplasts in their guard cells of stomata, on average. Obviously, this effect is not one of nitrogen as a nutrient.-Ammonium sulfate added to nitrophilous plants with low chloroplast numbers increased these numbers, whereas nitrophilous plants with high numbers and non-nitrophilous plants with any numbers did not react. Some species need more nitrogen for reproducing their chloroplasts in guard cells to the specific level than do other species.

Cell Sizes and Chloroplast Numbers per Cell of Hemiploid and Polyploid Plants.

Whereas guard cells of stomata of induced polyploids in the C0 generation show on average more than twice the volume of diploids, the volume is reduced in hemiploids to less than one half when compared with normaploid plants. The shortage cannot be explained as the result of an unspecific disorder of newly induced heteroploids because hemiploids and polyploids show opposite effects. Since the surplus disappears in later generations, both surplus and shortage might be overshoots on the way to new steady states. The chloroplast numbers of mature guard cells do not show such an effect; they therefore do not depend mainly on cell size. Rather, both characters depend on ploidy. Further evidence is discussed. In a double-logarithmic grid, chloroplast numbers and cell volumes in C0 generation are correlated linearly with ploidy, showing different slopes. Since nuclear and chloroplast DNA cooperate in the synthesis of many chloroplast proteins-and most envelope proteins are encoded in the nucleus-the nuclear DNA amount is coupled with the growth and division of plastids. Thus the continuity of plastids may result.