Ontogenetic changes in stomatal size and conductance of sunflowers

The ontogenetic changes in stomatal size, frequency and conductance (g_s) on abaxial and adaxial leaf surfaces of sunflower plants (Helianthus annuus L. Russian Mammoth) were examined under controlled environmental conditions. The stomatal frequency on the adaxial and abaxial leaf surfaces decreased with leaf ontogeny and insertion level. The ratio of adaxial to abaxial stomatal frequency did not change with leaf ontogeny and insertion level, and 42–44% of total stomata was apportioned to the adaxial surface. Ontogenetic changes in stomatal pore length were detected and increased with ontogenesis. The stomatal length of both leaf surfaces had linear relationships with leaf area. Ontogenetic changes in g_s were similar between the two surfaces. However the adaxial g_s was lower than abaxial g_s in leaves of higher insertion levels. Conductance had a linear relationship with width x frequency but not with pore area.     

Action of growth regulators on the cotyledonary stomata ofCucumis sativus L.: Structure and ontogeny

Anomocytic stomata and stomata with single subsidiary cells are commonly observed Sometimes a stoma appears anisocytic. Double cytoplasmic connections between nearby stomata and division of guard cells with persistent or degenerating nuclei are seen in GA. One or more divisions of guard cells, displaced guard cells and single guard cells with or without pore are noticed in SUC. Formation of single guard cells is a common feature in TIBA. Paracytic stomata, one and a half stomata and persistent stomatal initials are seen in SUL. COUM seems to be not inhibitory inCucumis sativus. In COL stomata with unequal guard cells, unequal stomatal cells with thickening in between but without intervening pore, stoma with double pores, persistent stomatal initials which may be solitary or in groups with varying shapes and with one or two nuclei of different shapes are noticed. The growth regulators affect the frequency of stomata, epidermal cells; stomatal index; size of guard and epidermal cells.     

Changes in leaf morphology and anatomy with tree age and height in the broadleaved evergreen species, Eucalyptus regnans F. Muell

Relatively little is known about changes in leaf attributes over the lifespan of woody plants. Knowledge of such changes may be useful in interpreting physiological changes with age. This study investigated changes in leaf morphology and anatomy with tree age and height in the broadleaved evergreen species, Eucalyptus regnans. Fully expanded leaves were sampled from the upper canopy of tree ages ranging from 6 to 240 years, and tree heights ranging from about 10–80 m. There were significant changes in leaf form with increasing tree age and height. Leaf size and specific leaf area (SLA; leaf area/leaf mass) decreased, leaf thickness increased, and leaves became narrower relative to their length, with increasing tree age and height. Cuticle thickness and leaf waxiness, including wax occlusion of the stomatal antechamber, increased with increasing age and height. By comparison, there were no clear trends in stomatal frequency or stomatal length with tree age, although there were curvilinear relationships between an index of total stomatal pore area per leaf lamina and both tree age and tree height. The results support the hypothesis that leaves of E. regnans become more xeromorphic with tree age and height. The results are discussed in relation to their significance for changes in water relations in the canopy with age.     

Artichoke Leaf Morphology and Surface Features in Different Micropropagation Stages

Artichoke (Cynara scolymus L.) leaf size and shape, glandular and covering trichomes, stomatal density, stomata shape, pore area and epicuticular waxes during micropropagation stages were studied by scanning electron microscopy (SEM) and morphometric analysis with the aim to improve the survival rate after transfer to greenhouse conditions. Leaves from in vitro shoots at the proliferation stage showed a spatular shape, ring-shaped stomata, a large number of glandular trichomes and juvenile covering hairs, but failed to show any epicuticular waxes. Leaves from in vitro plants at the root elongation stage showed a lanceolated elliptic shape with a serrated border, elliptical stomata, decreased pore area percentage, stomatal density, and mature covering trichomes. One week after transfer to ex vitro conditions, epicuticular waxes appeared on the leaf surface and stomata and pore area were smaller as compared to in vitro plants. Artichoke acclimatization may be improved by hormonal stimulation of root development, since useful morphological changes on leaves occurred during root elongation.     

Morphogenetic effects of various growth substances on the cotyledonary stomata of brinjal and tomato

The effect of different growth substances on the development of normal and abnormal stomata are presented. Anomocytic, paracytic, anisocytic and stoma with a single subsidiary cell are observed. Abnormal developments like persistent stomatal cells, degeneration of guard cells, unusual thickening, unequal guard cells, single guard cells and size and shape of the pore are noticed in various growth substances. The growth substances also affect the stomatal frequency, stomatal index, epidermal frequency and size of guard and epidermal cells in both the plants. The highest meristematic activity is found in MOR 100 ppm in brinjal and in GA 25 ppm in tomato. The largest size of stomata is found in COL 25 ppm in brinjal and in MH 50 ppm in tomato. The same growth substance responds differently in the two plants.     

Association genetics,geography and ecophysiology link stomatal patterning in Populus trichocarpa with carbon gain and disease resistance trade‐offs

Stomata are essential for diffusive entry of gases to support photosynthesis, but may also expose internal leaf tissues to pathogens. To uncover trade‐offs in range‐wide adaptation relating to stomata, we investigated the underlying genetics of stomatal traits and linked variability in these traits with geoclimate, ecophysiology, condensed foliar tannins and pathogen susceptibility in black cottonwood (Populus trichocarpa). Upper (adaxial) and lower (abaxial) leaf stomatal traits were measured from 454 accessions collected throughout much of the species range. We calculated broad‐sense heritability (H²) of stomatal traits and, using SNP data from a 34K Populus SNP array, performed a genome‐wide association studies (GWAS) to uncover genes underlying stomatal trait variation. H² values for stomatal traits were moderate (average H² = 0.33). GWAS identified genes associated primarily with adaxial stomata, including polarity genes (PHABULOSA), stomatal development genes (BRASSINOSTEROID‐INSENSITIVE 2) and disease/wound‐response genes (GLUTAMATE‐CYSTEINE LIGASE). Stomatal traits correlated with latitude, gas exchange, condensed tannins and leaf rust (Melampsora) infection. Latitudinal trends of greater adaxial stomata numbers and guard cell pore size corresponded with higher stomatal conductance (g_s) and photosynthesis (A_max), faster shoot elongation, lower foliar tannins and greater Melampsora susceptibility. This suggests an evolutionary trade‐off related to differing selection pressures across the species range. In northern environments, more adaxial stomata and larger pore sizes reflect selection for rapid carbon gain and growth. By contrast, southern genotypes have fewer adaxial stomata, smaller pore sizes and higher levels of condensed tannins, possibly linked to greater pressure from natural leaf pathogens, which are less significant in northern ecosystems.     

Foliar responses ofPeristrophe bicalyculata to coal smoke pollution

Study of the foliar responses ofPeristrophe bicalyculata (Reth) Nees to the pollution caused by thermal power plant emissions revealed that the stomatal size, pore length, density, and index, as well as the photosynthetic rate and total chlorophyll content were reduced inP. bicalyculata plants at the polluted site in pre-flowering, flowering as well as post-flowering stage of plant growth. Contrary to this, stomatal conductance increased at each stage. The intercellular level of carbon dioxide was raised in the pre-flowering and flowering stages but decreased later at the polluted site.     

Ecophysiological responses of almond (Prunus dulcis) seedlings to cadmium stress

Almond (Prunus dulcis L.) seedlings were exposed to 0, 25, 50, 100 and 150 μM of CdCl₂ in a solution culture under controlled conditions. The effects of cadmium (Cd) exposure on almond seedlings growth, stomatal architecture, gas exchange and physiological parameters were investigated. Under cadmium stress conditions, significant decrease in fresh and dry weight, length of shoot and chlorophyll content were observed. Stomatal conductance, transpiration and net photosynthetic rates were generally depressed by Cd stress, despite stomatal frequency values and stomatal pore size remained unchanged. Exposure to Cd severely restricted the starch content and increased soluble sugars.     

Changes in stomatal frequency, stomatal conductance and cuticle thickness during leaf expansion in the broad-leaved evergreen species, Eucalyptus regnans

Aspects of leaf anatomical and physiological development were investigated in the broad-leaved evergreen species, Eucalyptus regnans F.Muell. Newly emergent leaves were tagged in the field and measured for stomatal conductance while a subset was collected every 14 days for the measurement of stomata and cuticle over a 113-day period. Cuticle thickness increased during leaf expansion, the increase following a sigmoid curve. Stomatal frequency (no. mm⁻²) decreased from 56 to 113 days after leaf emergence. The frequency of both immature and intermediate developmental stages of stomata also decreased over this time, but the total number of stomata per leaf remained relatively constant. Stomatal conductance (g _s) of young expanding leaves increased during expansion, and was significantly linearly correlated with stomatal frequency (excluding immature stomata), and with cuticle thickness. The progressive increase in g _s in young developing leaves was contrary to the observed changes in structural characteristics (increased cuticle thickness and decreased stomatal frequency). This increase in g _s with development may be related to the progressive increase in number of mature stomata with larger apertures and, therefore, a higher total pore area in fully expanded leaves.     

A surrogate measure of stomatal aperture

It is proposed that a measurement of the peristomatal groove distance (PGD) of guard cells on surface impressions of leaf epidermis can act as a surrogate measure of stomatal aperture. To test this idea, investigations were carried out on two species, one in which it is possible to make direct measurements of pore width with relative ease (Commelina communis L.) and one whose stomata are so small that this is difficult (Phaseolus vulgaris L.). Leaf water vapour conductance measurements were first taken with a porometer, then, without delay, a silicone rubber impression of the leaf was made of the area directly under the porometer cup. From a positive replica of this impression, stomatal aperture, PGD and pore length were measured. The correlations between stomatal aperture and PGD and between PGD and stomatal conductance were positive and highly significant. Because a causal relationship between stomatal aperture and PGD is expected, linear regression was used to obtain equations for converting PGD measurements into estimates of stomatal aperture. These account for 91.7% of the variation of aperture in the case of C. communis and 70.7% in P. vulgaris, suggesting that PGD measurements have potential as an alternative measure of pore width in cases where direct measurements would be both difficult and subject to excessive measurement error or bias.     

The nature of heterogeneity in the stomatal behaviour of Phaseolus vulgaris L. primary leaves

The aim of this research was to investigate the nature of heterogeneity in stomatal conductance and, in particular, to determine whether the characteristic 'patchy' pattern of water infiltration is reflected in measurements on individual stomata. Silicone rubber replicas were made of primary leaves of glasshouse-grown Phaseolus vulgaris L. plants, and the leaves were then infiltrated with water at controlled sub-atmospheric gas pressures according to their estimated or measured stomatal conductance. Seven leaves examined in detail all showed patchy infiltration, and the mean sire of infiltrated areas was negatively correlated with the prevailing stomatal conductance. In four of the leaves, a one millimetre wide transect across the leaf was selected for further detailed study. Measurements of mean peristomatal groove distance (PGD) and stomatal frequency were made along the transect and related to the state of infiltration. Analysis of variance indicated that, in all four cases, variation in PGD among patches was highly significant, but there was no significant difference between patches of different infiltration categories. Thus, local (patch-level) variation in stomatal aperture appeared to bear no relation to the infiltration status of the patches. The dominant source of stomatal variability was between individual pores in the same locality, which accounted for 82% or more of the total variability. Taking into account variation in stomatal frequency, correlations between predicted stomatal conductance and the extent of infiltration were significant in only one out of the seven leaves studied. Possible reasons for these results are discussed. It is suggested that the infiltration method misrepresents the underlying state of the stomata as being either open or closed, when there is little evidence for this from measurements of stomatal dimensions. For these unstressed plants under relatively stable conditions, it is concluded that the 'unit of variability' in stomatal heterogeneity may rest at the individual pore ('micro') scale, rather than at the areolar patch ('macro') scale, or above.     

THE ULTRASTRUCTURAL CYTOLOGY OF THE DIFFERENTIATING GUARD CELLS OF VIGNA SINENSIS

Structural differentiation of the guard cells of Vigna sinensis results from the integration of the following interrelated processes: a) intense activity of ribosomes, dictyosomes, endoplasmic reticulum (ER) membranes and mitochondria and patterned organization of microtubules; b) unequal thickening and ordered micellation of their walls and opening of the stomatal pore; and c) the divergent differentiation of the plastids. In differentiating guard cells, microtubules appear anticlinally oriented and more or less evenly distributed along the unthickened part of the dorsal wall and in the middle part of the ventral wall where thickening of the future pore occurs. In periclinal walls, microtubules fan away from the margins of the increasing thickening of the ventral wall and, later, from the rims of the stomatal pore towards the dorsal walls, parallel to the depositing radial microfibrils. Microtubules may be the cytoplasmic elements underlying guard-cell morphogenesis. Although cell-plate organization in guard-cell mother cells does not seem to differ from that of other protodermal cells, the middle lamella of the ventral wall becomes electron-translucent. The stomatal pore develops schizogenously from the internal and/or external ends of the ventral wall and proceeds inwards, remaining incomplete in most of the stomata of plants grown for 30 days in darkness and in some malformed ones which were developed after a prolonged action of colchicine. The guard cell, when approaching maturity, loses its organelle complexity and plasmodesmata, but it keeps a significant portion of its cytoplasm and organelles. Perigenous stomata generally exceed the size of mesoperigenous and mesogenous ones, develop large vacuoles and appear able to induce oriented divisions in their vicinity.     

From reproduction to production,stomata are the master regulators

The best predictor of leaf level photosynthetic rate is the porosity of the leaf surface, as determined by the number and aperture of stomata on the leaf. This remarkable correlation between stomatal porosity (or diffusive conductance to water vapour g_s) and CO₂ assimilation rate (A) applies to all major lineages of vascular plants (Figure 1) and is sufficiently predictable that it provides the basis for the model most widely used to predict water and CO₂ fluxes from leaves and canopies. Yet the Ball–Berry formulation is only a phenomenological approximation that captures the emergent character of stomatal behaviour. Progressing to a more mechanistic prediction of plant gas exchange is challenging because of the diversity of biological components regulating stomatal action. These processes are the product of more than 400 million years of co‐evolution between stomatal, vascular and photosynthetic tissues. Both molecular and structural components link the abiotic world of the whole plant with the turgor pressure of the epidermis and guard cells, which ultimately determine stomatal pore size and porosity to water and CO₂ exchange (New Phytol., 168, 2005, 275). In this review we seek to simplify stomatal behaviour by using an evolutionary perspective to understand the principal selective pressures involved in stomatal evolution, thus identifying the primary regulators of stomatal aperture. We start by considering the adaptive process that has locked together the regulation of water and carbon fluxes in vascular plants, finally examining specific evidence for evolution in the proteins responsible for regulating guard cell turgor.     

Computer-based studies of diffusion through stomata of different architecture

BACKGROUND AND AIMS: The influence of stomatal architecture on stomatal conductance and on the developing concentration gradient was explored quantitatively by comparing diffusion rates of water vapour and CO(2) occurring in a set of three-dimensional stoma models. The influence on diffusion of an internal cuticle, a sunken stoma, a partially closed stoma and of substomatal chambers of two different sizes was considered. METHODS: The study was performed by using a commercial computer program based on the Finite Element Method which allows for the simulation of diffusion in three dimensions. By using this method, diffusion was generated by prescribed gas concentrations at the boundaries of the substomatal chamber and outside of the leaf. The program calculates the distribution of gas concentrations over the entire model space. KEY RESULTS: Locating the stomatal pore at the bottom of a stomatal antechamber with a depth of 20 microm decreased the conductance significantly (at roughly about 30 %). The humidity directly above the stomatal pore is significantly higher with the stomatal antechamber present. Lining the walls of the substomatal chamber with an internal cuticle which suppresses evaporation had an even stronger effect by reducing the conductance to 60 % of the original value. The study corroborates therefore the results of former studies that water will evaporate preferentially at sites in the immediate vicinity to the stomatal pore if no internal cuticle is present. The conductance decrease affects only water vapour and not CO(2). Increasing the substomatal chamber increases CO(2) uptake, whereas transpiration increases if an internal cuticle is present. CONCLUSIONS: Variation of stomatal structure may, with unchanged pore size and depth, profoundly affect gas exchange and the pathways of liquid water inside the leaf. Equations for calculation of stomatal conductance which are solely based on stomatal density and pore depth and size can significantly overestimate stomatal conductance.     

Effects of experimental warming on stomatal traits in leaves of maize (Zea may L.)

We examined the warming effects on the stomatal frequency, stomatal aperture size and shape, and their spatial distribution pattern of maize (Zea may L.) leaves using a light microscope, an electron scanning microscope, and geostatistic techniques. A field manipulative experiment was conducted to elevate canopy temperature by 2.08°C, on average. We found that experimental warming had little effect on stomatal density, but significantly increased stomatal index due to the reduction in the number of epidermal cells under the warming treatment. Warming also significantly decreased stomatal aperture length and increased stomatal aperture width. As a result, warming significantly increased the average stomatal aperture area and stomatal aperture circumference. In addition, warming dramatically changed the stomatal spatial distribution pattern with a substantial increase in the average nearest neighbor distance between stomata on both adaxial and abaxial surfaces. The spatial distribution pattern of stomata was scale dependent with regular patterns at small scales and random patterns at larger scales on both leaf surfaces. Warming caused the stomatal distribution to become more regular on both leaf surfaces with smaller L(t) values (Ripley's K‐function, L(t) is an expectation of zero for any value of t) in the warming plots than the control plots.     

A taxonomic tool for identifying needle remains of south‐western European Pinus species of the Late Quaternary

This work provides a tool whereby the needle remains of native, south‐western European Pinus spp. can be easily identified from species‐specific epidermal features. To construct this tool, the needles of P. uncinata, P. sylvestris, P. nigra, P. pinaster, P. pinea and P. halepensis were gathered across the Northern Hemisphere range of each taxon and compared with non‐indigenous trees growing in two South Australian Botanic Gardens. Three needles from each of these species were taken from three adult trees growing at three different localities. Light microscopy was used to observe the key epidermal and stomatal features of the needles. To improve interpretation, additional scanning electron microscopy samples were prepared. Epidermal features, including variation in the diameter of the epistomatal chamber aperture (pore), are described. A taxonomic key based on the size, shape and arrangement of the subsidiary cells of the stomatal complexes was constructed. This key enables the identification of pine needle fragments at the species level (except those belonging to the group P. gr. nigra‐uncinata). Despite their overlapping range, pore size was helpful in distinguishing between P. nigra and P. uncinata and between three groups of species. Isolated stomata were also observed. Cluster and discriminant analyses of stomatal variables described in earlier studies were performed. Overlap in guard cell variables hampers species‐level identification of isolated stomata. Species discrimination is improved if groups of ecological affinity are considered. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 282–298.     

Stomatal Characteristics at Different Ploidy Levels inCoffeaL.

Stomatal frequency, epidermal cell frequency, stomatal guardcell length and stomatal index were examined at different ploidylevels inCoffea. In general, stomatal and epidermal cell frequencyper unit leaf area decreased while stomatal guard cell lengthincreased with an increase in ploidy. The reduction in stomatalfrequency at higher ploidy levels was mainly a result of largerepidermal cells. In the case ofC. canephora(cultivar S.274)a significant reduction in stomatal frequency was noticed fromdiploid to tetraploid level which was due to both larger epidermalcell size and less stomatal differentiation at the tetraploidlevel. Besides the effect of ploidy on stomatal frequency andguard cell length, genotypic differences in stomatal frequencyand stomatal guard cell length were also observed among cultivarsof the same ploidy level. Although variation in stomatal frequencyamong cultivars was found to be associated with the differencein stomatal to epidermal cell ratio, variation in guard celllength was attributed to differential genetic architecture.In the present study a highly significant positive correlation(r=0.82) between stomatal and epidermal cell frequency and highnegative correlations between stomatal frequency and guard celllength (r=-0.91) and epidermal cell frequency and stomatal guardcell length (r=-0.93) were obtained. The study also indicatedthat stomatal frequency can be predicted with 83 and 87% accuracy,respectively, by measuring stomatal guard cell length in coffee.Copyright1997 Annals of Botany Company Coffea; ploidy level; stomatal characteristics     

The Effect of Epidermal Cell Water Potential on Stomatal Response to Illumination of Leaf Discs of Vicia faba

Illuminated leaf discs of Vicia faba were brought into equilibrium with a series of mannitol solutions. The width of stomatal aperture and the osmotic potential of guard cells and epidermal cells were determined. It was found that the maximal aperture was obtained when epidermal cells were at about incipient plasmolysis and that any increase in their turgor pressure brought about a decrease in stomatal aperture. These findings emphasize the importance of epidermal cells in determining the width of the stomatal pore.     

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.     

A comprehensive analysis of the physiological and anatomical components involved in higher water loss rates after leaf development at high humidity

To better understand the poor regulation of water loss after leaf development at high relative air humidity (RH), the relative importance of the physiological and anatomical components was analyzed focusing on cultivars with a contrasting sensitivity to elevated RH. The stomatal responsiveness to three closing stimuli (desiccation, abscisic acid feeding, light/dark transition), as well as several stomatal features (density, index, size and pore dimensions) and the cuticular transpiration rate (CTR) were determined in four rose cultivars, grown under moderate (60%) and high (95%) RH. Moreover, the effects of changes in stomatal density and pore dimensions on the stomatal conductance (g_s) were quantified using a modified version of the Brown and Escombe equation. Higher water loss, as a result of plant growth at high RH, was primarily caused by an increase in residual g_s, and to a lesser extent due to higher CTR. It was estimated that in leaflets subjected to desiccation the enhanced g_s in high RH- as compared to moderate RH-grown plants was mostly due to poor stomatal functionality and to a lesser extent the combined result of higher stomatal density and longer pore length. It is concluded that the reduced degree and, specially, the reduced rate of stomatal closure are the primary causes of the large genotypic variation in the control of water loss in high RH-grown plants. Furthermore, it was found that although changes in stomatal length have no influence on stomatal functionality, changed anatomical features per se represent a significant and direct contribution to the increased water loss.