The stomata of the cork-oak, Quercus suber. An ultrastructural approach

In the evergreen leaves of Quercus suber, stomata play a major role in adaptation to drought and temperature stress. The leaf is of zygostomic type and has about 430 stomata per square milimeter of abaxial leaf surface. The stomatal complex is of the anomocytic type. The guard cells protrude from the epidermal plane. The guard cell nucleus contains heterochromatin in small granules. The guard cell cytoplasm is characterised by a large number of well developed mitochondria, amyloplasts with stroma and grana, and a well developed cytoskeleton with a cortical array of microtubules oriented pa railed to the slit axis that persist even in mature cells. Guard cell walls are asymmetrically thickened and devoid of plasmodesmata. No area of cell walls was free of cuticle or covered by a thin cuticular layer and apparently no area of limited cuticular development provides evaporation when the stomata are closed.     

Stomatal Features in the Leaf of Aganosma dichotoma (Roth) K. Schum

Paracytic and anisocytic types of mature stomata are found inthe leaf of Aganosma dichotoma. Stomata with one guard cell,stomata with degenerated guard cells, and contiguous stomataare common. Stomata with arrested pore development are alsofound in certain cases. A single guard cell without any porehas not been designated as a stoma with one guard cell in thepresent investigation. Ontogeny of contiguous stomata have beentraced. Subsidiary cells are, morphologically, just like theircontiguous guard cells. Subsidiary cells may retain their shapeand contents even when their contiguous stoma becomes mature,or may change their shape and lose their contents. They mayor may not divide. Subsidiary cells form a whorl of more thantwo subsidiary cells around a stoma by their divisions. Degenerationof guard cell(s)— their contents and nuclei—havebeen traced. In certain cases guard cells divide forming morethan two guard cells associated to a single pore. Cytoplasmicconnections are found between two guard cells of nearby stomata,and between a guard cell and an epidermal cell. Near the wound,the epidermal cells over the veins become meristermatic givingrise to new epidermal cells but no meristemoid.     

Acid-Induced Stomatal Opening in Commelina communis and Vicia faba

The effects of H^$ and fusicoccin (FC) on stomatal opening inthe dark were investigated using epidermal strips of Commelinacommunis and Vicia faba cv. Ryosai Issun. Citrate-phosphatebuffer induced maximal opening of stomata at pH 3.0 when testedover the range of 2.7 to 5.0. HCl at 1 mM also induced stomatalopening without appreciable accumulation of K^$ in the guardcells. After 4 hr treatment with 10 µM FC, stomata openedwith concomitant accumulation of K^$ in the guard cells, although1–2 hr treatment caused opening without concomitant K^$increase. These results suggest that stomatal opening can be caused bysalt accumulation and/or changes of the physicochemical conditionsin the cell wall of the guard cells due to high acidity. ¹ Present address: Biological Laboratory, Faculty of Education,Nagasaki University, Nagasski 852, Japan. (Received April 30, 1982; Accepted July 17, 1982)     

Epidermal and Guard Cell Interactions on Stomatal Aperture in Epidermal Strips and Intact Leaves

Despite the observation first made by von Mohl in 1856, thatepidermal cells greatly influence stomatal aperture, subsequentstudies have failed to pay adequate attention to epidermal cellviability or to quantify the degree of its influence on aperturein epidermal strips and leaf sections. Using Vicia faba stripsand leaf sections we found the following: (i) a non-linear relationshipbetween aperture and guard cell contact with live epidermalcells; (ii) epidermal cell viability on isolated strips hada threshold at about 25 °C; (iii) epidermal strips withdead epidermal cells had wider apertures and lower variabilitythan strips with live cells or intact leaf sections; (iv) afterepidermal cell viability was accounted for, stomatal aperturesshowed no significant differences between isolated strips orstrips removed from leaf sections treated in the same manner;(v) highly variable apertures appeared to be the normal conditionof the intact leaf. Caution should therefore be used in interpretingstomatal behaviour from epidermal strips without first takinginto account mechanical interactions between the guard and surroundingepidermal cells. Vicia faba L, broad bean, epidermal strips, leaf impressions, stomata, guard cells, temperature effects     

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     

The Stomata of Gnetum

In both Gnetum gnemon and G. ula the atomatal development inthe collars and axes of the male and female cones and the outertwo envelopes of the ovule is of the haplocheilic type. In theleaf some stomata do show parallel subsidiary cells, but theyarise from the surrounding epidermal cells and do not seem tohave a common origin with the guard cells. This conflicts withearlier accounts and is of interest in relation to the typesof stomata met with in other gymnosperms, living and fossil.     

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     

Responses of Commelina communis Stomata In Vitro

Weyers, J. D. B. and Paterson, N. W. 1987. Responses of Commelinacommunis stomata in vitro.— J. exp. Bot. 38: 631–641. Analysis of the kinetics of movements of Commelina communisL. stomata in vitro revealed a sequence of opening and closingphases dependent on the incubation medium used and the physiologicalstate of the plant material. In buffer containing 50 mol m ^–3KC1 the sequence of aperture changes appeared to be influencedby equilibration of cell water potentials with that of the mediumand by solute fluxes (dependent and independent on metabolicactivity). The results indicate that the stomatal aperture afterseveral hours of incubation may not always provide a reliablequantitative estimate of the ability of the stomata to operate.As a consequence, modifications are suggested to the ways inwhich experiments using epidermal strips are carried out andreported. Key words: Epidermal strips, guard cells, hydroactive, hydropassive, kinetics, potassium chloride, mannitol, osmotic effects, solute fluxes.     

Major Element Composition of Epidermal and Mesophyll Tissues2Commelina Communis L. and Vicia Faba L.: Some Further Considerations of the Role of Ions in Stomatal Functioning

Epidermal and mesophyll tissues of Commelina communis L. andVicia faba L. were analysed by atomic absorption spectrometryfor the major plant inorganic cations and anions (K, Na, Ca,Mg, P, NO₃-N, Cl) when stomata of the leaf were open and closed.Water-soluble and residual levels of the elements were estimatedand a charge balance of the soluble fraction made. The major portion of K, Na, Cl, and P was extracted in the water-solublefraction of the epidermal and mesophyll tissues of both species.In both species the bulk of Ca remained in the insoluble residueof the epidermis whereas in mesophyll tissue it was equallydistributed be-between the two fractions in C. communis butmainly in the insoluble residue in V. faba. Magnesium was predominantlyfound in the water-soluble fraction of V. faba mesophyll tissueand distributed approximately equally between the two fractionsin the epidermal tissue. In C. communis Mg was slightly moreabundant in the water-soluble fraction of both mesophyll andepidermis. In both species no statistically significant differences inthe levels of the elements could be detected between epidermaland mesophyll tissues from leaves with open stomata and thesame tissues from leaves with closed stomata, suggesting thatthere was no major flux of ions between mesophyll and epidermisduring stomatal movements. Regardless of whether the stomata were open or closed, therewere considerably more water-soluble inorganic cations thananions present in all tissues of both species with K being themajor cation and Cl being the major anion. In V.faba and C-communis epidermis there was 49–53 per cent and 56%68per cent excess cation respectively. In the mesophyll tissuethe excess cation was 63–75 per cent and 75%78 per centin V.faba and C. communis respectively. When the partitioning of the levels of the elements betweenepidermis and mesophyll of a leaf is considered, except forNO₃-N in both species and Na in V. faba, 20 per cent or lessof each element was present in the epidermis.     

Direct Measurements of Turgor Pressure Potentials of Guard Cells, I.

Measurements were made of pressures applied in subsidiary andguard cells which caused closure and opening of stomatal pores.These pressures were lower than would be expected from plasmolyticallydetermined osmotic potentials of guard cell saps. The pressuresneeded in guard cells to open almost closed stomata were practicallythe same as those required to close open stomata when appliedin adjacent subsidiary cells. Completely closed stomata couldnot be opened by this technique. The implications for the understandingof the mechanism of guard cell deformation, the Spannungsphase,and the pressure potentials of guard cells are discussed.     

Rhythmic Growth Rings of Wood and Their Relationship with the Foliage in Oak Seedlings Grown in a Favourable Environment

The anatomy of wood was studied inQuercus roburL. andQuercussuberL. seedlings exhibiting 3–8 units of extension, eachwith a tier of photosynthesizing leaves in their upper parts,generated as the result of rhythmic shoot growth under favourableconditions. At all the axis levels examined (i.e. the base ofeach of the different units of extension, four other equidistantlevels within the first unit of extension and the upper partof the taproot), the wood displayed rings when treated withWiesner reagents. This indicated cinnamaldehyde groups presentin lignins. No rings appeared when the Maüle reaction wasused for specific detection of syringyl subunits in lignins.A trend towards a periodical arrangement of xylem parenchymabands was also found when sections were treated with I₂/KI.The number of rings coincided with the number of leaf tiersabove the level of measurement, and did not vary inside thefirst unit of extension. Thus, the rings are called rhythmicgrowth rings. In sections of the first and the second unitsof extension, and in the taproot, the area and width of a givenrhythmic growth ring were highly correlated with the total areaof leaves present above the level of measurement at the presumedtime of growth ring formation. Moreover, stem diameter at thebase of the units of extension was highly correlated with theleaf area above. These results indicate that differentiationof xylem, particularly its lignification, varies rhythmicallyin oak seedlings. They imply that wood production is linkedto the photosynthesizing and/or transpiring area of the plant.Thus, during a growth cycle ofQ. roburandQ. suberseedlings,there appears to be integration of the primary metabolic activitieswith the laying down of rhythmic growth rings.Copyright 1998Annals of Botany Company Allometric relationship, juvenile wood, leaf area, lignification, oak seedling, periodic structure,Quercus roburL.,Quercus suberL., rhythmic growth ring, unit of extension.     

Cellular Interactions in the Development of Stomatal Patterns in Vinca major L

The development of the leaf epidermis of Vinca major L. wasfollowed in situ by epi-illumination microscopy and evidencewas sought for cellular interactions. Stomata were often foundto be initiated in adjoining cells. The epidermal cells whichseparated such stomata when they had matured were formed fromthe same cells as the stomatal complexes themselves. The presenceof developing and mature stomata may influence only the orientationof divisions in neighbouring cells, and not the initiation andmaturation of stomata. There is great variability in the relativeorientation, timing and number of divisions which intervenebetween the first unequal division and the maturation of a stomaas well as the location of stomata relative to the spongy mesophylland minor veins. The results indicate that continuous short-rangeinteractions between the future guard cells and the adjoiningcells, rather than interactions between future stomata or afixed programme of development, are essential for the formationof the pattern of functional stomata in the mature leaf. Vinca major L., cell lineage, cellular interactions, development of stomata, epi-illumination microscopy, meristemoids, patterned differentiation, stomata     

An Electrophysiological Study of the Stomatal Complex of Tradescantia virginiana

Measurements of electrical potential difference (PD) and potassiumactivity were carried out on the intact leaf of Tradescantiavirginiana. PD gradients across the stomatal complex were observedwith both open and closed stomata. The guard cell PD appearedto be linearly related to stomatal aperture. With the stomataopen a gradient of potassium activity across the stomatal complexwas observed which became reversed on stomatal closure. Calculationof the driving forces on potassium suggested that it was distributedpassively between the vacuoles of the cells of the stomatalcomplex. The electrophysiological data obtained from this investigationenabled potassium activity in the apoplast to be calculated.The results showed that on stomatal closure there was a massiveincrease in the potassium activity in the guard cell wall. Key words: Stomata, Ionic gradients, Electrical potentials     

Metabolic Inhibitors Block ABA-Induced Stomatal Closure

Closure of stomata of Commelina communis L. leaf epidermis causedby abscisic acid (ABA) was inhibited by sodium azide, potassiumcyanide and hypoxic conditions. Azide was more effective thancyanide at low concentrations, but the cyanide effect couldbe enhanced by addition of salicylhydroxamic acid, providingindirect evidence for cyanide-resistant respiration in epidermaltissue. Azide also inhibited ABA-induced closure of ‘isolated’ stomata and shrinkage of guard cell protoplasts.The results indicate that metabolic energy is required for ABAaction involving solute loss from the guard cells. Possiblemechanisms of action are discussed.     

Epidermal Structure and Ontogeny of Stomata in Vegetative and Reproductive Organs of Ephedra and Gnetum

The epidermal structure and development of stomata in vegetativeand reproductive organs of Ephedra foliata and Gnetum ula isdescribed. The epidermal cells are polygonal, isodiametric,or elongated with thick or thin straight, arched, or slightlysinuous anticlinal walls. The cuticle is thin or thick. Papilla-likeunicellular outgrowths are present in Ephedra foliata. The maturestomata are orientated parallel to the longitudinal axes orirregularly. The mature stomata are anomocytic, paracytic, witha single subsidiary cell, cyclocytic, and actinocytic. Arresteddevelopment, contiguous stomata, and stomata with aborted guardcells have been observed. The ontogeny of stomata on differentorgans of these two plants is typically haplocheilic or perigenousbut the stomatal apparatus varies from organ to organ.     

The function of guard cells does not require an intact array of cortical microtubules

The development of stomatal guard cells is known to require cortical microtubules; however, it is not known if microtubules are also required by mature guard cells for stomatal function. To study the role of microtubules in guard cell function, epidermal peels of Vicia faba were subjected to conditions known to open or close stomata in the presence or absence of microtubule inhibitors. To verify the action of the inhibitors, microtubules in appropriately treated epidermal peels were localized by cryofixation followed by freeze substitution and embedding in butyl-methyl methacrylate. Mature guard cells had a radial array of microtubules, focused toward the thick cell wall of the pore, and the appearance of this array was the same for stomata remaining closed in darkness or induced to open by light. Treatment of epidermal peels with 1 mM colchicine for 1 h depolymerized nearly all cortical microtubules. Measurements of stomatal aperture showed that neither 1 mM colchicine nor 20 M taxol affected any of the responses tested: remaining closed in the dark, opening in response to light or fusicoccin, and closing in response to calcium and darkness. We conclude that intact microtubule arrays are not invariably required for guard cell function.     

Abnormal Stomatal Behaviour and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: EFFECT OF ABSCISIC ACID AND AUXIN ON STOMATAL BEHAVIOUR AND PEROXIDASE ACTIVITY

The wilty tomato mutant flacca and the normal variety RheinlandsRuhm were compared in terms of: (1) potassium transport intoand out of the guard cells, (2) cell wall properties which includeprotein, hydroxyproline and peroxidase activity, and (3) activityof indol-3yl-acetic acid oxidase. Also studied were the effectsof auxin on stomatal behaviour and peroxidase activity whenapplied to normal plants during development, and the short-termeffect of abscisic acid on the resistance of flacca stomatato closure under plasmolysis. Potassium transport, wall protein and hydroxyproline all seemedto be equal in mutant and normal plants. Peroxidase activitywas higher in the soluble and wall fractions of the mutant,and decreased toward normal in the mutant treated with abscisicacid. More stomata were open and peroxidase activity was higherin normal plants treated with auxin during development. Thepercentage of open stomata under plasmolysis was lower and theiraperture size was smaller in the epidermal strips taken fromabscisic-acid-treated mutant plants than from control mutantplants.     

Water Control and Survival of Ipomoea batatas Grown Photoautotrophically under Forced Ventilation and Photomixotrophically under Natural Ventilation

Water loss and its determining factors were compared in leavesof in vitro -grown sweet potato (Ipomoea batatas L. Lam, ‘Beniazuma’)plantlets cultured either photoautotrophically under forcedventilation (scaled-up vessel) or photomixotrophically undernatural ventilation. Functional stomata, a greater amount ofwax deposition on the leaves and smaller epidermal conductancewere observed in plantlets cultured photoautotrophically underforced ventilation. When transplanted directly into soil inthe greenhouse (without ex vitro hardening), plantlets grownphotoautotrophically under forced ventilation survived betterand grew faster. This was because they were better able to controltranspiration and thus lost less water and showed no signs ofwilting. In contrast, plantlets cultured under photomixotrophicconditions had open stomata which were not functional, a highertranspiration rate, and uncontrolled and rapid water loss immediatelyafter transfer ex vitro. Thus, wilting and severe desiccationwith irreversible tissue damage were observed in the leaves,which died within a few days. Copyright 2000 Annals of BotanyCompany Epicuticular wax, hardening, stomata, sweet potato, transpiration, water control     

Stomata and Structure of Tetraploid Apple Leaves cultured in Vitro

Leaves of anther-derived tetraploid apple (Malus pumila Mill.)shoots were examined by low-temperature scanning electron microscopy(LT-SEM). Leaves were serrate and wide with an undulating adaxialsurface due to convex epidermal cells, apparently without crystallineepicuticular wax. Stomata were absent from the adaxial surface,except for the marginal teeth which exhibited 40-60 stomataper leaf; they probably originated from residual mitotic activity.One third of abaxial stomata was occluded by the residual cuticleof the mother guard cell across the stomatal pore which rupturedwhen the stomata became functional. The stomatal index was 7·2(± 1·6) with 60-75 stomata mm^-2, i.e. abaxialstomata of tetraploid leaves expanded in vitro were less frequentthan those in triploid leaves either cultured in vitro (475-575stomata mm^-2) or grown on the tree (320-390 stomata mm^-2) wherethe stomatal index was 21 (± 4). Freeze-fracture transsectionsshowed that the tetraploid in vitro leaves were composed ofa layer of adaxial epidermal cells, followed by a single layerof palisade cells and four to five layers of spongy mesophyllcells and the abaxial layer of epidermal cells, in contrastto juvenile seedling-grown apple leaves in which the two layersof palisade cells comprised the majority (52-60%) of the leafvolume. The same morphological features, such as wide and lesspointed leaves, reduced stomatal density and stomatal index,and increased stomatal size that were previously reported fortree-grown tetraploid leaves were also expressed in vitro. Thus,causes of the stomatal deformation in tissue-cultured Rosaceaeare interpreted to be in part genetic and not purely environmental.Copyright1994, 1999 Academic Press Malus pumila Mill., apple, biotechnology, breeding, cryo-preservation, CO₂, juvenile, low temperature-scanning electron microscopy (LT-SEM), micropropagation, ploidy, stomata, tissue-culture, transpiration     

Some Observations on the Diversity of Stomata and Trichomes in Six Species of Dioscorea

Stomata and trichomes are described on the leaves of six speciesof Dioscorea. As many as six types were found in D. bulbiferaand D. oppositifolia, four types in D.hispida and D. wallichii,three types in D. belophylla, but only two types in D. alata.Although there is a diversity of stomata even on the same surface,the predominant type is anomocytic in all the species exceptD. bulbifera in which it is tricytic. Rarely a stoma is alsocyclocytic in D. bulbifera. An increase in the number of subsidiarycells in paracytic, tricytic, or diacytic stomata takes placeby the wall formation in them. Similarly a reduction in thenumber of subsidiary cells of a tetracytic stoma is the resultof lateral subsidiary cells assuming the form of epidermal cells.Abnormalities such as a stoma with one guard cell, degenerationof guard cells, and contiguous stomata are also met with. Theorganization of different types of stomata is studied in D.bulbifera and D. wallichii and shown to be perigenous. Capitateglandular hairs were seen on the leaves of D. belophylla, D.bulbifera, D. hispida, and D. wallichii but non-glandular, uniseriate,3-celled trichomes were observed only in D. hispida.