Further Investigations into the Physiological Relationship Between an Epiphyllous Liverwort and its Host Leaves

Experiments were carried out to investigate the physiologicalrelationship between an epiphyllous liverwort, Radula flaccidaLbg. et G., and its host leaves. The osmotic potential of theepiphylla cell sap (-30 to -35 bar) was found to be much lowerthan that of the host cells (-10 to -12 bar). There is thereforea good physiological basis for the movement of water from thehost leaves to the epiphylla. The amount of light incident onthe host leaf which is intercepted by even the heaviest epiphyllacolony investigated was less than 2 per cent and this was foundto produce no measurable difference between the chlorophyllcontents of epiphylla-colonized and uncolonized parts of thehost leaf. ¹⁴CO₂ light fixation products were found not to movebetween the host leaf and the epiphylla in either direction.It is concluded that the dependence (parasitism) of R.flaccidaon its host leaves is partial and does not include the derivationof organic food substances.     

Silica Deposition in the Grass Leaf in Relation to Transpiration and the Effect of Dinitrophenol

Characteristic opal phytolith (‘silica body’) formationwas demonstrated in detached leaves of Sieglingia decumbens(Heath Grass), cultured in 100 ppm dissolved silicon (silicondioxide), previously, the leaves were free from intracellulardeposits as a result of silica-minimal tiller growth. The formertechnique allowed the study of the leaf deposition processesindependently of apical and root tissues, under growth-cabinetand glasshouse conditions. Deposition in excised leaves wascompletely suppressed by a surface, monomolecular coating, thusindicating that total net water loss was a limiting factor,however, evaporation from the recipient, epidermal tissues perse was not a requirement for this in situ deposition Generally,apart from an apparent, cell site shift in one treatment, phytolith-formationwas unaffected by the presence of the metabolic inhibitor 2,4-dinitrophenol. Also, some evidence was obtained of the influxof germanium dioxide into epidermal ldioblasts, which indicateda non-specificity of the host cell for silica. These results and those of earlier studies suggest that passive,non-metabolic mechanisms could account for the transport, influx,and cell lumen polymerization of silica in the grass leaf.     

Structural Aspects of the Neo-formation of Floral Buds on Leaf Discs of Streptocarpus nobilis Cultured in vitro

Leaf discs of Streptocarpus nobilis were cultured in vitro underconditions leading to flowering. The histo-logical aspects ofthe in vitro flower bud development were studied. It was foundthat in some instances flower buds develop from meristematiccells differentiated from wound tissue, and in others they arisedirectly from epidermal and sub-epidermal leaf blade tissues.     

Infection and Root-Nodule Development in Stylosanthes Species by Rhizobium

Root nodules of the tropical forage legume Stylosanthes occurredonly at lateral root junctions and resulted from direct invasionby rhizobia through spaces between epidermal cells. No infectionthreads were present in either the root hairs or nodules. Invasionof the host cortical cells was through structurally alteredcell walls. The bacteria reached the site of nodule initiationin the lateral root cortex by progressive collapse of the initiallyinvaded cells which were compressed by neighbouring cells toform intercellular thread-like infection zones. The bacteriamultiplied in the invaded cells of the nodule initial whichdivided repeatedly to form the nodule. Bacteroids formed onlywhen the host cells ceased to divide. Some abnormal associations occurred in S. capltata and S. hamata40264A. Division of invaded cells was restricted in S. capitataand the bacteria became enlarged and grossly deformed. In S.hamata restricted cell division was immediastely followed bythe brcakdown of the host cells and, although the bacteria multiplied,no bacteroids were formed. Bacteria isolated from these nodulesformed both effective and abnormal nodules when inoculated ontothe same host.     

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     

Potassium Loss from Stomatal Guard Cells at Low Water Potentials

The potassium content of guard cells and the resistance to viscousflow of air through the leaf were determined in sunflower (Helianthusannuus) subjected to low leaf water potentials under illuminatedconditions. In intact plants desiccated slowly by withholdingwater from the soil, large losses in guard cell K occurred asleaf water potentials decreased. Leaf viscous resistance increased,indicating stomatal closure. Similar results were obtained whendetached leaf segments were desiccated rapidly. Upon rehydrationof leaves, no stomatal opening was observed initially, despiteleaf water potentials at predesiccated levels. After severalhours, however, re-entry of K occurred and stomata became fullyopen. Turgid leaf segments floated on an ABA solution showedlosses of guard cell K and closure of stomata as rapidly andcompletely as those brought about by desiccation. It is concludedthat stomatal closure at low water potentials under illuminatedconditions is not controlled solely by water loss from the tissuebut involves the loss of osmoticum from the guard cells as well.This in turn decreases the turgor difference between the guardcells and the surrounding cells, and closing occurs.     

The Absorption Lag, Epidermal Turgor and Stomata

Simultaneous measurements of the opening response of stomatato illumination, the development of the Absorption Lag and changesin leaf thickness, showed that the accelerated opening of stomataduring part of the Motorphase coincided with the attainmentof the peak of the Absorption Lag and the beginning of a decreasein leaf thickness. The latter could be attributed in part toa loss of epidermal turgor. These results are discussed in connectionwith attempts to correlate stomatal movements in leaves understress with changes in bulk leaf water properties. Key words: Absorption Lag, epidermal turgor, stomata     

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.     

Characterization of Plant Epidermal Lens Effects by a Surface Replica Technique

Optical replicas of leaf surfaces were made for characterizingthe lens properties of individual epidermal cells. Using a dentallatex, moulds were made of leaf surfaces and subsequently usedto produce agarose replicas. The replicas focused light in amanner similar to intact epidermal cells and it was possibleto measure both focal lengths and intensifications within leafreplicas of Thermopsis montana, Mahonia repens, and Smilacinastellata which had epidermal cells of different diameter. Focallengths ranged from 74—130 µm which indicated thatlight was concentrated within the underlying photosynthetictissues of these leaves. Focal intensifications were measuredsensiometrically and were 1.5 for T. montana and 2-6 for theother species. These values compare favourably with calculatedfocal lengths and measurements taken from isolated epidermallayers. The results indicate that the epidermis can concentratelight within the leaf to amounts well in excess of ambient light.Furthermore, the replicas faithfully reproduced fine anatomicaldetails from a wide variety of leaves and they provide a non-destructiveway to reproduce surface characteristics for anatomical andphysiological studies.     

Involvement of auxin and a homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens

Differentiation of epidermal cells is important for plants because they are in direct contact with the environment. Rhizoids are multicellular filaments that develop from the epidermis in a wide range of plants, including pteridophytes, bryophytes, and green algae; they have similar functions to root hairs in vascular plants in that they support the plant body and are involved in water and nutrient absorption. In this study, we examined mechanisms underlying rhizoid development in the moss, Physcomitrella patens, which is the only land plant in which high-frequency gene targeting is possible. We found that rhizoid development can be split into two processes: determination and differentiation. Two types of rhizoids with distinct developmental patterns (basal and mid-stem rhizoids) were recognized. The development of basal rhizoids from epidermal cells was induced by exogenous auxin, while that of mid-stem rhizoids required an unknown factor in addition to exogenous auxin. Once an epidermal cell had acquired a rhizoid initial cell fate, expression of the homeodomain-leucine zipper I gene Pphb7 was induced. Analysis of Pphb7 disruptant lines showed that Pphb7 affects the induction of pigmentation and the increase in the number and size of chloroplasts, but not the position or number of rhizoids. This is the first report on the involvement of a homeodomain-leucine zipper I gene in epidermal cell differentiation.     

Epicuticular Phenolics Over Guard Cells: Exploitation for in situ Stomatal Counting by Fluorescence Microscopy and Combined Image Analysis

Guard cells emit an alkali-induced, blue fluorescence upon excitationby ultraviolet radiation (emission maximum energy at 365 nm).Fluorescence emission of guard cells was brighter than thatof the neighbouring epidermal cells in a number of wild andcultivated plants including conifers, but the relative fluorescenceintensity and quality was species-dependent. Three representativeplants possessing stomatal complexes which differed morphologicallywere studied: Olea europaea, Vicia faba and Triticum aestivum.Immersing leaves of these plants in chloroform for 30 s (therebyremoving epicuticular waxes) significantly reduced the intensityof the fluorescence emitted by guard cells. This indicates thatguard cell fluorescence could be due to either an increasedconcentration of fluorescing compounds (probably wax-bound phenolics),or a thicker cuticular layer covering the guard cells. Giventhat the alkali-induced blue fluorescence of the guard cellsis a common characteristic of all plants examined, it couldbe used as a rapid and convenient method for in situ measurementsof the number, distribution and size of stomatal complexes.The proposed experimental procedure includes a single coatingof the leaf surface by, or immersion of the whole leaf in, a10% solution of KOH for 2 min, washing with distilled water,and direct observation of the leaf surface under the fluorescencemicroscope. Fluorescence images were suitable for digital imageanalysis and methodology was developed for stomatal countingusing Olea europaea as a model species. Copyright 2001 Annalsof Botany Company Cuticle, epicuticular waxes, fluorescence microscopy, image analysis, phenolics, stomata     

Water Relations of the Mistletoe Amyema fitzgeraldii and its Host Acacia acuminata

Water relations of the mistletoe Amyema fitzgeraldii and itshost Acacia acuminata were studied near Geraldton, Western Australia.Transpiration rates of host and parasite under unstressed winterconditions varied more than 300–fold between day and nightwhile leaf water potential gradients between the partners remainedwithin the range 0·4–0·6 MPa. Plots of transpirationagainst leaf water potential indicated closely similar fluidphase resistances in host and parasite during daytime but divergentbehaviour at night due to an apparently large increase in resistanceof the haustorial junction between the partners. Data for summerand winter studies across a full range of light intensitiesshowed the parasite to transpire, on average, 1·4 timesfaster and to exhibit noticeably lower water use efficienciesthan its host. Experiments following restorative changes atnight in leaf water potentials of host and parasite on detachedhost branches supplied through their cut ends with water indicatedthat the haustorium offered a major resistance to water uptakeby the parasite. Restoration of leaf water potentials by theparasite lagged markedly behind that of the host, especiallyduring winter, leading to a rapid build up in potential gradientbetween partners. A phase of rapid flow into the parasite thenfollowed, presumably motivated by lowering of the haustorialresistance. Reversal of the potential gradient between hostand parasite was recorded in a night-time study involving abagged (non-transpiring) mistletoe attached to a host branchfrozen at the base to prevent further water uptake. Mechanismsare proposed to account for the apparently highly variable natureof the resistance of the haustorium. Key words: Mistletoe, transpiration, haustorial resistance, Amyema fitzgeraldii, Acacia acuminata     

Cellular basis for dimethipin-induced loss of leaf turgor and desiccation

Dimethipin-induced increase in transpiration from kidney bean leaves (Phaseolus vulgaris L. cv. Black Valentine) was not correlated with stomatal aperture. From analysis of the kinetics of water loss from excised kidney bean leaves, it was concluded that the increase in transpiration was due almost entirely to an increase in the cuticular component. Both light and scanning electron microscopic analysis of dimethipin-treated leaves indicated that the first cells to be affected by dimethipin were the epidermal cells. These results suggest that dimethipin causes a loss of leaf turgor and desiccation by disrupting epidermal cells, thereby removing a major barrier for water loss from the leaf.Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Vapour Loss through Stomatal Pores with the Mesophy II Tissue 1

Measurements of sustained rates of vapour loss via stomatalpores in epidermal strips show that these rates compare withtranspiration rates of intact leaves. The water supply pathwithin the epidermal tissue is thus capable of sustaining ahigh rate of evaporation from subsidiary and guard cells.     

Anatomy of Ricciocarpus natans (L.) Corda, Studied by Scanning Electron Microscopy

The anatomy of the vegetative thallus in the life-forms of theamphibious liverwort Ricciocarpus natans was studied by scanningelectron microscopy. Particular attention was given to the structureof the epidermal stomatoid pores and the minute internal poresin the aerenchyma. The aquatic and terrestrial life-forms werefound to differ in (1) number and size of ventral scales, (2)presence of rhizoids and (3) width of the thallus margin. scanning electron microscopy, Ricciocarpus natans (L.) Corda, Bryophyta, liverwort, stomatoid pores, aerenchyma, structural adaptations     

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     

Light Gradients and the Transverse Distribution of Chlorophyll Fluorescence in Mangrove andCamelliaLeaves

The light gradient and transverse distribution of chlorophyllfluorescence in mangrove andCamellialeaves, which have differentmorphological characteristics, were examined using a micro-fluorescenceimaging system reported previously (Takahashiet al., Plant,Cell and Environment17: 105–110, 1994). Epidermal cellsscattered light strongly, resulting in an increase in the fluencerate in epidermal cells. For theCamellialeaf, a light gradientwas formed by absorption of light by photosynthetic pigmentsassociated with the induction of chlorophyll fluorescence. Forthe mangrove leaf, a light gradient was formed by backward scatteredlight within a thick layer of non-assimilatory cells. Lightwith a low absorption coefficient (515 nm) penetrated deeperthan that with a higher absorption coefficient (477 nm and 488nm) in theCamellialeaf, while light of both wavelengths showedsimilar profiles in the mangrove leaf. In the mangrove leaves,scattered light declined significantly in the non-assimilatorycell layer which is in front of the assimilatory cells. Light,the intensity of which was reduced to approx. 10% of the maximum,was well scattered and induced a considerable amount of chlorophyllfluorescence in the assimilatory cells, which appear to be wellorganized to capture weak light.Copyright 1998 Annals of BotanyCompany fluorescence, intact leaf, light gradient, mangrove (Rhizophora mucronataLamk.),Camellia japonicaL.     

Effect of the Nature of the Substratum on the Force Required to Detach a Common Littoral Alga

It has been shown that the force required to detach the holdfastsof Fucus vesiculosus depends upon the nature of the substratum;adhesion is less to barnacle shells. The loweradhesion appearsto be due to a partial dissolution of the shell by acidic material,probably exndates. Growth of germlings on various substrataindicates that thalli and primary rhizoids develop better oncalcareous substrata. Some ecological consequences of the phenomenaare examined.     

Role of Cell-wall-degrading Enzymes in the Development of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

Extracts of limited and spreading lesions caused by Mycosphaerellapinodes on detached pea leaflets contained proteolytic, cellulolytic,and pectolytic enzymes although only in spreading lesions wasthere much degradation of cell walls. The brown tissue fromlimited M. pinodes lesions was resistant to maceration by enzymesfrom spreading lesions. Limited lesions contained water-soluble,95 per cent ethanol insoluble, partially dialysable, inhibitorsof pectin transeliminase which is probably the macerating enzyme. Green, spreading M. pinodes lesions developed only on leafletsfloating on water. Growth of these lesions was accompanied bycontinous loss of phenolic substances to the water while thephenol content in infected tissue remained similar to that inuninoculated controls. In contrast, the phenol content in mature,limited M. pinodes lesions on leaflets suspended just abovethe water level was about four times that in healthy tissue.It is suggested that loss of phenolics from floating leafletsprevents tissue browning and the development of resistance ofthe cell walls to maceration. But this type of resistance doesnot appear to be a major factor in the limitation of lesionson suspended tissue. Extracts of limited Ascochyta pisi lesions on leaflets floatingon water contained pectolytic and hemicellulolytic enzymes.Some cellulase (Cx) activity was detected although there waslittle evidence of cellulose degradation in cell walls in infectedtissue. The nature of the macerating factor remains uncertainbut it was found that extracts from lesions contained inhibitorsof pectic enzymes and that tissue just beyond that colonizedby the fungus was resistant to maceration; this resistance isprobably important in restricting the growth of the pathogenin the leaf.     

Experimental Studies of the Factors Controlling Transpiration: II. THE RELATION BETWEEN TRANSPIRATION RATE AND LEAF WATER CONTENT

Data are presented which show, when stomatal control is eliminated,that wheat leaves may lose 5–6 per cent. and Pelargoniumleaves 10–12 per cent. of their water without any reductionin the transpiration rate. Experiments in which Pelargonium and wheat leaves, with stomatalcontrol present, were submitted to cycles of changing watercontent also failed to establish any direct relation betweentranspiration rate and leaf water content. It is concluded that leaf water content over the range of 70–100per cent. of that present in the turgid state has no significanteffect in determining the rate of water loss from leaves. A repetition of Knight's experiment showed that stomata openedin still air and closed in moving air. This was not recordedby Knight, who used a porometer cup permanently attached tothe leaf. It is concluded that the higher transpiration raterecorded by Knight after a period of still air was due to widerstomatal aperture and not to the higher leaf water content assuggested by him.