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     

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.     

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.     

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.     

Structure and Development of Stomata in Vegetative and Floral Organs of Three Species of Kalanchoe

The structure and ontogeny of stomata in vegetative and floralorgans of three species of Kalanchoe is described. The matureanisocytic stomata are mono-cyclic or completely or incompletelyamphicyclic, rarely paracytic, transitional between paracyticand anisocytic and with a single subsidiary cell. The developmentof all the types is syndetocheilic or mesogenous from organto organ but the mature stomatal apparatus varies from organto organ as regards the number and arrangement of subsidiarycells. Abnormal stoma with a single guard cell and arresteddevelopment were observed on all organs. An abnormal stoma witha single guard cell develops directly from the meristemoid.     

The Permeability of the Guard Cell Plasma Membrane and Tonoplast

Uptake experiments and efflux compartmental analysis of planthormones, osmotica and toxins using ‘isolated’ guardcells of Valerianella locusta and guard cell protoplasts (GCP)of Vicia faba were performed in order to study the permeabilityproperties of guard cell plasma membrane and tonoplast. Theplasma membrane of guard cells exhibits a higher permeabilitythan plasma membranes of mesophyll cells for most solutes investigated.The permeability coefficients (P_s calculated for the guard cellplasma membranes are also significantly higher than the P_s valuesfor the guard cell tonoplast. This applies also for protonatedABA. We suppose that the high permeability for ABAH could bepart of the target cell properties. A Collander analysis demonstratesa linear correlation between P_s, values and the ratio K_r/M_r^1,5for both plasma membrane (r = 0.87) and for the tonoplast (r=0.93). Because of deviations from the observed correlations,the permeation of some solutes (ABA, GA, IAA through the tonoplast;methylamine through the plasma membrane) seems to be facilitatedby an additional transport mechanism. The Collander analysisof the plasma membrane of GCP shows very similar results tothe analysis of the plasma membrane of ‘isolated’guard cells, indicating that isolation of protoplasts does notalter the permeability of the guard cell plasma membrane. Key words: Permeability coefficient, guard cells, plasma membrane, tonoplast     

The Azolla-Anabaena azollae Relationship : XII. Nitrogenase Activity and Phycobiliproteins of the Endophyte as a Function of Leaf Age and Cell Type

Nitrogenase activity was measured in leaves along the main stem axes of Azolla pinnata R. Br. The activity was negligible in leaves of the apical region, rapidly increased to a maximum as leaves matured, and declined in aging leaves. In situ absorption and fluorescence emission spectra were obtained for individual vegetative cells and heterocysts in filaments of the A. pinnata and Azolla caroliniana endophytes removed from the cavities of progressively older leaves. These spectra unequivocally demonstrate the occurrence of phycobiliproteins in the two cell types of both endophytes at the onset of heterocyst differentiation in filaments from young leaves, during the period of maximal nitrogenase activity in filaments from mature leaves, and in filaments from leaves entering senescence. Phycobiliproteins of the A. caroliniana endophyte were purified and extinction coefficients determined for the phycoerythrocyanin, phycocyanin, and allophycocyanin. The phycobiliprotein content and complement of sequential leaf segments from main stem axes and of vegetative cell and heterocyst preparations were measured in crude extracts. There was no obvious alteration of the phycobiliprotein complement associated with increasing heterocyst frequency of the endophyte in sequential leaf segments and the phycobiliprotein complement of heterocysts was not appreciably different from that of vegetative cells. These findings indicate that the phycobiliprotein complement of the vegetative cell precursor is retained in the heterocysts of the endophyte.     

Differential response of antioxidant enzymes to salinity stress in two varieties of Azolla (Azolla pinnata and Azolla filiculoides)

The activity and modulation of antioxidant components were comparatively analyzed in two varieties of Azolla (Azolla pinnata and Azolla filiculoides) under different concentrations of NaCl. The total superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity increased in A. pinnata, whereas both enzyme activities decreased in A. filiculoides. The plants of A. pinnata exposed to 30 mM NaCl contained a lower amount of Na⁺ ions and showed a lower electrolyte leakage than A. filiculoides. Our studies indicate a differential response of antioxidant enzymes in relation to salt tolerance in Azolla plants. On the basis of our comparative analysis, A. pinnata has been ranked salt tolerant as compared with A. filiculoides, which is salt sensitive.     

The response of rice to the growth and nitrogen fixation inAzolla caroliniana andAzolla pinnata at varying rates of phosphate fertilization

The response of rice toAzolla caroliniana, newly introduced in India, was compared with the reponse to the local isolate ofAzolla pinnata at varying rates of phosphate fertilizer (4.4–8.8 kg P ha^–1) during a wet and a dry season.Fresh weight, dry weight and fixed N were more for both species 21 DAI (days after inoculation) than 14 DAI, but acetylene reduction activity (ARA) was higher 14 DAI than 21 DAI. Dry weight of Azolla and fixed N were less 14 DAI forA. caroliniana than forA. pinnata during the wet season. Twenty-one DAI, fresh weight ofA. caroliniana was 62.1 and 27.6% higher than that ofA. pinnata during the wet and dry season, respectively. However, dry weight and fixed N were more 21 DAI inA. caroliniana than inA. pinnata during only the wet season. The ARA was higher inA. caroliniana both 14 and 21 DAI, irrespective of season. The presence of either species in the rice field increased grain yield, straw yield, number of panicles m^–2, number of grains per panicle and reduced percentage sterility during both the wet and the dry season. Phosphate application significantly increased fresh weight, dry weight, ARA and fixed N for both species as well as grain and straw yields of rice. The responses to phosphate fertilizer were similar for both Azolla species and for rice grown with either one of the Azolla species.     

Pigment distribution and nitrogen fixation inAnabaena azollae

Summary The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity.In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N₂-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla).Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O₂ evolution and the absence of photo-fixation of CO₂ in the cyanobionts.Pigment composition and N₂-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility.It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.     

The growth of four species of Azolla as affected by temperature

The growth of 22 strains of Azolla pinnata R. Br., 3 strains of A. filiculoides Lam. and one strain each of A. mexicana Presl and A. caroliniana Willd. was tested separately in liquid culture media kept in controlled, artificial light (30 klux) growth cabinets. Three temperature levels were used: 33°C (37/29°C day/night), 29°C (33/25°C) and 22°C (26/18°C)/ Photoperiod was 12 h a day.For most A. pinnata strains (except three) and an A. mexicana strain the maximum weekly relative growth rate was higher at 33°C than at 22°C, but not for A. filiculoides and A. caroliniana. The highest value of maximum relative growth rate corresponded to 1.9 doubling days and in most strains this occurred in the first week. As the plants grew, the growth rate slowed down more severely at higher temperatures. The maximum biomass was higher at 22°C than at 33°C in all strains. At 22°C, it took 30–50 days to attain maximum biomass and the highest value was 14 g N m^?2 or 320 g dry m^?2 by A. caroliniana, followed by 12 g N m^?2 or 290 g dry wt. m^?2 by one strain of A. filiculoides. At 29°C, the maximum biomass was attained in 20–35 days. The highest value was 6.3 g N m^?2 or 154 g dry wt. m^?2 by A. caroliniana. At 33°C, most A. pinnata strains gave a maximum biomass of less than 4 g N m^?2 after 13–23 days, while some strains grew up to 30 days, resulting in a higher maximum biomass. The highest maximum biomass at 33°C was 5.5 g N m^?2 or 140 g m^?2 dry wt. by A. pinnata from Cheng Mai while the maximum biomass of A. filiculoides and A. caroliniana was much less. Azolla filiculoides requires lower temperature than other species for its growth. Azolla pinnata has the best tolerance to high temperatures among the four species. Azolla mexicana could not be discriminated from A. pinnata in its response to temperature. Azolla caroliniana may keep an intermediate position between A. filiculoides and A. pinnata in temperature response.The formation of ammonia in the medium was examined and it occurred mostly under stationary growth conditions, but, at 33°C, some strains of A. pinnata and A. mexicana released or formed ammonia at 0.3–0.8 μg N ml^?1 per week during their initial exponential growth stage.     

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     

Direct Measurements of Turgor Pressure Potentials of Guard Cells: II. THE MECHANICAL ADVANTAGE OF SUBSIDIARY CELLS, THE SPANNUNQSPHASE, AND THE OPTIMUM LEAF WATER DEFICIT

Evidence of the mechanical advantage of subsidiary cells wasobtained by simultaneous measurements of turgor pressure potentialsin adjacent subsidiary and guard cells using injection circuitswith two separate needles. In Tradescantia virginiana the mechanicaladvantage approaches two. Using the same technique evidencewas obtained that the Spannungsphase is, in the first place,a turgor relations phenomenon due to the mechanical advantageof epidermal or subsidiary cells. In addition, the evidenceindicated that the elastic properties of guard cell walls mayundergo changes during the Spannungsphase when potassium iontransport commences. During these measurements it was confirmedthat the optimum leaf water deficit for maximum stomatal openingoccurs when the epidermal turgor is near zero. Under these conditionsthe width of the stomatal pore is a function of the turgor pressureof the guard cells, since at zero turgor of the subsidiary cellstheir mechanical advantage has disappeared.     

Studies of the Azolla pinnata—Anabaena azollae symbiosis: Concurrent growth of Azolla with rice

The growth of Azolla pinnata R. Br. under a rice crop was monitored throughout the growing season. Growth rate and N₂-fixation of Azolla decreased markedly after a 4–6 week culture period, a consequence of the low nutrient level in the floodwater rather than shading from the rice plants. The study also showed that Azolla growing concurrently with rice, increased grain yield significantly. The total N released by the local strain of Azolla pinnata, under 60% moisture and flooded soil conditions, was 1 mg g^?1 fresh weight of Azolla.     

Elevated carbon dioxide affects the patterning of subsidiary cells in Tradescantia stomatal complexes

The influence of elevated CO₂ concentration (670 ppm) on thestructure, distribution, and patterning of stomata in Tradescantialeaves was studied by making comparisons with plants grown atambient CO₂. Extra subsidiary cells, beyond the normal complementof four per stoma, were associated with nearly half the stomatalcomplexes on leaves grown in elevated CO₂. The extra cells sharedcharacteristics, such as pigmentation and expansion, with thetypical subsidiary cells. The position and shape of the extrasubsidiary cells in face view differed in the green and purplevarieties of Tradescantia. Substomatal cavities of complexeswith extra subsidiary cells appeared larger than those foundin control leaves. Stomatal frequency expressed on the basisof leaf area did not differ from the control. Stomatal frequencybased on cell counts (stomatal index) was greater in leavesgrown in CO₂-enriched air when all subsidiary cells were countedas part of the stomatal complex. This difference was eliminatedwhen subsidiary cells were included in the count of epidermalcells, thereby evaluating the frequency of guard cell pairs.The extra subsidiary cells were, therefore, recruited from theepidermal cell population during development. Stomatal frequencyin plants grown at elevated temperature (29 C) was not significantlydifferent from that of the control (24 C). The linear aggregationsof stomata were similar in plants grown in ambient and elevatedCO₂. Since enriched CO₂ had no effect on the structure or patterningof guard cells, but resulted in the formation of additionalsubsidiary cells, it is likely that separate and independentevents pattern the two cell types. Plants grown at enrichedCO₂ levels had significantly greater internode lengths, butleaf area and the time interval between the appearance of successiveleaves were similar to that of control plants. Porometric measurementsrevealed that stomatal conductance of plants grown under elevatedCO₂ was lower than that of control leaves and those grown atelevated temperature. Tradescantia was capable of regulatingstomatal conductance in response to elevated CO₂ without changingthe relative number of stomata present on the leaf. Key words: Elevated CO₂, stomata, subsidiary cells, patterning     

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     

Technical Note: Phytoremediation of Hg and Cd from Industrial Effluents using an Aquatic Free Floating Macrophyte Azolla Pinnata

The level of heavy metal pollution in Singrauli, an industrial region in India, was assessed and the phytoremediation capacity of a small water fern, Azolla pinnata R.Br (Azollaceae), was observed to purify waters polluted by two heavy metals, i.e., mercury (Hg) and cadmium (Cd) under a microcosm condition. Azolla pinnata is endemic to India and is an abundant and easy-growing free-floating water fern usually found in the rice fields, polluted ponds, and reservoirs of India. The fern was grown in 24 40-L aquariums containing Hg²⁺ and Cd²⁺ ions each in concentrations of 0.5, 1.0, and 3.0 mgL^?1 during the course of this study. The study revealed an inhibition of Azolla pinnata growth by 27.0–33.9% with the highest in the presence of Hg (II) ions at 0.5 mgL^?1 in comparison to the control. After 13 days of the experiment, metal contents in the solution were decreased up to 70–94%. In the tissues of Azolla pinnata, the concentration of selected heavy metals during investigation was recorded between 310 and 740 mgKg^?1 dry mass, with the highest level found for Cd (II) treatment at 3.0 mgL^?1 containing a metal solution.     

GAL4 GFP enhancer trap lines for analysis of stomatal guard cell development and gene expression

To facilitate the monitoring of guard cells during developmentand isolation, a population of 704 GAL4 GFP enhancer trap lineswas screened and four single insert lines with guard cell GFPexpression and one with developmentally-regulated guard cellGFP expression were identified. The location of the T-DNA inserts,the expression of the flanking genes, and the promoter activityof the genomic DNA upstream of the T-DNA were characterized.The results indicated that the GFP expression pattern in atleast one of the lines was due to elements in the intergenicDNA immediately upstream of the T-DNA, rather than due to theactivity of the promoters of genes flanking the insert, andprovide evidence for the involvement of Dof elements in regulatingguard cell gene expression. It is shown further that the GAL4GFP lines can be used to track the contribution of guard cellmaterial in vitro, and this method was used to assess the purityof guard cell samples obtained using two methods of guard cellisolation. Key words: Arabidopsis, development, enhancer trap, GFP, guard cells, stomata, T-DNA Received 21 July 2008; Revised 7 October 2008 Accepted 16 October 2008     

Jasmonates Induce Intracellular Alkalinization and Closure ofPaphiopedilumGuard Cells

Jasmonates (jasmonic acid or methyl jasmonate) promote stomatalclosure inPaphiopedilum Supersuk(RHS, 1973) andP. tonsum(Rchb.f)Stein. Studies on guard cells loaded with pH dependent fluorescentdyes show that jasmonates cause intracellular alkalinizationof up to 0.5 pH units within 5 to 15 min. Jasmonate-inducedalkalinization always preceded stomatal closure and where alkalinizationwas not detected no closure occurred. Propionic acid inhibitedjasmonate-induced stomatal closure, suggesting that jasmonate-inducedintracellular alkalinization is involved in guard cell movements. BCECF; confocal microscopy; cytosolic pH; guard cells; jasmonic acid; methyl jasmonate; Paphiopedilum Supersuk(R. H. S.); Snarf-1; stomatal movements     

In vivo-Microspectrophotometric Characterization of Flavonol Glycosides in Vicia faba Guard and Epidermal Cells

Cytological observations by fluorescence and U.V.-absorptionmicroscopy together with in vivo spectrophotometric analysesof stomata, guard cell protoplasts and epidermal cells of Viciafaba have shown that kaempferol 3,7-O-glycosides are localizedin the vacuoles. The alkaline-induced emission spectra recordedwith guard and epidermal cells after NH₄OH-treatment were identical,exhibiting an emission maximum at 525 nm; the spectra correlatedwith that of reference flavonols after exposure to NH₄OH Theexcitation spectra of both cell types are typical of these flavonolsshowing two maxima at 290 nm and 390 nm. In agreement, two absorptionmaxima were recorded for guard cells at 270 nm and 330 nm, withoutalkali, which shifted bathochromically to 275 nm and 380 nm,respectively, after NH₄OH treatment. The fluorescence intensitymeasured at 525 nm demonstrates a photostability in epidermalcells whereas it increases by a factor of about five with theexcitation time up to 30 min in guard cells. For the latter,several possible processes are discussed. Key words: Alkaline-induced fluorescence, emission, excitation, U.V.-absorption spectra, kaempferol glycosides, cell specificity