Responses of Commelina communis L. Guard Cell Protoplasts to Abscisic Acid

Fitzsimons, P. J. and Weyers, J. D. B. 1987. Responses of Commelinacommunis L. guard cell protoplasts to abscisic acid.—J.exp. Bot. 38: 992–1001. Guard cell protoplasts (GCPs) isolated from the leaf epidermisof Commelina communis L. responded to abscisic acid (ABA) ina manner which was qualitatively and quantitatively similarto that of intact stomata. ABA inhibited swelling of GCPs underlow-CO₂ conditions and swollen GCPs responded to the hormoneby shrinking. Both the absolute volume decrease and the initialrate of shrinking were commensurate with the extent and ratesof solute loss computed for ABA-treated intact, open stomata.This indicates that GCPs represent a suitable experimental systemfor studies of ABA-mediated solute fluxes. A radiotracer equilibrationmethod was developed for the rapid estimation of GCP osmoticvolume changes. Using this technique it was found that, on average,82% of the reduction in solute content caused by ABA treatmentwas due to the loss of K⁺. It is envisaged that electroneutralitymight be maintained during ABA-induced shrinkage of GCPs bynet inward proton movement leading to acidification of the vacuole. Key words: Abscisic acid, Commelina communis L., guard cells, protoplasts     

The Structure and Orientation of Guard Cells in Plants Showing Stomatal Responses to Changing Vapour Pressure Difference

Transmission electron micrographs revealed that a substantialpart of the guard cell wall of both Quercus robur L. and Populusnigra ‘italica’ L. was either free of cuticle orcovered with a greatly reduced cuticular layer. In Quercus thestructure of the guard cell was such that the area of limitedcuticular development would be exposed to the evaporating powerof the atmosphere even when the stomata were closed. Lanthanumstaining confirmed that this area might be an important siteof evaporation. A similar evaporation site was identified inthe guard cell wall of Pinus sylvestris L. Light micrographsrevealed that this area could also be exposed on the outsideof the leaf when the stomata were closed. It appears that guardcell orientation with respect to the epidermal plane dependsupon epidermal turgor. Changes in orientation of the guard cellcoupled with the exact location of the cuticle-free area inthe guard cell wall may explain the nature of the stomatal responseof individual species to changing VPD and the effect of othervariables, e.g. water deficit, on this response. Quercus robur L, oak, Populus nigra L, poplar, stomata, guard cells, cuticle, evaporation, vapour pressure difference     

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.     

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     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

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     

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.     

Structure and Development of Stomata in Some Leptosporangiate Ferns

The structure and development of stomata are described for 17species of leptosporangiate ferns. In these species the maturestomata are anomocytic, diacytic, Pyrrosia(applied), tetracytic,suspended, or floating type. Stomata are present on both surfacesof floating as well as sub merged leaves of Azolla and Marsilea.In a few species twin stomata, arrested development, and persistentstomatal initials are present Floating stomata result from disintegrationof the anticlinal suspending wall in Pleopeltis, and by detachmentand displacement in Azolla pinnata. The development of stomatais haplocheilic, syndetocheilic, or syndetohaplocheilic. InCyathea spinulosa the guard cell nuclei divide amitoticallyand the resulting two daughter nuclei occupy the opposite polesof the guard cells     

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     

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.     

Involvement of Calyculin A- and Okadaic Acid-sensitive Protein Phosphatase in the Blue Light Response of Stomatal Guard Cells

Calyculin A (CA) and okadaic acid (OA), inhibitors of proteinphosphatases, inhibited blue light (BL)-dependent H⁺pumpingin Vicia guard cell protoplasts at half-inhibitory concentrationsof 4.5 nM and 400 nM, respectively. Light-induced stomatal openingin Viciaepidermis was completely suppressed by CA at 100 nMand by OA at 1 µM. These results suggest that CA- andOA-sensitive protein phosphatase is involved in the BL responseof stomatal guard cells. (Received June 27, 1997; Accepted September 2, 1997)     

Stomata on the Primary Root of Pisum sativum L.

The first record of stomata on a non-specialized root was obtainedby scanning electron microscopy of 4-d-old Pisum sativum L.In some cases subsidiary cells were trichoblasts. Stomata andthe root triarch vascular structure were simultaneously presentin transverse sections through the root. Pisum sativum, pea, root stomata, guard cells, trichoblasts     

Studies in Stomata of Chilli and Brinjal

Ontogeny and development of stomata in chilli (Capsicum annuumL.) and brinjal (Solanum melongena L ) are described Paracytic,anomocytic, and anisocyuc stomata are present Abnormalitiessuch as stoma with a single guard cell and degenerated stomatalcells are common. Degeneration of guard cell is traced in chilliJuxtaposed, superposed, and obliquely placed contiguous stomataare described. An incompletely differentiated menstemoid wasfound contiguous with a normal stoma Rarely a guard cell dividesto form a menstemoid. Cytoplasmic connections between the guardcells of adjacent or juxtaposed contiguous stomata are observedin chilli Extruded nucleoh in the guard cells of chilli arecommon Extruded nucleoh may enlarge The wounded epidermis ofchilli lose sinuosity of their walls and the degeneration ofguard cells is common.     

Properties of the Signal Transduction Pathway in the Blue Light Response of Stomatal Guard Cells of Vicia faba and Commelina benghalensis

Blue light-dependent proton extrusion in guard cell protoplastsfrom Vicia faba and light-dependent stomatal opening in theepidermis of Commelina benghalensis are inhibited by the calmodulin(CaM) antagonist, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfononamide(W-7) and the myosin light chain kinase (MLCK) inhibitor, 1-(5-iodonaphthalene-1-sulfonyl)-lH-hexahydro-1,4-diazepine (ML-7) [Shimazaki, K., Kinoshita, T.and Nishimura, M. (1992) Plant Physiol. 99: 1416]. We now suggestthat the inhibition occurs in the blue light signaling pathwaywithout affecting the proton pump. Addition of fusicoccin (FC),an activator of H⁺-ATPase, to the protoplasts and the epidermiswhose blue light-dependent proton extrusion and light-dependentstomatal opening had been inhibited by W-7 and ML-7, inducedboth proton extrusion and stomatal opening, respectively. Bluelight-dependent proton extrusion was inhibited by K-252a, awide-range inhibitor of protein kinases, and KT5926, a selectiveinhibitor of MLCK. FC induced proton extrusion in the presenceof K-252a and KT5926. In contrast, phenylmercuric acetate (PMA),carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and N, N'-dicyclohexylcarbodiimide(DCCD) inhibited both the proton extrusion and stomatal opening,but FC did not induce the responses. These results suggest thatW-7, ML-7, K-252a and KT5926 inhibit the signal transductionprocess by which the perception of blue light is transducedinto activation of the proton pump in guard cells, and thatMLCK or MLCK-like protein is involved in the blue light responseof stomata. The possibility that calcium-dependent, calmodulinindependent protein kinase [Harper, J.F. et al. (1991) Science252: 951] functions rather than MLCK in the blue light responseof stomata should be noted, however. (Received July 23, 1993; Accepted September 30, 1993)     

Ferricyanide Reduction by Guard Cell Protoplasts

Guard cell protoplasts of Commelina communis L. reduced exogenousferricyanide at pH values lower than 5?0; upon addition of NADH,reduction of ferricyanide by guard cell protoplasts was stimulatedover the pH range 4?0 to 9?0 with two peaks of activity at pH5?0 and between pH 8?0 and pH 9?0. Calcium chloride (1?0 molm^–3) and MgCl2 (1?0 mol m^–3) increased the NADH-stimulatedreduction of ferricyanide. Superoxide dismutase and cyanidehad little effect on the NADH-stimulated reduction of ferricyanideby guard cell protoplasts, but, salicylhydroxamic acid completelyinhibited this activity. The NADH-stimulated reduction of ferricyanidealso occurred in the cell-free supernatant. Horseradish peroxidasedid not reduce ferricyanide in the absence of NADH over a broadrange of pH (4?0 to 9?0). However, in the presence of NADH,horseradish peroxidase reduced ferricyanide over the pH range5?0 to 9?0 with maximal activity at pH 8?0. The NADH-stimulatedreduction of ferricyanide by horseradish peroxidase showed similarproperties to those observed with guard cell protoplasts. Mannitol,superoxide dismutase, and cyanide did not inhibit the NADH-stimulatedreduction of ferricyanide by horseradish peroxidase; SHAM, however,completely inhibited the reduction of ferricyanide by horseradishperoxidase. Catalase inhibited the NADH-stimulated reductionof ferricyanide by horseradish peroxidase by 20%, while absenceof oxygen in the assay medium stimulated this activity over60%. We propose that the reduction of ferricyanide in the presenceof NADH by guard cell protoplasts, can be explained in termsof peroxidase activity associated with the plasma membrane andsecreted to the extracellular medium. However, the capacityof guard cell protoplasts to reduce ferricyanide at acid pHvalues where little peroxidase activity occurs may indicatethe presence of a plasma membrane redox system in guard cellsof C. communis. Key words: Commelina, guard cell protoplasts, ferricyanide reduction, peroxidase, redox system     

An Unusual Feature of Stomatal Microanatomy in Certain Taxonomically- related Eucalyptus spp

The line of closure in the stoma in fully grown adult leavesof certain eucalypts is formed by special ridges of cuticlecalled ‘stomatal bars’ developed at the line ofclosure itself or from upgrowth of the cuticle of the lowersurface of the guard cells. Stomatal bars, previously discoveredin three members of the informal group ‘Bisectae’are shown to be restricted to certain species in that group.Possession of stomatal bars may affect stomatal performancebut does not appear to be a general adaptive response to thehabitats of the species which possess them. More probably, thepossession of stomatal bars is an inherited character with taxonomicvalue. The presence of stomatal bars in other genera is discussed. Eucalyptus spp, stomata, microanatomy, guard cells, stomatal bars     

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)     

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.     

Anatomy and Transpiration of the Avocado Inflorescence

Structure and function of the inflorescence of cv. 'Hass' and'Fuerte' avocado (Persea americana Mill.) were examined by scanningelectron microcopy (SEM) and by porometry. Sepals and petalscould not be distinguished by their position in the flower,by visual gross morphology or by microscopic surface structureand were hence designated as tepals. These tepals were arrangedin two whorls of three, followed by two whorls of three outerand three inner stamens, each opposite a tepal. The most conspicuousfeature of tepals, developing leaves and peduncles was the densecover of hair which were most frequent on the adaxial tepalsurface (925-1200 trichomes mm^-2), followed by their abaxialsurface (625-1000 mm^-2) and peduncles (375-655 mm^-2). Stomatawere absent from the adaxial surfaces of both tepal and leaves,as well as peduncles. On the tepals, abaxial stomata appearedfunctional, small (8-9 x 11-13 µm) and scarce with 2·8-3·4stomata mm^-2, i.e. very low relative to avocado leaves (350-510stomata mm^-2) or young fruit (50-75 stomata mm^-2. However, flowersincluding tepals transpired 1·2-1·3 mmol underfield conditions in Southern California (1·6-2 kPa),i.e. in excess of leaves (0·7-1·1 mmol) and peduncles(0·6-0·8 mmolH₂O m^-2 s^-1). This situation wasattributed to the few small but functional abaxial stomata onthe tepal, in contrast to 80% closed stomata and dense epicuticularwax cover in form of rodlets on young and dendritic crystalson old leaves including the guard cells, and absence of stomatafrom the peduncle.Copyright 1993, 1999 Academic Press Persea americana Mill., avocado, bioenergetics, flower, fruit, leaf, peduncle, scanning electron microscopy, stomata, transpiration, petals, sepals, tepals