Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: I. Root Effect and Kinetin-like Activity

The wilty tomato mutant, flacca, and the normal variety, Rheinlands Ruhm, were compared for kinetin-like activity in ontogeny. The mutant wilts easily because its stomata resist closure. This stomatal resistance decreases with age. The occurrence of a root factor which induces stomatal opening was inferred from grafting experiments. It was hypothesized that the excessive stomatal openings in the mutant may result from excess of kinetin-like activity in the leaf of that plant. In addition, it was suggested that the closure of stomata in the aging mutant is due to a decrease of kinetin-like activity with age. Kinetin-like activity in the leaf was determined by incorporation of labeled leucine. The concentration of cytokinins in root exudate and leaf extract was determined by the soybean callus assay. Evidence was presented of higher kinetin-like activity in the leaves of the mutant and higher cytokinin concentration in its root exudate. Cytokinin concentration in the shoot was found to be only slightly higher in the mutant than in the normal plants. Kinetin-like activity in the leaf and cytokinin concentration of root exudate decreased with age in both mutant and normal plants. Kinetin-like activity in the leaves of mutant plants, which phenocopy the normal variety as a result of continuous application of abscisic acid, was lower than in control mutant plants. The significance of these findings per se and in connection with stomatal behavior is discussed.     

Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: IV. Effect of Abscisic Acid and Water Content on RNase Activity and RNA

Plants of the wilty tomato (Lycopersicum esculentum) mutant, flacca, and of the normal cultivar Rheinlands Ruhm growing under either “normal” or high humidity were used in this research. Under normal humidity, RNase activity was much higher in mutant plants in which abscisic acid (ABA) and water content were lower than in the normal plant. The mutant also contained less RNA and protein per cell and less soluble RNA relative to ribosomal RNA as compared with the normal genotype. In ABA-treated mutant plants, RNase activity decreased while RNA, protein, the ratio of soluble to ribosomal RNA and water content increased.     

Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: V. Effect of Abscisic Acid on Indoleacetic Acid Metabolism and Ethylene Evolution

The wilty tomato mutant flacca, the normal cultivar Lycopersicon esculentum Mill. Rheinlands Ruhm, and abscisic acid-induced phenotypic revertants were compared with respect to ethylene evolution, activity of tryptophan aminotransferase, and [1-¹⁴C]indoleacetic acid decarboxylation.     

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.     

Gas Exchange, Stomatal Behavior, and deltaC Values of the flacca Tomato Mutant in Relation to Abscisic Acid

The relationship between stomatal conductance and capacity for assimilation was investigated in flacca, a mutant of tomato (Lycopersicon esculentum Mill.) that has abnormal stomatal behavior and low abscisic acid (ABA) content. The assimilation capacity, determined by measuring assimilation rate as a function of intercellular CO₂ pressure, did not differ in leaves of flacca and its parent variety, Rheinlands Ruhm (RR). On the other hand, stomatal conductance of flacca leaves was greater than that of RR, and could be phenotypically reverted by spraying with 30 micromolar ABA. Stomatal conductance of flacca leaves was also reduced by increasing CO₂ pressure, increasing leaf to air vapor pressure difference, and decreasing quantum flux, irrespective of ABA treatment.

The high conductance of flacca leaves resulted in a high intercellular CO₂ pressure. This allowed greater discrimination against ¹³CO₂, as evidenced by more negative δ ¹³C values for flacca as compared to RR. The δ ¹³C values of both flacca and RR plants as influenced by ABA treatment were consistent with predictions based on gas exchange measurements, using a recent model of discrimination.

     

Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Abnormal Stomatal Behaviour and Leaf Anatomy in Capsicum annuum, scabrous diminutive, a Wilty Mutant of Pepper

An attempt is made to explain the tendency to excessive wiltingin scabrous diminutive, a pepper mutant. For this, mutant andnormal plants were compared with respect to leaf anatomy, transpirationof whole plants and detached drying leaves, density and openingof stomata, staining of potassium in epidermal cells and rootpressure. A much greater proportion of intracellular space was found inthe mutant leaf which contains fewer and smaller mesophyll cellsthan the normal plant. The anticlinal walls of the epidermisof the mutant leaf were almost straight whereas those of thenormal were wavy. Transpiration per unit leaf area of wholeplants, percentage of stomata open both day and night, and waterloss from detached drying leaves were all higher in the mutant.Potassium staining in guard cells was similar in both planttypes, slightly less in darkness and marginally higher in light.The subsidiary cells of normal leaves did not stain at all,but those of the mutant leaves stained heavily in both lightand darkness. Root pressure was lower in the mutant. Possible explanations for the tendency of the pepper mutantto wilt are discussed.     

Water Relations and Growth of the flacca Tomato Mutant in Relation to Abscisic Acid

The flacca mutant in tomato (Lycopersicon esculentum Mill. cv Rheinlands Ruhm) was employed to examine the effects of a relatively constant diurnal water stress on leaf growth and water relations. As the mutant is deficient in abscisic acid (ABA) and can be phenotypically reverted to the wild type by applications of the growth substance, inferences can be made concerning the involvement of ABA in responses to water stress. Water potential and turgor were lower in leaves of flacca than of Rheinlands Ruhm, and were increased by ABA treatment. ABA decreased transpiration rates by causing stomatal closure and also increased the hydraulic conductance of the sprayed plants. Osmotic adjustment did not occur in flacca plants despite the daily leaf water deficits. Stem elongation was inhibited by ABA, but leaf growth was promoted. It is concluded that, in some cases, ABA may promote leaf growth via its effect on leaf water balance.     

Ion Imbalance in Capsicum annum scarbrous diminutive a Wilty Mutant of pepper: II.RUBIDIUM FLUX

Root tips of the wilty pepper mutant scarbrous diminutive accumulateless rubidium than those of the normal genotype. This phenomenonwas evident in root tips excised from plants maintained for2 d in CaSO₄ solution (low salt plants), especially in the lowerexternal concentration range (0.1– 1.0 mM) of RbCl. Theefflux rate of Rb⁺ from mutant root tips was twice as high asin normal root tips. These results indicate that the ability of the mutant rootsto absorb and accumulate Rb⁺ and K⁺ is impaired. This defectcould be a consequence of either an impaired Na⁺/K⁺ carriersystem, or increased leakiness of mutant membranes, or both. The fact that the normal roots can accumulate Rb⁺ much fasterthan mutant roots supports the first alternative, i.e. thatthe high affinity carrier system was impaired in the mutantroots. However, the higher efflux rate of Rb⁺ from the mutantroots suggests that membrane leakiness was also affected.     

Ion Imbalance in Capsicum annuum, scabrous diminutive, a Wilty Mutant of Pepper I. SODIUM FLUXES

Leaves of the wilty pepper mutant, scabrous diminutive, accumulatemore Na+ than those of the normal genotype, when both grow inmedium containing N⁺. It seems that the regulation of Na⁺ fluxin the mutant root was modified. Net uptake of Na⁺ was muchhigher and efflux of ²²Na⁺ was lower in the mutant roots thanin the normal ones. Two possible explanations for these differencesbetween mutant and normal plants are discussed, namely (a) achange in membrane permeability and (b) a change in the mechanismof Na⁺ extrusion.     

The Tomato Fruit Cell Wall : II. Polyuronide Metabolism in a Nonsoftening Tomato Mutant

A nonsoftening tomato (Lycopersicon esculentum L.) variety, dg, was examined to assess the physiological basis for its inability to soften during ripening. Total uronic acid levels, 18 milligrams uronic acid/100 milligrams wall, and the extent of pectin esterification, 60 mole%, remained constant throughout fruit development in this mutant. The proportion of uronic acid susceptible to polygalacturonase in vitro also remained constant. Pretreatment of heat-inactivated dg fruit cell walls with tomato pectinmethylesterase enhances polygalacturonase susceptibility at all ripening stages. Pectinesterase activity of cell wall protein extracts from red ripe dg fruit was half that in extracts from analogous tissue of VF145B. Polygalacturonase activities of cell wall extracts, however, were similar in both varieties. Diffusion of uronic acid from tissue discs of both varieties increased beginning at the turning stage to a maximum of 2.0 milligrams uronic acid released/gram fresh weight at the ripe stage. The increased quantity of hydrolytic products released during ripening suggests the presence of in situ polygalacturonase activity. Low speed centrifugation was employed to induce efflux of uronide components from the cell wall tree space. In normal fruit, at the turning stage, 2.1 micrograms uronic acid/gram fresh weight was present in the eluant after 1 hour, and this value increased to a maximum of 8.2 micrograms uronic acid/gram fresh weight at the red ripe stage. However, centrifuge-aided extraction of hydrolytic products failed to provide evidence for in situ polygalacturonase activity in dg fruit. We conclude that pectinesterase and polygalacturonase enzymes are not active in situ during the ripening of dg fruit. This could account for the maintenance of firmness in ripe fruit tissue.     

Stomatal Closure in Flooded Tomato Plants Involves Abscisic Acid and a Chemically Unidentified Anti-Transpirant in Xylem Sap

We address the question of how soil flooding closes stomata of tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants within a few hours in the absence of leaf water deficits. Three hypotheses to explain this were tested, namely that (a) flooding increases abscisic acid (ABA) export in xylem sap from roots, (b) flooding increases ABA synthesis and export from older to younger leaves, and (c) flooding promotes accumulation of ABA within foliage because of reduced export. Hypothesis a was rejected because delivery of ABA from flooded roots in xylem sap decreased. Hypothesis b was rejected because older leaves neither supplied younger leaves with ABA nor influenced their stomata. Limited support was obtained for hypothesis c. Heat girdling of petioles inhibited phloem export and mimicked flooding by decreasing export of [14C]sucrose, increasing bulk ABA, and closing stomata without leaf water deficits. However, in flooded plants bulk leaf ABA did not increase until after stomata began to close. Later, ABA declined, even though stomata remained closed. Commelina communis L. epidermal strip bioassays showed that xylem sap from roots of flooded tomato plants contained an unknown factor that promoted stomatal closure, but it was not ABA. This may be a root-sourced positive message that closes stomata in flooded tomato plants.     

Dormancy and Germination of Abscisic Acid-Deficient Tomato Seeds : Studies with the sitiens Mutant

The role of abscisic acid (ABA) in the dormancy induction of tomato (Lycopersicon esculentum) seeds was studied by comparison of the germination behavior of the ABA-deficient sitiens mutant with that of the isogenic wild-type genotype. Freshly harvested mutant seeds, in contrast to wild-type seeds, always readily germinate and even exhibit viviparous germination in overripe fruits. Crosses between mutant and wild-type and self-pollination of heterozygous plants show that in particular the ABA fraction of embryo and endosperm is decisive for the induction of dormancy. After-ripened wild-type seeds fully germinate in water but are more sensitive toward osmotic inhibition than mutant seeds. Germination of both wild-type and mutant seeds is equally sensitive toward inhibition by exogenous ABA. ABA content of mature wild-type seeds is about 10-fold the level found in mutant seeds. Nevertheless, it is argued that the differences in dormancy between the seeds of both genotypes are not a result of actual ABA levels in the mature seeds or fruits but a result of differences in ABA levels during seed development. It is hypothesized that the high levels of ABA that occur during seed development in wild-type seeds induce an inhibition of cell elongation of the radicle that can still be observed after long periods of dry storage.     

Germination and Dormancy of Abscisic Acid- and Gibberellin-Deficient Mutant Tomato (Lycopersicon esculentum) Seeds (Sensitivity of Germination to Abscisic Acid,Gibberellin, and Water Potential)

Germination responses of wild-type (MM), abscisic acid (ABA)-deficient (sitw), and gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds to ABA, GA4+7, reduced water potential ([psi]), and their combinations were analyzed using a population-based threshold model (B.R. Ni and K.J. Bradford [1992] Plant Physiol 98: 1057-1068). Among the three genotypes, sitw seeds germinated rapidly and completely in water, MM seeds germinated more slowly and were partially dormant, and gib-1 seeds did not germinate without exogenous GA4+7. Times to germination were inversely proportional to the differences between the external osmoticum, ABA, or GA4+7 concentrations and the corresponding threshold levels that would either prevent ([psi]b, log[ABAb]) or promote (log[GAb]) germination. The sensitivity of germination to ABA, GA4+7, and [psi] varied widely among individual seeds in the population, resulting in a distribution of germination times. The rapid germination rate of sitw seeds was attributable to their low mean [psi]b (-1.17 MPa). Postharvest dormancy in MM seeds was due to a high mean [psi]b (-0.35 MPa) and a distribution of [psi]b among seeds such that some seeds were unable to germinate even on water. GA4+7 (100 [mu]M) stimulated germination of MM and gib-1 seeds by lowering the mean [psi]b to -0.75 MPa, whereas ABA inhibited germination of MM and sitw seeds by increasing the mean [psi]b. The changes in [psi]b were not due to changes in embryo osmotic potential. Rather, hormonal effects on endosperm weakening opposite the radicle tip apparently determine the threshold [psi] for germination. The analysis demonstrates that ABA- and GA-dependent changes in seed dormancy and germination rates, whether due to endogenous or exogenous growth regulators, are based primarily upon corresponding shifts in the [psi] thresholds for radicle emergence. The [psi] thresholds, in turn, determine both the rate and final extent of germination within the seed population.     

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-1

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EF_s particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O₂ evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes.     

Photosystem II Activity, Plastoquinone A Levels, and Fluorescence Characterization of a Virescens Mutant of Barley

Chloroplasts isolated from seedlings of a virescens mutant of barley (Hordeum vulgare L cv Gateway) grown for 6 days under continuous illumination had lower levels of photosystem II activities on a chlorophyll basis than wild-type seedlings. After 8 days, however, the photosystem II rates of the mutant and wild-type were approximately equal. Lower levels of the photosystem II activities in the mutant were correlated with a smaller functional plastoquinone pool size as determined by room temperature fluorescence induction. Higher levels of extractable plastoquinone A on a chlorophyll basis, however, were obtained from mutant chloroplasts. An increased room temperature fluorescence yield in the mutant was shown to be due to a higher level of initial fluorescence. A decreased sigmoidicity in the room temperature fluorescence induction transient in the presence of diuron and an increased 77 K fluorescence emission at 680 nanometers lead us to believe that a certain population of the light harvesting chlorophyll protein complex in the mutant membranes is unconnected to photo-system II reaction centers. Although photochemical activities of the mutant approach wild-type values as the mutant develops, the population of dissociated light harvesting complexes does not appear to change.     

Stomatal Behavior and Water Status of Maize, Sorghum, and Tobacco under Field Conditions: II. At Low Soil Water Potential

Diurnal changes in the vertical profiles of irradiance incident upon the adaxial leaf surface (I), leaf resistance (r₁), leaf water potential (ψ), osmotic potential (π), and turgor potential (P) were followed concurrently in crops of maize (Zea mays L. cv. Pa602A), sorghum (Sorghum bicolor [L.] Moench cv. RS 610), and tobacco (Nicotiana tabacum L. cv. Havanna Seed 211) on several days in 1968 to 1970 when soil water potentials were low. The r₁, measured with a ventilated diffusion porometer, of the leaves in the upper canopy decreased temporarily after sunrise [~0530 hours Eastern Standard Time] as I increased, but then r₁ increased again between 0700 and 0830 hr Eastern Standard Time as the ψ, measured with a pressure chamber, decreased rapidly from the values of −7, −4 and −6 bars at sunrise to minimal values of −18, −22 and −15 bars near midday in the maize, sorghum, and tobacco, respectively. The π, measured with a vapor pressure osmometer, also decreased after sunrise, but not to the same degree as the decrease in ψ, so that a P of zero was reached in some leaves between 0730 and 0800 hours. The lower (more negative) π of leaves in the upper canopy than those in the lower canopy gave the upper leaves a higher P at a given ψ than the lower leaves in all three species; leaves at intermediate heights had an intermediate P. This difference between leaves at the three heights in the canopy was maintained at all values of ψ. The r₁ remained unchanged over a wide range of P and then increased markedly at a P of 2 bars in maize, −1 bar in sorghum, and near zero P in tobacco: r₁ also remained constant until ψ decreased to −17, −20, and −13 bars in leaves at intermediate heights in maize, sorghum, and tobacco, respectively. In all three species r₁ of leaves in the upper canopy increased at more negative values of ψ than those at the base of the canopy, and in tobacco, leaves in the upper canopy wilted at more negative values of ψ than those in the lower canopy.     

Ineffectiveness of Abscisic Acid in Stomatal Closure of Yellow Lupin, Lupinus luteus var. Weiko III

Stomata of yellow lupin leaves are remarkably insensitive toabscisic acid (ABA). Stomatal resistance was monitored usingboth a viscous now porometer and a diffusion porometer. Resultswere confirmed with scanning electron microscopy. When exogenousABA solutions were supplied via petioles, 10^–6 M solutionshad no effect on stomatal resistance. Upper (adaxial) stomatawere not affected by 10^–5 M ABA but lower stomata showed3-fold more resistance after 2 h. Stomata of both surfaces closedafter 30 min in 10^–4 M ABA. Isolated epidermal peels of lupin leaves were floated on ABAsolutions yet upper surface peels showed no stomatal closingin 10^–4 M ABA, while lower surface stomata closed to abarely significant extent. Stomata of intact leaves were not very sensitive to darkness,showing at most a doubling in resistance after 6 h darkness.Complete stomatal closure, however, was readily produced bywilting leaves. Hence, lupin stomata are physically capableof closing. Endogenous ABA levels of water-stressed leaves increased approximately10-fold, which corresponds to concentrations below 10 µMABA. It is concluded that ABA is unlikely to play a role incontrolling short-term stomatal response of lupins.