Guard Cell Chloroplasts Research Articles

Genomic constancy during the development of Lathyrus odoratus cultivars

The sweet pea, Lathyrus odoratus, has a well-documented history of the development of its cultivars in various parts of the world. Measurements of genome size, using flow cytometry, and observations on the karyotypes, as well as the analysis of meiotic pairing of F1 hybrids (between the wild sweet pea and two modern cultivars), all show that this development has not been accompanied by changes in karyotype or genome size. However, the cultivars do differ in pollen size and guard cell chloroplast number, both characteristics that have been suggested to be controlled by the nucleotype; however, this is clearly not the case in L. odoratus.

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.

Polyploidy and Aneuploidy in the Tamarillo, Cyphomandra betacea (Cav.) Sendt. (Solanaceae).

AbstractThe occurrence of spontaneous polyploidy was quantified in the tamarillo (Cyphomandra betacea [Cav.] Sendt.), a diploid (2n = 2x = 24) solanaceous fruit crop cultivated in New Zealand. Polyploids of this species were recognized by their small, almost seedless fruit, these types occurring at a frequency of 0.24 % in seedling orchards. Parallel spindles were seen on rare occasions during meiotic anaphase II in diploid tamarillos and were the probable cause of spontaneous polyploidy in this crop. Pollen volume and viability, seed number per fruit, seed weight, fruit size, stomatal length and guard cell chloroplast number were measured in diploids and polyploids. The polyploids typically showed the manifestation of the gigas effect, with thicker leaves and larger flowers. Pollen volume, stomatal length and guard cell chloroplast number also increased with higher ploidy. Pollen viability was markedly lower in the polyploids compared to the diploids and in combination with post‐zygotic abnormalities caused a reduction in seed set and hence fruit size. Improvements in fertility and thus fruit size would be needed if the polyploids were to have commercial potential.

Low temperature acclimation of guard cell chloroplasts by the arctic plant Saxifraga cernua L.

Abstract. The potential for thermal acclimation of photosynthetic electron transport by guard cell chloropiasts (GC ch) was assessed in epidermal peels taken from the abaxial side of Saxifraga cernua leaves grown at 20°C and 10°C. Chlorophyll a fluorescence induction kinetics measured in pairs of guard cells in individual stomata from tissue grown at 10 °C demonstrated a rise in the fluorescence to a maximum and a larger amplitude in variable fluorescence when measured at temperatures below 18°C than was seen in GC ch from tissue grown at 20°C. The rates of fluorescence quenching in 10°C-grown tissue were also faster than in 20°C-grown tissue when measured at temperatures below 18°C. State 1-State 2 transitions by GC ch were measured at selected temperatures between 5 and 25 °C as changes in the magnitude of the fluorescence emission maxima at 685, 695 and 730nm (F685, F695 and F730) measured at 77K. At measuring temperatures of 5 and 10°C, GC ch in tissue grown at 10 °C showed a greater transition to State 2 (a larger F730/F695 ratio) than did GC ch in tissue grown at 20 °C. At measuring temperatures of 20 and 25 °C, there was no difference in either the kinetics or the magnitude of the State 1 to State 2 transition in the two tissues. The ultrastructure of GC ch from tissues grown at 10 and 20 °C was also examined using transmission electron microscopy. Less than half (48%) of the grana from the higher temperature grown tissue had more than nine thylakoids/grana. Grana in GC ch which had developed at 10 °C showed a dramatic reduction in stacking, such that 85% of the grana contained no more than two thylakoids. The reduction in grana stacking was also accompanied by a decrease in the degree of appression of thylakoid membranes. The results demonstrate a capacity for thermal acclimation of GC ch function to low temperatures. This acclimation is associated with alterations in the chloroplast ultrastructure.

Blue Light-Modulation of Chlorophyll a Fluorescence Transients in Guard Cell Chloroplasts

Chlorophyll a fluorescence transients from mesophyll and single guard cell pairs of Vicia faba were measured by microspectrofluorometry. In both chloroplast types, fluorescence induction (O to P) was similar under actinic blue and green light. In slow transients from mesophyll cell chloroplasts, blue and green light induced identical, typical rapid quenching from P to S, and the M peak. In contrast, the P to S transient from guard cell (GC) chloroplasts irradiated with blue light showed a much slower quenching rate, and the P to T transition showed no M peak. Actinic green light induced mesophyll-like transients in GC chloroplasts, including rapid quenching from P to S and the M peak. Detection of these transients in single pairs of GC and isolated protoplasts ruled out mesophyll contamination as a signal source. Green light induced a rapid quenching and the M peak in GC chloroplasts from several species. The effect of CO(2) concentration on the fluorescence transients was investigated in the presence of HCO(3) (-) at pH 6.8 and 10.0. In transients induced by green light in both chloroplast types, a pH increase concomitant with a reduction in CO(2) concentration caused an increase in the initial rate of quenching and the elimination of the M peak. Actinic blue light induced mesophyll-like transients from GC chloroplasts in the presence of 10 micromolar KCN, a concentration at which the blue light-induced stomatal opening is inhibited. Addition of 100 to 200 micromolar phosphate also caused large increases in fluorescence quenching rates and a M peak. These results indicate that blue light modulates photosynthetic activity in GC chloroplasts. This blue light effect is not observed in the absence of transduction events connected with the blue light response and in the presence of high phosphate concentrations.

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L

A mass spectrometric method combining (16)O/(18)O and (12)C/(13)C isotopes was used to quantify the unidirectional fluxes of O(2) and CO(2) during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O(2) uptake and CO(2) evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O(2) (61 micromoles of O(2) per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O(2) per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO(2) in darkness at a rate of 27 micromoles of CO(2) per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O(2) evolution and CO(2) fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO(2) evolution by guard cell protoplasts was sharply decreased (37%), while O(2) uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO(2) assimilation and may be dissipated for other purposes such as ion uptake.

Number of Chloroplasts in Haploids and Diploids Produced via Anther Culture in Brassica campestris.

Microspore-derived plants were produced via anther culture in Brassica campestris. The cytological analysis of these plants revealed the existence of diploids, tetraploids as well as haploids. In these plants, the number of chloroplasts in guard cells were clearly related to the ploidy level. The number of chloroplasts in mesophyll protoplasts was also related to the ploidy level, though the relationship was not as clear as in guard cells. The number of chloroplasts in both types of cells increased after the number of chromosomes was artificially doubled with colchicine.

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.

The Use of Stomatal Chloroplast Number for Rapid Determination of Ploidy Level in Maize

AbstractThe usefulness of using the chloroplast number in epidermal guard cells as an indirect ploidy indicator was evaluated on seed‐grown and tissue culture‐derived maize plants. For seed‐grown plants, two maize genotypes (B89 and R75) which had both diploid and tetraploid seeds available were used as experimental materials. The ploidy levels of seed‐grown plants were confirmed by flow cytometric analysis of nuclear suspensions from these plants. For regenerated plants, haploid and diploid levels were examined and the ploidy levels of these plants were determined by chromosome counts of cells from root tips. A positive relationship between the chloroplast number and ploidy level was observed for both seed‐grown and regenerated plants. The stomatal guard cells of tetraploid plants had nearly double the number of chloroplasts as the diploid plants. Similar results were found from the regenerated plants. The differences in the mean chloroplast number between diploid and tetraploid seed‐grown plants and between haploid and diploid regenerated plants were highly significant. The results of this study demonstrate that counting chloroplasts in guard cells can be an efficient means of screening a large number of plants for ploidy levels. In addition, this study suggests the possibilities of using this method for detecting contaminated seed lots by different ploidy seed and for distinguishing plants of different genotypes.

Light perception in guard cells

Abstract. Guard cells perceive light via two photoreceptor systems: a blue‐light‐dependent photosystem and the guard cell chloroplast. Chloroplasts stimulate stomatal opening by transducing photosynthetic active radiation into proton pumping at the guard cell plasma membrane. In addition, guard cell chloroplasts fix CO2 photosynthetically. Sugar from guard cell photosynthesis can contribute to the osmotic build‐up required for opening. The blue‐light‐dependent photosystem activates proton pumping at the guard cell plasma membrane and stimulates starch hydrolysis. Available information on the photobiological properties of guard cells makes it possible to describe stomatal function in terms of the cellular components regulating stomatal movements. The blue light response is involved in stomatal opening in the early morning and stomatal responses to sunflecks. The guard cell chloroplast is likely to be involved in stomatal adaptations to sun, shade and to temperature. Interactions between these photosystems, a third photoreceptor in guard cells, phytochrome, and other mechanisms transducing stomatal responses such as VPD and carbon dioxide, provide the cellular basis for stomatal regulation.

The control of chloroplast number in Solanum muricatum Ait. and Cyphomandra betacea (Cav.) Sendt. and its value as an indicator of polyploidy

Guard cell chloroplast numbers were counted in the tamarillo (Cyphomandra betacea (Cav.) Sendt.), the pepino (Solanum muricatum Ait.), and in wild relatives and polyploids of these species. Chloroplast number was found to be a reliable indicator of the euploid level in both species, but in aneuploids the effects of different chromosomes were variable. Some had no effect but in others the effects werepositive and sometimes significantly different from the diploids. Comparison of DNA amounts and plastid numbers in wild Cyphomandra species did not reveal a high correlation between these two characters. These results argue against a purely nucleotypic control of chloroplast number in guard cells in favour of a more complex genotypic system.

COMPARISON OF TETRAPLOID AND SINGLE GENE‐INDUCED GIGAS VARIANTS IN CHICKPEA (CICER ARIETINUM). I. ORIGIN AND GENETIC CHARACTERIZATION

Two genetic variants with increased organ size were independently derived from diploid (2n = 2x = 16) chickpea (Cicer arietinum L.) line ICC 640. Radiation‐induced mutant PM1101 had greatly enlarged leaves, leaflets, and pods, and an elongated stem with longer internodes but fewer nodes than ICC 640. F1, F2, and F3 data from crosses with ICC 640 showed that the mutant characteristics of PM1101 were the pleiotropic effects of a single, recessive genetic factor. For purposes of comparison, tetraploid derivatives of ICC 640 were produced by colchicine treatment of seed. In the tetraploids, leaflets and pods were enlarged, but less dramatically than in PM1101. Enlarged pollen grains and stomatal guard cells, and increased guard cell chloroplast number were found in tetraploids but not in PM1101, while both variants produced fewer seeds than ICC 640. Mutant PM1101 and the tetraploids represent two very different manifestations of gigantism in chickpea.

Tentoxin Suppresses Stomatal Opening by Inhibiting Photophosphorylation

Ten toxin and, to a lesser extent, dihydrotentoxin (both at lOmmol m~3) reduce stomatal opening in epidermal strips of Commelina communis in the light but not in darkness. This effect was significantly greater in normal air than in C02-free air. Fusicoccin overcame the tentoxin effect. However, tentoxin did not inhibit stomatal opening in the light in epidermal strips of Paphiopedilum harrisianum, a species which lacks guard cell chloroplasts. It is concluded that tentoxin exerts its action on stomata not by an ionophorous effect in the plasmalemma of guard cells but by the inhibition of photophosphorylation in their chloroplasts. The effects of DCMU and tentoxin on guard cells are discussed in terms of their effects on chloroplasts and the extent to which energy is supplied from this organelle during stomatal opening in the light. The results indicate that neither photophosphorylation nor non-cyclic electron transport in guard cell chloroplasts are essential for stomatal opening.

Calvin Benson Cycle Enzymes in Guard-cell Protoplasts and Their Role in Stomatal Movement

Calvin-Benson cycle enzymes were found in Vicia guard-cell protoplasts. On the basis of enzyme activities and rates of photosynthetic O2 evolution in guard-cell and mesophyll protoplasts, we conclude that guard-cell chloroplasts utilize largely the phosphoglycerate (PGA)/dihydroxyacetone phosphate (DHAP) shuttle for the transfer of ATP and reducing equivalents from chloroplasts to the cytosol. In guard cells, PGA formation by photosynthetic CO2 fixation may act as a starter of the PGA/DHAP shuttle across the chloroplast envelope in synchronization with the photophosphorylation, thereby driving the stomatal opening.

Calvin-Benson Cycle Enzymes in Guard-Cell Protoplasts from Vicia faba L

Activities of Calvin-Benson cycle enzymes were found in protoplasts of guard cells from Vicia faba L. The activities of NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD) and ribulose-1,5-bisphosphate carboxylase (RuBPC) were 2670 and 52 micromoles per milligrams chlorophyll per hour, respectively. Activities of NADP-GAPD and RuBPC in guard cells were increased by red light illumination, and the light activations were inhibited completely by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. Enzymes related to the Calvin-Benson cycle such as 3-phosphoglycerate kinase (PGAK), triose phosphate (TP) isomerase, and fructose-1,6-bisphosphatase (FBPase) were shown to be present in guard-cell chloroplasts. From these results, we conclude that the photosynthetic carbon reduction pathway is present in guard-cell chloroplasts of Vicia faba. We compared these enzyme activities in guard cells with those in mesophyll cells. The activities of NADP-GAPD and PGAK were more than several-fold higher and that of TP isomerase was much higher in guard-cell chloroplasts than in mesophyll chloroplasts. In contrast, activities of RuBPC and FBPase were estimated to be roughly half of those in mesophyll chloroplasts. High activities of PGAK, NAD-GAPD, and TP isomerase were found in fractions enriched in cytosol of guard cells. Illumination of guard-cell protoplasts with red light increased the cellular ATP/ADP ratio from 5 to 14. These results support the interpretation that guard cells utilize a shuttle system (e.g. phosphoglycerate [PGA]/dihydroxyacetone phosphate [DHAP] shuttle) for an indirect transfer of ATP and reducing equivalents from chloroplasts to the cytosol.

Photosystem II in Guard Cells of Vicia faba: Immunological Detection

Antibodies were raised against individual polypeptides of the oxygen-evolving photosystem II (PSII) complex from mesophyll chloroplasts of Vicia faba (Long Pod). These antibodies were used to probe immunologically for the presence of the main structural components of the PSII complex in guard cell chloroplasts, using both immunofluorescence microscopy and Western blotting. Immunofluorescence of epidermal peels with antibodies raised against the extrinsic 33 kilodalton polypeptide, as well as the 47 and the 44 kilodalton subunits and the light-harvesting chlorophyll a/b protein, resulted in intense fluorescence indicating the presence of these polypeptide components in guard cell chloroplasts. Results obtained with Western blot analysis showed that the relative amounts of the 33 kilodalton and light-harvesting complex protein polypeptides are between 60 and 80% of that found in mesophyll cells (on chlorophyll basis). These results provide evidence for the existence of structural components associated with PSII activity in guard cell similar to those of mesophyll chloroplasts.

Immunogold Localization of Chloroplast Protein in Spinach Leaf Mesophyll, Epidermis, and Guard Cells

AbstractImmunogold labelling on ultrathin sections of Lowicryl K4M embedded lower epidermis of spinach (Spinacia oleracea L.) with adhering mesophyll cells has been applied to localize five proteins, namely ribulose‐1,5‐bisphosphate carbocylase/oxygenase (EC 4.1.1.39), chloroplast coupling factor (EC 3.6.1.34), NADP‐malate dehydrogenase (EC 1.1.1.82), chloroplast fructose‐1,6‐bisphosphatase (EC 3.1.3.11) and thioredoxin m by electron microscopy in stomata. Comparative data of labelling densities of the localized proteins in chloroplasts of spongy parenchyma, epidermis and guard cells are presented. All five proteins could be detected in guard cell chloroplasts at levels little different from those found in chloroplasts of spongy parenchyma and of epidermis cells. The two light‐activated enzymes NADP‐malate dehydrogenase and fructose‐1,6‐bisphosphatase are reduced in stomata to about one third of the mesophyll concentration. Aldehyde‐sensitivity of the inspected enzymes and suitability of cryosections for immunolocalization studies on plant tissues, in comparison with resin‐embedded specimens, was investigated. Fixation with 2% glutaraldehyde resulted in no detectable decrease of antigenicity. Cryosections were found to be less suitable for immunogold labelling, since labelling densities were lower than on resin. Only labelling of the thylakoid‐bound coupling factor was comparable using both techniques.

Light Quality and Osmoregulation in Vicia Guard Cells

Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K(+) content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of -0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K(+) content or starch degradation in guard cell chloroplasts. At 10 micromoles per square meter per second, blue light caused a net 6.8 micrometer increase in aperture over 5 hours and there was a substantial decrease in starch content of chloroplasts but no increase in guard cell K(+) content. At 25 micromoles per square meter per second of blue light, apertures increased faster (net gain of 5.7 micrometers after 1 hour) and starch content decreased. About 80% of guard cells had a higher K(+) content after 1 hour of incubation but that fraction decreased to 10% after 5 hours. In the absence of KCl in the incubation medium, stomata opened slowly in response to 25 micomoles per square meter per second of blue light, without any K(+) gain or starch loss. In dual beam experiments, stomata irradiated with 50 micomoles per square meter per second of red light for 3 hours opened without detectable starch loss or K(+) gain; addition of 25 micomoles per square meter per second of blue light caused a further net gain of 4.4 micometers in aperture accompanied by substantial K(+) uptake and starch loss. Comparison of K(+) content in guard cells of opened stomata in epidermal peels with those induced to open in leaf discs showed a substantially higher K(+) content in the intact tissue than in isolated peels. These results are not consistent with K(+) (and its counterions) as the universal osmoticum in guard cells of open stomata under all conditions; rather, the data point to sugars arising from photosynthesis and from starch degradation as additional osmotica. Biochemical confirmation of these findings would indicate that osmoregulation during stomatal opening is the result of three key metabolic processes: ion transport, photosynthesis, and sugar metabolism.

Is ribulose bisphosphate carboxylase present in guard cell chloroplasts

Evidence for and against the presence of ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in guard cell chloroplasts is presented. Methods to investigate this question, including immunocytochemistry, are compared.

Enhancement of the Stomatal Response to Blue Light by Red Light, Reduced Intercellular Concentrations of CO2, and Low Vapor Pressure Differences

The effects of environmental parameters on the blue light response of stomata were studied by quantifying transient increases in stomatal conductance in Commelina communis following 15 seconds by 0.100 millimole per square meter per second pulses of blue light. Because conductance increases were not observed following red light pulses of the same or greater (30 seconds by 0.200 millimole per square meter per second) fluences, the responses observed could be reliably attributed to the specific blue light response of the guard cells, rather than to guard cell chlorophyll. In both Paphiopedilum harrisianum, which lacks guard cell chloroplasts, and Commelina, the blue light response was enhanced by 0.263 millimole per square meter per second continuous background red light. Thus, the blue light response and its enhancement do not require energy derived from red-light-driven photophosphorylation by the guard cell chloroplasts. In Commelina, reduction of the intercellular concentration of CO(2) by manipulation of ambient CO(2) concentrations resulted in an enhanced blue light response. In both Commelina and Paphiopedilum, the blue light response was decreased by an increased vapor pressure difference. The magnitude of blue-light-specific stomatal opening thus appears to be sensitive to environmental conditions that affect the carbon and water status of the plant.