Effects of Age and Water Stress on Endogenous Levels of Plant Growth Regulators in Euphorbia lathyrus L.

The effect of droughting and waterlogging on changes in endogenouslevels of hormones have been studied in Euphorbia lathyrus L.with particular reference to plant age and leaf age. Younger tissues (i.e. immature leaves and young plants) synthesizedsignificantly greater amounts of ABA per unit fresh weight thandid corresponding older tissue. The increased ABA levels correlatedwell with corresponding increases in water saturation deficit(WSD) and stomatal resistance in a given treatment but the relationshipbetween the absolute values of these three parameters variedgreatly between seedlings of different ages. Endogenous ethylenelevels in leaves and stems were unaffected by droughting irrespectiveof age. Imposition of waterlogging led to marked increases in ABA levelsin young plants though this was not strictly related to changesin WSD or stomatal resistance. Endogenous levels of ethyleneincreased in response to waterlogging, this being marked inthe older seedlings which also had a higher incidence of senescenceand abscission. The more complex effects of waterlogging, the significance ofage of tissue on hormonal responses to stress, and the adaptivevalue of such responses to younger tissues are discussed.     

Hormones in Relation to Stress Recovery in Populus robusta Cuttings

The relationships between changes in WSD,² stomatal resistance,and hormone levels during stress and stress recovery have beeninvestigated in a perennial plant, Populus robusta, over anextended stress period, involving a number of stress cyclesof differing duration. There were marked increases in WSD, stomatal resistance, freeABA levels, and ethylene levels in leaves and stems during stressimposition. However, during stress recovery, WSD declined mostrapidly, while decreases in other parameters were related tothe duration of the preceding stress. In general, the declinein stomatal resistance and free ABA levels to control valueswas protracted. Although there were increases in bound ABA levels,above control values, there was no close correlation with changesin free ABA levels. The concentrations of bound ABA observedin the latter phases of these experiments were higher than freelevels. No clear pattern of change in IAA concentration emerged in responseto stress or stress recovery. Although ethylene concentrations rise in response to initialstress imposition and fall during relief, subsequent stressand stress relief periods were without significant effect. The results are discussed in relation to the role of endogenousgrowth regulators in the response of plants to stress and inparticular the fate of stress-induced ABA.     

Characterisation of Non-Uniform Photosynthesis Induced by Abscisic Acid in Leaves Having Different Mesophyll Anatomies

The effects of abscisic acid (ABA) on photosynthesis in leavesof Helianthus annuus L. were compared with those in leaves ofVicia faba L. After the ABA treatment, the response of photosyntheticCO₂ assimilation rate, A, to calculated intercellular partialpressure of CO₂, P_i, (A(p_i) relationship) was markedly depressedin H. annuus. A less marked depression was also observed inV.faba. However, when the abaxial epidermes were removed fromthese leaves, neither the maximum rate nor the CO₂ responseof photosynthetic oxygen evolution was affected by the applicationof ABA. Starch-iodine tests revealed that photosynthesis was not uniformover the leaves of H. annuus treated with ABA. The starch contentwas diffferent in each bundle sheath extension compartment (thesmallest subdivision of mesophyll by veins with bundle sheathextensions, having an area of ca. 0.25 mm² and ca. 50 stomata).In some compartments, no starch was detected. The distributionof open stomata, examined using the silicone rubber impressiontechniques, was similar to the pattern of starch accumulation.In V.faba leaves, which lack bundle sheath extensions, distributionof starch was more homogeneous. These results indicate that the apparent non-stomatal inhibitionof photosynthesis by ABA deduced from the depression of A(pⁱ)relationship is an artifact which can be attributed to the non-uniformdistribution of transpiration and photosynthesis over the leaf.Intercellular gaseous environment in the ABA-treated leavesis discussed in relation to mesophyll anatomy. ¹ Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received September 30, 1987; Accepted January 13, 1988)     

Stomatal responses of Vicia faba L. to indole acetic acid and abscisic acid

Evidence is presented that stomata in isolated epidermal peelsof Vicia faba L. open in darkness in response to the externalpresence of indole acetic acid (IAA) in the incubation medium.The effect of IAA is found to be overcome completely in thepresence of either TRIS or MES buffers. In the absence of buffer,V. faba stomata are shown to be influenced by IAA in a concentration-dependenttrend which reached a maximum at an [IAA] of 10^–3 molm^–3. Further investigations reveal that stomata in thisspecies can be shown to respond to the presence of IAA and anotherphytohormone, abscisic acid (ABA). IAA and ABA are demonstratedto be antagonistic in their effects provided the incubationconditions are suitable. The data are discussed in relationto stomatal responses of other species in different treatmentconditions. Recommendations are made with respect to standardizationof incubation media during epidermal peel experiments. Key words: Vicia faba, stomata, indole acetic acid, abscisic acid, buffers     

Abscisic Acid Accumulation by in Situ and Isolated Guard Cells of Pisum sativum L. and Vicia faba L. in Relation to Water Stress

Isolated guard cells, prepared by sonication of epidermal peels, were used to investigate the endogenous level of abscisic acid (ABA) in the guard cells of turgid and stressed leaves of Vicia faba L. and the argenteum (arg) mutant of Pisum sativum L. The guard cells of V. faba and arg were found to contain 18 and 8 times more ABA, respectively, when isolated from stressed leaves than from turgid leaves. Isolated guard cells of V. faba were also directly stressed with the osmoticum Aquacide III. These guard cells were capable of producing stress-induced ABA to at least 3 times their ABA level when non-stressed.     

An Early Cytochemical Marker of Commitment to Stelar Differentiation in Meristems from Dicotyledonous Plants

A cytochemical study of root apices from Vicia faba and Pisumsativum showed esterase activity to be present in the stele,root cap and rhizodermis, but almost completely absent fromthe developing cortex and quiescent centres. The meristem cellsgiving rise to the cortex were almost negative whilst thosegiving rise to the stele were positive for esterase activity.Cambia from roots, shoots and petioles of a number of dicotyledonousspecies were all positive for esterase activity. It is proposedthat esterase activity may be used as an early marker of commitmentto differentiation into stele in roots of dicotyledonous plants,and that the cambia are fully committed meristems. Pisum sativum L., Vicia fabaL., garden pea, broad bean, meristems, stelar differentiation, esterase activity, xylem differentiation, cytochemistry, cambium     

Abscisic Acid-Induced Stomatal Closure in Vicia faba Epidermal Strips. Excretion of Solutes from Guard Cells and Increase in Elastic Modulus of Guard Cell Wall

The effects of abscisic acid (ABA) on the size of the apertureof stomata on epidermal strips of Vicia faba were studied inincubation media with different pH values. The osmotic potentialof guard cells, as determined by the limiting plasmolysis method,was higher at pH 4.0 than at pH 6.0, although the size of thestomatal apertures was almost identical at both pH values. AtpH 4.0, ABA effectively caused stomatal closure but had onlya small effect on the osmotic potential, whereas, at pH 6.0,ABA significantly increased the osmotic potential. ABA promotedthe efflux of Cl^– and malate from epidermal strips intothe incubation medium, an effect which was more marked at pH6.0, with a concomitant efflux of K⁺ to balance the charge onthe exported anions. From these results, it is suggested thatABA may cause an increase in the elastic modulus of the cellwalls of guard cells. ³ Present address: Nagano Prefectural Vegetable and OrnamentalCrops Experimental Station, 2206 Oomuro, Matsusiro-machi, Nagano381-12, Japan (Received September 30, 1986; Accepted January 9, 1987)     

Characterization of a wilty sunflower (Helianthus annuus L.) mutant II. Water relations, stomatal conductance, abscisic acid content in leaves and xylem sap of plants subjected to water deficiency

The response of w-1, a wilty sunflower (Helianthus annuus L.)mutant, to water stress is described in comparison with thecontrol line (W-1). Detached leaves of w-1 strongly dehydratedduring the first 30 min without significant changes in leafconductance, whereas W-1 responded rapidly to water loss byreducing stomatal aperture. After 2 h stress ABA increased slightlyin w-1, while W-1 leaves showed a 20-fold increase. When waterstress was imposed to potted plants by water withholding, w-1quickly dehydrated, and lost turgor, while W-1 maintained positiveturgor values for a longer period. Wild-type plants respondedto small changes in leaf water potential by accumulating ABAand by closing stomata, whereas in the mutant significant changesin ABA content and in stomatal conductance were found only atvery low water potentials. In another experiment in which waterwas withheld under high relative humidity, when soil water contentstarted to decrease W-1 rapidly closed stomata in the absenceof any change in leaf water status and the reduction in conductancewas paralleled by a rise in xylem sap ABA concentration. Bycontrast the mutant started to accumulate ABA in the xylem sapand to close stomata when soil water content and leaf waterpotential were dramatically reduced. The low endogenous ABAlevels and the inability to synthesize the hormone rapidly eitherin the leaves or in the roots seem to be responsible for thehigh sensitivity of w-1 to water stress. Key words: ABA, Helianthus annuus L, water relations, stomatal conductance, drought, wilty mutant     

Identification of Shoot Apical Meristem Cells Committed to Form Vascular Elements in Pisum sativum L. and Vicia faba L.

A cytochemical study of naphthol AS-D esterases in vegetativeshoot apices of Pisum sativum and Vicia faba L. has shown thepresence of carboxyl esterases (E.C. 3.1.1.1 [EC] .) in those meristemcells already committed to form vascular elements. These cellsform a sequence linking the morphologically identifiable procambiumto the cells of the tunica layers at a site either already identifiableas the next primordium or which will form the next primordium.The implications of this result are briefly discussed in relationto the control of primordia formation and procambial cell development. Pisum sativum, Vicia faba, determination, vascular tissue, shoot apex, cytochemistry     

Leaf Nucleic Acids:: II. METABOLISM DURING SENESCENCE AND THE EFFECT OF KINETIN

The nucleic acids in the green and in the senescent leaves ofthree types of plant have been studied. High and low molecularweight RNA of the chloroplast is not present in senescent leavesof Xanthium pensylvanicum, but both cytoplasmic and chloroplasticfractions are found in yellow leaves of Vicia faba and Nicotianatabacum. RNA is more rapidly degraded than DNA in the leavesof these plants when they are detached, and kinetin treatmenttemporarily arrests the loss of chlorophyll and nucleic acid.Once X. pensylvanicum leaves are yellow and senescent they cannotbe re-greened, whereas those of Nicotiana spp., and to someextent those of V. faba, can be rejuvenated. We suggest thatthe retention of chloroplast RNA in yellow leaves may be a majorfactor determining their ability to re-green and that the patternof organelle senescence prior to the first stages of leaf autolysisand dehydration is species-specific.     

Changes in abscisic acid and its glucose ester in Phaseolus vulgaris L. during chilling and water stress

Levels of free and conjugated abscisic acid (ABA) were determined in leaves and roots of intact bean (Phaseolus vulgaris L., cv. Mondragone) seedlings under chilling (3C) and drought as well as during recovery from stress. Abscisic acid-glucose ester (ABAGE) was the only conjugate releasing free ABA after alkaline hydrolysis of the crude aqueous extracts. During the first 20–30 h chilled plants rapidly dehydrated and wilted without any change in ABA and ABAGE levels. Subsequently, leaf and root ABA levels increased and plants regained turgor. ABAGE concentration showed a slight increase in leaves but not in roots. Upon recovery from chilling a transient, but significant, rise in leaf ABA content was observed, while no appreciable change in ABAGE was found. Drought triggered ABA accumulation in leaves and roots, while a rise in ABAGE content was detected only in leaf tissues. Recovery from stress caused a drop in ABA levels without a correspondent increase in ABAGE concentration. We conclude that ABAGE is not a source of free ABA during either chilling or water stress and that only a small proportion of the ABA produced under stress is metabolised to ABAGE during recovery.Abbreviations ABA = abscisic acid - ABAGE = abscisic acid-glucose ester - DW = dry weight - FW = fresh weight - RIA = radioimmunoassay - RWC = relative water content - w = water potential - o = osmotic potential - p = turgor potential     

In Vitro Culture and Plant Regeneration in Vicia faba subsp. Equina (var. Spring Blaze)

Explants of stem, leaves, roots, and cotyledons from etiolatedaxenically grown Vicia faba seedlings were cultured on a rangeof media. Shoot organogenesis was only obtained with nodal stemand cotyledonary node explants when cultured on MS medium with3% sucrose, 2.0 mg 1^–1 BAP and 02 mg 1^–1 NAA. Callusproliferation accompanied shoot organogenesis from nodal stemexplants. Successive subculture of nodal stem callus resultedin proliferation of regenerative callus which contained severalshoot bud initials. The capacity for shoot regeneration fromthis callus was maintained for 9 months. Histological studiesreveal de novo formation of meristematic centres in callus andtheir further development into bud primordia. High frequencyrooting of these adventitious shoots was obtained on half-strengthMS medium with 1.5% sucrose, 0.1 mg 1^–1 NAA and 0.5 mg1^–1 kinetin. Key words: Vicia faba, adventitious shoots, axillary shoots, de novomeristem formation, organogenesis, tissue culture     

The Effects of Exogenous Amino Acid on Acetylene Reduction Activity of Vicia faba L. cv. Fiord

The feed back control mechanism proposed to explain the inhibitionof N₂ fixation by N was investigated using Vicia faba cv. Fiord.Plants were grown under controlled conditions without mineralN in coarse river sand. Asparagine was supplied to plants activelyfixing N₂ by absorption through cut roots and via a wick ordirect injection into the stem just above the bottom leaf. Responsesin N₂ fixation were measured by acetylene reduction (AR). Feedingplants with [¹⁴C]-labelled asparagine showed that the amidewas taken up when exogenously applied. Asparagine (10 mM) suppliedby the above procedures resulted in a 50-70% inhibition of ARby 48 h. Glutamine produced a similar effect. The cut root methodallowed higher levels of these amides to be supplied but theinhibition observed with 10 mM asparagine was only increasedslightly with higher levels of the amide. The antibiotic Securopenprevented bacterial contamination of root solutions of asparagineand glutamine and had no effect on nodule activity. It is concludedthat accumulation of asparagine of glutamine or the resultantincrease in the pool of soluble N in the plant cause a feedbackeffect on the activity of nitrogenase.Copyright 1993, 1999 AcademicPress Vicia faba, faba bean, asparagine, inhibition of N₂ fixation     

Effect of Exogenous DNA on Acid Deoxyribonuclease Activity in Intact Roots of Vicia faba L.

Exogenous DNA has been administered to roots of Vicia faba inwhich this DNA stimulates an increase in acid deoxyribonucleaseactivity. This enzyme activity is associated with vesicles whichfollow whatappears to be the cytological pathway of foreignDNA in host tissues.     

Effect of CO2 on Volume Change of Guard Cell Protoplast from Vicia faba L

Guard cell protoplasts (GCP) were isolated from epidermal stripsof Vicia faba L. by enzymatic digestion. The presence of non-osmoticvolume in the protoplast was suggested by the relationship betweenprotoplast volume and the mannitol concentration of the suspendingmedium. Light illumination caused swelling of GCP only whenKCl was present in the suspending medium. Dark treatment causedshrinking of GCP irrespective of the presence of 10 mM KCl.In the presence of 10 µM abscisic acid (ABA), GCP shrank.Light-induced swelling was suppressed at concentrations of ambientCO₂ higher than that in normal air. Promotion of swelling wasnot always observed at lower CO₂ concentration. These volumechange responses to light, ABA and CO₂ suggest that GCP retainsits physiological activity as a guard cell. The osmotic contributionof K⁺ to volume increase was lower than expected. Ambient CO₂seems to have some effect on the contribution of K⁺ to osmoregulationof GCP. (Received January 30, 1982; Accepted June 25, 1982)     

Effects of high osmotic potential of a medium on mitotic cycle in roots ofVicia faba L

The present paper deals with the effects of osmotic potential of a medium on cell reproduction and elongation of the roots ofVicia faba L. andVicia sativa L. As the osmoticum polyethylene glycol (PEG 4000) in various concentrations ranging from 5 % to 25 % (i.e. fromca.-0.11 up to -1.27 MPa) has been used. The results show that at higher concentrations than 7.5 %, the growth of roots is slowed down and at the concentration of 25 % PEG this decrease in growth rate is as much as 6 fold compared with the control. The mitotic cycle is prolonged, however, only 1.86 times. Thus, the inhibition of root growth is caused mainly by the inhibition of cell elongation. Concerning the effect of high osmotic potential on mitotic cycle it was found that the roots after immersion into 25% PEG are able to overcome this osmotic stress and after 6 h to renew the mitotic activity. The S phase of the cycle is the most sensitive to this factor and even after mitotic activity was renewed it showed a slower rate in comparison with the control.     

Stress-related levels of abscisic acid in guard cell protoplasts of Vicia faba L.

Levels of abscisis acid (ABA) were determined in isolated guard cell (GCP) and mesophyll cell (MCP) protoplasts of Vicia faba L. in relation to water stress. Incubation of GCP and MCP in 0.4 M or 0.8 M mannitol resulted in an average increase in the level of free abscisic acid (ABA) in the cells of 34% (GCP) and 38% (MCP) within 15–60 min. It is concluded that guard cell protoplasts form ABA in response to osmotic stress.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - GCP guard cell protoplasts - MCP mesophyll cell protoplasts - MES [2-(N-morpholino)-ethanesulfonic acid] - TLC thin layer chromatography Part 20 in the series, Use of Immunoassay in Plant Science     

The Effect of Osmotic Stress on the Solutes in Guard Cells of Vicia faba L.

We investigated the changes in the levels of solutes in guardcells under osmotic stress. Epidermal strips peeled from Viciafaba L. leaflets were sonicated and incubated in 0.4 M mannitolsolution (osmotic stress) in either light or dark. Stomata wereclosed by osmotic stress. Under osmotic stress, malate accumulatedlight-dependently and sucrose accumulated light-independentlyin the guard cells. The level of K⁺ in guard cells increasedslightly under osmotic stress in the light, although withoutstatistical significance. The levels of all these solutes werereduced by 10 µM ABA treatment. These results suggestthat osmotic stress affects carbon metabolism in guard cells;this metabolic change is different from that caused by ABA alone.Respiratory activity of guard cells decreased under osmoticstress. Therefore, the accumulation of malate and sucrose maybe caused by reduced respiration under osmotic stress. Accumulationof solutes in guard cells by osmotic stress may result in increasedosmotic pressure of guard cells and may play a role in protectionof guard cells from osmotic stress. (Received December 17, 1998; Accepted May 28, 1999)     

Abscisic-acid contents and concentrations in protoplasts from guard cells and mesophyll cells ofVicia faba L.

The abscisic-acid (ABA) contents of isolated guard-cell protoplasts and mesophyll-cell protoplasts fromVicia faba were determined by high-pressure liquid chromatography followed by gas chromatography. The amounts of ABA found immediately after preparation of the protoplasts varied from 90 to 570 amol per guard-cell protoplast, and from 75 to 100 amol per mesophyll-cell protoplast. These contents correspond to concentrations between 36 and 230 mol per liter in guard-cell protoplasts and between 2.7 and 3.3 mol per liter in mesophyll-cell protoplasts. During exposure of protoplasts to betaine concentrations of 0.3, 0.5, and 0.8 mol·l^-1 at 0° and 20°C for 30 min, ABA contents as well as the fractions of ABA that leaked into the medium remained constant for both protoplast types. There was no evidence for net production of ABA in isolated protoplasts subjected to osmotic stress.Abbreviation ABA abscisic acid     

Lateral Root Anlage Development in Excised Roots of Vicia faba L., Pisum sativum L., Zea mays L. and Phaseolus vulgaris L.

The development of lateral root primordia has been investigatedin excised roots of Vicia faba, Pisum sativum, Zea mays andPhaseolus vulgaris cultured in White's medium supplemented with2 per cent sucrose and compared with previously published dataon such development in primaries of the corresponding intactplants (control roots). Primordia were produced in each batchof excised roots over the 6 day culture period but at a lowerrate (number day^–1) than in the controls. Such primordia in cultured roots of Zea and Phaseolus completedtheir development and grew out as lateral roots over a periodsimilar in length to that found in the controls, but with acell number of only about 33 per cent of that attained at thetime of secondary emergence in the primaries of the latter roots.These lower cell numbers were at least partly a reflection ofincreases in mean cell doubling time over the period of anlagedevelopment investigated in the excised roots relative to thecorresponding values found in the controls. Primordia initiated in excised roots of Pisum and Vicia didnot complete their development in culture, i.e. no lateral rootsemerged and arrest took place with cell numbers of only 37 (Pisum)and 17 (Vicia) per cent of the numbers determined at the timeof secondary root emergence in the controls. Such arrested primordiahad few nuclei in S and none in mitosis. Moreover, at leastin Pisum, the frequency distribution of the relative DNA contentof the nuclei in the latter primordia approximated that foundin the apical meristem of primary roots following the establishmentof the stationary phase under conditions of carbohydrate starvation. It has also been demonstrated in the course of these investigationsthat lateral root primordium development in all four speciesis at least biphasic and possibly triphasic. Vicia faba L., broad bean, Pisum sativum L., garden pea, Zea mays L., maize, Phaseolus vulgaris L., dwarf bean, root primordia, anlage, cell doubling time, lateral root emergence