Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels

Controversies regarding the function of guard cell chloroplasts and the contribution of mesophyll in stomatal movements have persisted for several decades. Here, by comparing the stomatal opening of guard cells with (crl‐ch) or without chloroplasts (crl‐no ch) in one epidermis of crl (crumpled leaf) mutant in Arabidopsis, we showed that stomatal apertures of crl‐no ch were approximately 65–70% those of crl‐ch and approximately 50–60% those of wild type. The weakened stomatal opening in crl‐no ch could be partially restored by imposing lower extracellular pH. Correspondingly, the external pH changes and K⁺ accumulations following fusicoccin (FC) treatment were greatly reduced in the guard cells of crl‐no ch compared with crl‐ch and wild type. Determination of the relative ATP levels in individual cells showed that crl‐no ch guard cells contained considerably lower levels of ATP than did crl‐ch and wild type after 2 h of white light illumination. In addition, guard cell ATP levels were lower in the epidermis than in leaves, which is consistent with the observed weaker stomatal opening response to white light in the epidermis than in leaves. These results provide evidence that both guard cell chloroplasts and mesophyll contribute to the ATP source for H⁺ extrusion by guard cells.     

Structural and functional aspects of stomata

Differential cell wall thickening in developing guard cells of Polypodium vulgare L. has been studied with particular reference to guard cell protoplast deformation and the eventual formation of the stomatal pore. Concomitant studies on the development of guard cell chloroplasts and their starch inclusions during ontogeny of the stomatal complex have provided data which have been incorporated into a model to account for the formation of the pore. Guard cell starch inclusions reach a maximum density per unit volume at the same time as the guard cell walls achieve maximum differential thickening. These events coincide with the development of the pore. It is suggested that, whilst pore formation is initiated enzymatically, the mechanical forces required to bring about the separation of the two guard cells are of an osmotic nature derived from starch hydrolysis. The development of the mesophyll in relation to the epidermis is examined in respect of the formation of substomatal chambers.     

Ultrastructure of stomatal development in Arabidopsis (Brassicaceae) leaves

Stomatal development was studied in wild-type Arabidopsis leaves using light and electron microscopy. Development involves three successive types of stomatal precursor cells: meristemoid mother cells, meristemoids, and guard mother cells (GMCs). The first two types divide asymmetrically, whereas GMCs divide symmetrically. Analysis of cell wall patterns indicates that meristemoids can divide asymmetrically a variable number of times. Before meristemoid division, the nucleus and a preprophase band of microtubules become located on one side of the cell, and the vacuole on the other. Meristemoids are often triangular in shape and have evenly thickened walls. GMCs can be detected by their roughly oval shape, increased starch accumulation, and wall thickenings on opposite ends of the cells. Because these features are also found in developing stomata, stomatal differentiation begins in GMCs. The wall thickenings mark the division site in the GMC since they overlie a preprophase band of microtubules and occur where the cell plate fuses with the parent cell wall. Stomatal differentiation in Arabidopsis resembles that of other genera with kidney-shaped guard cells. This identification of stages in stomatal development in wild-type Arabidopsis provides a foundation for the analysis of relevant genes and of mutants defective in stomatal patterning, cell specification, and differentiation.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.     

Slow photosynthetic induction and low photosynthesis in Paphiopedilum armeniacum are related to its lack of guard cell chloroplast and peculiar stomatal anatomy

Paphiopedilum and Cypripedium are close relatives in the subfamily Cypripedioideae. Cypripedium leaves contain guard cell chloroplasts, whereas Paphiopedilum do not. It is unclear whether the lack of guard cell chloroplasts affects photosynthetic induction, which is important for understory plants to utilize sunflecks. To understand the role of guard cell chloroplasts in photosynthetic induction of Paphiopedilum and Cypripedium, the stomatal anatomy and photosynthetic induction of Paphiopedilum armeniacum and Cypripedium flavum were investigated at different ratios of red to blue light. The highest stomatal opening and photosynthesis of intact leaves in P. armeniacum were induced by irradiance enriched with blue light. Its stomatal opening could be induced by red light 250 µmol m^?2 s^?1, but the magnitude of stomatal opening was lower than those at the other light qualities. However, the stomatal opening and photosynthesis of C. flavum were highly induced by mixed blue and red light rather than pure blue or red light. The two orchid species did not differ in stomatal density, but P. armeniacum had smaller stomatal size than C. flavum. The stomata of P. armeniacum were slightly sunken into the leaf epidermis, while C. flavum protruded above the leaf surface. The slower photosynthetic induction and lower photosynthetic rate of P. armeniacum than C. flavum were linked to the lack of guard cell chloroplasts and specific stomatal structure, which reflected an adaptation of Paphiopedilum to periodic water deficiency in limestone habitats. These results provide evidence for the morphological and physiological evolution of stomata relation for water conservation under natural selection.     

PECTATE LYASE LIKE12 patterns the guard cell wall to coordinate turgor pressure and wall mechanics for proper stomatal function in Arabidopsis

Plant cell deformations are driven by cell pressurization and mechanical constraints imposed by the nanoscale architecture of the cell wall, but how these factors are controlled at the genetic and molecular levels to achieve different types of cell deformation is unclear. Here, we used stomatal guard cells to investigate the influences of wall mechanics and turgor pressure on cell deformation and demonstrate that the expression of the pectin-modifying gene PECTATE LYASE LIKE12 (PLL12) is required for normal stomatal dynamics in Arabidopsis thaliana. Using nanoindentation and finite element modeling to simultaneously measure wall modulus and turgor pressure, we found that both values undergo dynamic changes during induced stomatal opening and closure. PLL12 is required for guard cells to maintain normal wall modulus and turgor pressure during stomatal responses to light and to tune the levels of calcium crosslinked pectin in guard cell walls. Guard cell-specific knockdown of PLL12 caused defects in stomatal responses and reduced leaf growth, which were associated with lower cell proliferation but normal cell expansion. Together, these results force us to revise our view of how wall-modifying genes modulate wall mechanics and cell pressurization to accomplish the dynamic cellular deformations that underlie stomatal function and tissue growth in plants.     

Chlorophyll a Fluorescence Transients in Mesophyll and Guard Cells : MODULATION OF GUARD CELL PHOTOPHOSPHORYLATION BY CO(2)

Chlorophyll fluorescence transients from mesophyll and guard cell chloroplasts of variegated leaves from Chlorophytum comosum were compared using high resolution fluorescence spectroscopy. Like their mesophyll counterparts, guard cell chloroplasts showed the OPS fluorescence transient indicating the operation of the linear electron transport and the possible generation of NADPH in these organelles. They also showed a slow fluorescence yield decrease, equivalent to the MT transition in mesophyll, suggesting the formation of the high energy state and photophosphorylation. Unlike the mesophyll chloroplasts, the fluorescence from guard cell chloroplasts lacked the increment of the SM transition, indicating that the two types of chloroplasts have some metabolic differences. The presence of CO₂ (supplied as bicarbonate, pH 6.7) specifically inhibited the MT-equivalent transition while its absence accelerated it. These observations constitute the first specific evidence of a guard cell chloroplast response to CO₂. Control of photosynthetic ATP levels in the guard cell cytoplasm by CO₂ may provide a mechanism regulating the availability of high energy equivalents at the guard cell plasmalemma, thus affecting stomatal opening.     

Overexpression of the Arabidopsis ��-expansin gene AtEXPA1 accelerates stomatal opening by decreasing the volumetric elastic modulus

Guard cell walls of stomata are highly specialized in plants. Previous research focused on the structure and anatomy of guard cell walls, but little is known about guard cell regulation during stomata movement. In this work, we investigate the possible biological role of the Arabidopsis expansin gene AtEXPA1 in stomatal opening. The AtEXPA1 promoter drove the expression of the GUS reporter gene specifically in guard cells. Light-induced stomatal opening was accelerated in 35S::AtEXPA1 lines, whereas the anti-AtEXPA1 antibody decelerated light-induced stomatal opening. The inhibition of the anti-AtEXPA1 antibody on stomatal opening was largely dependent on the environmental pH. The volumetric elastic modulus (ε) was measured as an indicator of changes in the cell wall. The ε value of guard cells in 35S::AtEXPA1 lines was smaller than in the wild types. The putative role of AtEXPA1 as controller of stomatal opening rate and its regulation are discussed.     

Origin and development of stomatal microanatomy in two species ofEucalyptus

Summary Development of the stomata ofEucalyptus orbifolia (in which they are relatively superficial) andE. incrassata (in which they are deeply sunken) is described from light microscopy of thin sections of resin-embedded material. The envelope of the guard mother cell is retained intact while in the daughter cells (guard cells) the inner and outer thickenings are formed. The mother cell envelope may even remain discrete and intact during early stages of formation of the separation spaces, precursors of the future stomatal pore, between the thickenings. Remnants of the guard mother cell wall may be retained as parts of at least the inner stomatal ledges. Likewise, remnants of the wall which divides the mother cell persist on the maturing guard cells.Sudan III-positive materials, probably cutin, are removed from the cuticle over the mother cell soon after it is formed. The cuticle above the guard cell is finally perforated by enzymic attack forming, inE. incrassata, a large cavity outside the developing stoma into which the outer stomatal ledges grow as extensions of the upper guard cell walls.The termostiole is suggested for the aperture in the cuticle. The flanges of cuticle seen in section to bound it are termedostiolar ledges. The ostiolar ledges are to be distinguished from the outer stomatal ledges, which develop from the upper thickenings of the guard cell initials. The distinction is clear inE. incrassata (and other species with deeply sunken stomata) but not in mesophytic plants or species with superficial stomata such asE. orbifolia in which the outer stomatal ledges are fused with the cuticle.Growth of the outer stomatal ledges inE. incrassata involves transport of wall materials through an annular space, the equivalent of an ectocythode.The relevance of the observations to stomatal development in other genera is discussed.     

Fluorescence Properties of Guard Cell Chloroplasts: EVIDENCE FOR LINEAR ELECTRON TRANSPORT AND LIGHT-HARVESTING PIGMENTS OF PHOTOSYSTEMS I AND II

The presence of chloroplasts in guard cells from leaf epidermis, coleoptile, flowers, and albino portions of variegated leaves was established by incident fluorescence microscopy, thus confirming the notion that guard cell chloroplasts are remarkably conserved. Room temperature emission spectra from a few chloroplasts in a single guard cell of Vicia faba showed one major peak at around 683 nanometers. Low-temperature (77 K) emission spectra from peels of albino portions of Chlorophytum comosum leaves and from mesophyll chloroplasts of green parts of the same leaves showed major peaks at around 687 and 733 nanometers, peaks usually attributed to photosystem II and photosystem I pigment systems, respectively. Spectra of peels of V. faba leaves showed similar peaks. However, fluorescence microscopy revealed that the Vicia peels, as well as those from Allium cepa and Tulipa sp., were contaminated with non-guard cell chloroplasts which were practically undetectable under bright field illumination. These observations pose restrictions on the use of epidermal peels as a source of isolated guard cell chloroplasts. Studies on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive variable fluorescence kinetics of uncontaminated epidermal peels of C. comosum indicated that guard cell chloroplasts operate a normal, photosystem II-dependent, linear electron transport. The above properties in combination with their reported inability to fix CO₂ photosynthetically may render the guard cell chloroplasts optimally suited to supply the reducing and high-energy phosphate equivalents needed to sustain active ion transport during stomatal opening in daylight.     

A conserved functional role of pectic polymers in stomatal guard cells from a range of plant species

Guard cell walls combine exceptional strength and flexibility in order to accommodate the turgor pressure-driven changes in size and shape that underlie the opening and closing of stomatal pores. To investigate the molecular basis of these exceptional qualities, we have used a combination of compositional and functional analyses in three different plant species. We show that comparisons of FTIR spectra from stomatal guard cells and those of other epidermal cells indicate a number of clear differences in cell-wall composition. The most obvious characteristics are that stomatal guard cells are enriched in phenolic esters of pectins. This enrichment is apparent in guard cells from Vicia faba (possessing a type I cell wall) and Commelina communis and Zea mays (having a type II wall). We further show that these common defining elements of guard cell walls have conserved functional roles. As previously reported in C. communis, we show that enzymatic modification of the pectin network in guard cell walls in both V. faba and Z. mays has profound effects on stomatal function. In all three species, incubation of epidermal strips with a combination of pectin methyl esterase and endopolygalacturonase (EPG) caused an increase in stomatal aperture on opening. This effect was not seen when strips were incubated with EPG alone indicating that the methyl-esterified fraction of homogalacturonan is key to this effect. In contrast, arabinanase treatment, and incubation with feruloyl esterase both impeded stomatal opening. It therefore appears that pectins and phenolic esters have a conserved functional role in guard cell walls even in grass species with type II walls, which characteristically are composed of low levels of pectins.     

Functional Stomata in a Variegated Leaf Chimera of Pelargonium zonale L. without Guard Cell Chloroplasts

Fluorescence microscopy indicated that chlorophyll was absentfrom epidermal and guard cells overlying all white areas andgreen areas (of certain leaves) in variegated leaves of Pelargoniumzonale, cv. Chelsea Gem. Stomata with chlorophyll-free guardcells, in general, responded normally to light and CO₂ as gaugedby direct measurements of stomatal aperture and by transpirationalwater loss studies, although stomata from white regions of variegatedleaves were more reluctant to open than stomata from green regionsof the leaves. Thus, functional stomata without guard cell chloroplastshave been discovered in another genus, namely Pelargonium, besidesthat originally discovered in Paphiopedilum. When stomata withchlorophyll-free guard cells opened, K⁺ accumulated in the guardcells. This indicates that chloroplasts are not essential forthe normal functioning of stomata and that the energy sourcefor driving stomatal movements can come from sources other thanphotophosphorylation. Key words: Guard cell chloroplasts, Leaf chimera, Pelargonium, Stomata     

Absence of Variable Fluorescence from Guard Cell Chloroplasts of Stenotaphrum secundatum

The lack of detectable variable fluorescence from guard cell chloroplasts in both the albino and green portions of variegated leaves of St. Augustine grass (Stenotaphrum secundatum var variegatum A.S. Hitchc.) is reported. Fluorescence was measured either with a highly sensitive, modified fluorescence microscope which was capable of recording fluorescence induction curves from single chloroplasts, or with a spectrofluorometer. Both fast and slow fluorescence transients from S. secundatum guard cells showed a rapid rise and then remained at a steady level. Neither variable fluorescence increase (induction) nor decrease (quenching), properties normally associated with photosystem II, was observed from these chloroplasts. These fluorescence kinetics did not change either with alterations of the specimen preparation procedure or with alterations of the excitation light intensities and wavelengths. These results indicate that guard cell chloroplasts in this variety of S. secundatum do not conduct normal photosystem II electron transport. Light regulation of stomatal conductance in intact leaves of this plant did occur, however, and was similar to light regulation observed in other species. The conductance of the green portion of the leaves was much greater in the light than in the dark, and was much greater than the conductance of the albino portion of the leaves. Stomata in the green portion of the leaves also showed greater opening in blue light than in red light. These results provide evidence that stomatal regulation in this variety of S. secundatum does not rely on photosystem II electron transport in guard cell chloroplasts.     

Tracing the ontogeny of stomatal clusters in Arabidopsis with molecular markers

The origin and process by which the mosaic of the different cell types is established during the development of the leaf epidermis in Arabidopsis are largely unknown, although the recent characterization of two mutants which develop stomatal clusters (four lips (flp) and too many mouths (tmm)) has opened up the possibility for genetic dissection of the stomata spacing. By using growth conditions which limit gas exchange with the open atmosphere, stomatal clusters that look like phenocopies of flp and tmm have been induced, suggesting that stomata spacing is under environmental as well as genetic control in Arabidopsis. The origin of these clusters has been addressed by following promoter activity for genes that are markers for competence for cell division (cdc2aAt), mitotic activity (cyc1aAt), and guard mother cell and developing guard cell identity (rha1). Their different expression patterns in the various cell types during epidermal differentiation and the asynchrony in the development of the various stomata that constitute each cluster suggest that these stomatal clusters derive from a single protodermal cell through a process that involves changes in cell fate in a subset of subsidiary cells. It was also found that guard cells express cdc2aAt and cyc1aAt, supporting the idea that they may remain competent for cell division.     

Tentoxin Suppresses Stomatal Opening by Inhibiting Photophosphorylation

Tentoxin and, to a lesser extent, dihydrotentoxin (both at 10mmol m^–3) reduce stomatal opening in epidermal stripsof Commelina communis in the light but not in darkness. Thiseffect was significantly greater in normal air than in CO₂-freeair. Fusicoccin overcame the tentoxin effect. However, tentoxindid not inhibit stomatal opening in the light in epidermal stripsof Paphiopedilum harrisianum, a species which lacks guard cellchloroplasts. It is concluded that tentoxin exerts its actionon stomata not by an ionophorous effect in the plasmalemma ofguard cells but by the inhibition of photophosphorylation intheir chloroplasts. The effects of DCMU and tentoxin on guardcells are discussed in terms of their effects on chloroplastsand the extent to which energy is supplied from this organelleduring stomatal opening in the light. The results indicate thatneither photophosphorylation nor non-cyclic electron transportin guard cell chloroplasts are essential for stomatal opening. Key words: Commelina, epidermal strips, Paphiopedilum, photophosphorylation, stomata, tentoxin     

Ultrastructural and histochemical studies on guard cells

Serial thick sections of guard cells from Vicia faba L., Nicotiana tabacum L., Allium cepa L., Zea mays L. and Beta vulgaris L. were obtained systematically (600–800 nm) and viewed with the transmission electron microscope in an effort to demonstrate the presence or absence of a symplastic transport pathway within the stomatal complex. Eight to ten stomata from each species were examined, and no continuous plasmodesmata were found connecting guard cells to sister guard cells or to adjacent epidermal or subsidiary cells. Continuous plasmodesmata were observed in immature guard cells, but were sealed (truncated) during the development of the mature cell wall. Histochemical stains, phosphotungstic acid and silver methenamine, were used to demonstrate differentiation within the mature guard-cell wall. The structural differentiation of the stomatal apoplastic region is discussed in relation to fanctional specialization. Plasma-membrane elaborations or plasmalemmasomes were identified in the guard cells of Zea, and it is suggested that these structures may function in ion transport.Abbreviations PTA-HCl phosphotungstic acid and hydrochloric acid - SM silver methenamine - UA-LC uranyl acetate and lead citrate     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

Structure of the stomatal complex of the monocot Flagellaria indica

The structure of the mature stomatal complex of Flagellaria indica L. was studied since the Flagellariaceae is reported to be one of a handful of nongrass families with a grass-type stoma, and since relatively little is known about stomatal ultrastructure in monocots other than grasses. Both the grass guard cell and its nucleus are dumbbell-shaped, and the walls that separate adjacent grass guard cells are perforated. Electron and fluorescence microscopy reveal that the Flagellaria guard cell lacks these features. Instead, the Flagellaria guard cell is neither dumbbellnor kidney-shaped, its nucleus is roughly kidney-shaped, and the end walls are thickened and imperforate. Additional structural features of the stomatal apparatus of Flagellaria include: 1) the subsidiary cells have a protuberance that underlies the middle of the guard cell and that forms an additional and innermost aperture of the pore; 2) guard and subsidiary cell walls are thickened differentially and are layered; and 3) organelles in both cell types appear to be confined to specific domains. Although Flagellaria is closely related to grasses, it does not have a grass or dumbbell-shaped type of stomate. This suggests that the grass type of stomate may be less widespread than reported.     

STRUCTURE AND DEVELOPMENT OF WALLS IN FUNARIA STOMATA

The development and structure of the guard cell walls of Funaria hygrometrica Hedw. (Musci) were studied with the light and electron microscopes. The stoma consists of only one, binucleate guard cell as the pore wall does not extend to the ends of the cell. The guard cell wall is thinnest in the dorsal wall near the outer wall but during movement is most likely to flex at thin areas of the outer and ventral walls. The mature wall contains a mottled layer sandwiched between two, more fibrillar layers. The internal wall layer has sublayers with fibrils in axial and radial orientations with respect to the pore. During substomatal cavity formation, the middle lamella is stretched into an electron dense network and into strands and sheets. After stomatal pore formation, the subsidiary cell walls close to the guard cell become strikingly thickened. The functional implications of these results are discussed.     

Stomatal Responses to CO(2) in Paphiopedilum and Phragmipedium: Role of the Guard Cell Chloroplast

A role of the guard cell chloroplasts in the CO₂ response of stomata was investigated through a comparison of the leaf gas exchange characteristics of two closely related orchids: Paphiopedilum harrisianum, which lacks guard cell chloroplasts and Phragmipedium longifolium, which has chlorophyllous guard cells. Leaves of both species had an apparent quantum yield for assimilation of about 0.05, with photosynthesis saturating at 0.300 to 0.400 millimoles per square meter per second. CO₂ curves were obtained by measuring steady-state assimilation and stomatal conductance under 0.180 or 0.053 millimoles per square meter per second white light, or darkness, at 0 to 400 microliters per liter ambient CO₂. The response of assimilation to changes in CO₂ was similar in the two species, but the response of conductance was consistently weaker in Paphiopedilum than in Phragmipedium. The data suggest involvement of guard cell chloroplasts in the stomatal response to CO₂ and in the coupling of assimilation and conductance in the intact leaf.