The pH of xylem sap from tomato (Lycopersicon esculentum) plants increased from pH 5.0 to 8.0 as the soil dried. Detached wild-type but not flacca leaves exhibited reduced transpiration rates when the artificial xylem sap (AS) pH was increased. When a well-watered concentration of abscisic acid (0.03 &mgr;m) was provided in the AS, the wild-type transpirational response to pH was restored to flacca leaves. Transpiration from flacca but not from wild-type leaves actually increased in some cases when the pH of the AS was increased from 6.75 to 7.75, demonstrating an absolute requirement for abscisic acid in preventing stomatal opening and excessive water loss from plants growing in many different environments.

The flacca tomato (Lycopersicon esculentum) mutant displays a wilty phenotype as a result of abscisic acid (ABA) deficiency. The Mo cofactor (MoCo)-containing aldehyde oxidases (AO; EC 1.2.3.1) are thought to play a role in the final oxidation step required for ABA biosynthesis. AO and related MoCo-containing enzymes xanthine dehydrogenase (XDH; EC 1.2.1.37) and nitrate reductase (EC 1.6.6.1) were examined in extracts of the flacca tomato genotype and of wild-type (WT) roots and shoots. The levels of MoCo were found to be similar in both genotypes. No significant XDH or AO (MoCo-containing hydroxylases) activities were detected in flacca leaves; however, the mutant exhibited considerable MoCo-containing hydroxylase activity in the roots, which contained notable amounts of ABA. Native western blots probed with an antibody to MoCo-containing hydroxylases revealed substantial, albeit reduced, levels of cross-reactive protein in the flacca mutant shoots and roots. The ABA xylem-loading rate was significantly lower than that in the WT, indicating that the flacca is also defective in ABA transport to the shoot. Significantly, in vitro sulfurylation with Na2S reactivated preexisting XDH and AO proteins in extracts from flacca, particularly from the shoots, and superinduced the basal-level activity in the WT extracts. The results indicate that in flacca, MoCo-sulfurylase activity is impaired in a tissue-dependent manner.

The hypothesis that ABA produced by roots in drying soil is responsible for stomatal closure was tested with grafted plants constructed from the ABA-deficient tomato mutants, sitiens and flacca and their near-isogenic wild-type parent. Three types of experiments were conducted. In the first type, reciprocal grafts were made between the wild type and sitiens or flacca. Stomatal conductance accorded with the genotype of the shoot, not the root. Stomates closed in all of the grafted plants in response to soil drying, regardless of the root genotype, i.e. regardless of the ability of the roots to produce ABA. In the second type of experiment, wild-type shoots were grafted onto a split-root system consisting of one wild-type root grafted to one mutant (flacca or sitiens) root. Water was withheld from one root system, while the other was watered well so that the shoots did not experience any decline in water potential or loss of turgor. Stomates closed to a similar extent when water was withheld from the mutant roots or the wild-type roots. In the third type of experiment, grafted plants with wild-type shoots and either wild-type or sitiens roots were established in pots that could be placed inside a pressure chamber, and the pressure increased as the soil dried so that the shoots remained fully turgid throughout. Stomates closed as the soil dried, regardless of whether the roots were wild type or sitiens. These experiments demonstrate that stomatal closure in response to soil drying can occur in the absence of leaf water deficit, and does not require ABA production by roots. A chemical signal from roots leading to a change in apoplastic ABA levels in leaves may be responsible for the stomatal closure.

The molybdenum cofactor (MoCo)-containing enzymes aldehyde oxidase (AO; EC 1.2.3.1) and xanthine dehydrogenase (XDH; EC 1.2.1.37) require for activity a sulfuration step that inserts a terminal sulfur ligand into the MoCo. The tomato flacca mutation was originally isolated as a wilty phenotype due to a lack of abscisic acid (ABA) that is related to simultaneous loss of AO and XDH activities. An expressed sequence tag candidate from tomato was selected on the basis of homology to sulfurases from animals, fungi and the recently isolated Arabidopsis genes LOS5/ABA3. The tomato homologue maps as a single gene to the bottom of chromosome 7, consistent with the genetic location of the flacca mutation. The structure of FLACCA shows a multidomain protein with an N-terminal NifS-like sulfurase domain; a mammal-specific intermediate section; and a C-terminus containing conserved motifs. Prominent among these are molybdopterin oxidoreductases and thioredoxin redox-active centre/iron-sulfur-binding region signatures which may be relevant to the specific sulfuration of MoCo. Indeed, the molecular analysis of flacca identifies the mutation in a highly conserved motif located in the C-terminus. Activity gel assays show that FLACCA is expressed throughout the plant. Transient and stable complementation of flacca and the Arabidopsis aba3 mutants with Aspergillus nidulans hxB and FLACCA yielded full, partial and tissue-specific types of Mo-hydroxylase activities. Restoration of activity in the root alone is sufficient to augment plant ABA content and rectify the wild-type phenotype. Thus the pleiotropic flacca phenotype is due to the loss of activity of enzymes requiring a sulfurated MoCo.

An attempt was made to explain the excessive wilting tendency of 3 tomato mutants, notabilis, flacca, and sitiens. The control varieties in which these mutations were induced are Rheinlands Ruhm for flacca and sitiens and Lukullus for notabilis. Although all 3 mutants are alleles of separated loci, they seem to react similarly to water stress. The mutants wilt faster than the control plants when both are subjected to the same water stress. It was demonstrated by measurements of water loss from whole plants that all 3 mutants have much higher rates of transpiration than the control varieties, particularly at night. The extent of cuticular transpiration was compared in both kinds of plants by measuring the rate of water loss from detached drying leaves coated with vaseline on the lower surface. The difference in cuticular transpiration between the mutant and the control plants seems to be negligible. However, various facts point to stomata as the main factor responsible for the higher rates of water loss in the mutant plants. The stomata of the latter tend to open wider and to resist closure in darkness, in wilted leaves, and when treated with phenylmercuric acetate. Stomata of the 2 extreme mutants, sitiens and flacca, remain open even when the guard cells are plasmolyzed. The stomata of the mutants also are more frequent per unit of leaf surface and vary more in their size.

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-(14)C]indoleacetic acid decarboxylation.The level of ethylene evolution was higher in flacca plants than in the normal cultivar. Ethylene evolution was reduced to the wild type level in abscisic acid-induced phenotypic revertants and to a lesser extent in mutant plants grown under humid conditions. Leaf epinasty, which characterized flacca plants in the present experiments, did not appear in absciscic acid-treated mutant plants, but did appear under high humidity. Tryptophan aminotransferase activity, similar to ethylene evolution, was higher in flacca plants and was reduced to the normal level by abscisic acid treatment. Indoleacetic acid decarboxylation was similar in mutant and normal plants, but was increased by abscisic acid treatment. The relationships among ethylene, auxin, and the morphological symptoms which characterize the mutant are discussed.

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 CO(2) 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 CO(2) 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 CO(2) pressure. This allowed greater discrimination against (13)CO(2), as evidenced by more negative delta (13)C values for flacca as compared to RR. The delta (13)C 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.

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.

Lycopersicon esculentum Mill. cv Rheinlands Ruhm (RR) and cv Moneymaker and the three wilty mutants flacca (flc), sitiens (sit), and sitiens(w) (sit(w)), together with most reciprocal grafts, were grown in pots and in solution culture. Detached leaflets, and control and steam-girdled intact plants, were left turgid or were wilted in air. Detached leaflets and the leaflets and roots of the intact plants were analyzed for their abscisic acid (ABA) content. Turgid RR leaflets contained about 2.9 ng ABA per milligram dry weight. On average, the flc and sit leaflets contained 33 and 11% of this amount, respectively. The lack of ABA approximately correlated with the severity of the mutant phenotype. Mutant roots also contained less ABA than wild-type roots. Wild-type scions on mutant stocks (wild type/mutant) maintained the normal phenotype of ungrafted plants. Mutant scions grafted onto wild-type stocks reverted to a near wild-type phenotype. After the wild-type leaves were excised from solution culture-grown mutant/wild-type plants, the revertive morphology of the mutant scions was maintained, although endogenous ABA levels in the leaflets fell to typical mutant levels and the leaflets became wilty again. When stressed in air, both leaflets and roots of RR plants produced stress-induced ABA, but the mutant leaflets and roots did not. The roots and leaflets of the grafted plants behaved according to their own genotype, with the notable exception of mutant roots grown with wild-type scions. Roots of flc and sit(w) recovered the ability to accumulate stress-induced ABA when grafted with RR scions before the stress was imposed.

Extracts prepared from the turgid and water-stressed leaves of wild-type tomato (Lycopersicon esculentum Mill cv Ailsa Craig) and the wilty mutants sitiens, notabilis, and flacca were tested for their ability to metabolize xanthoxin to ABA. Extracts from wild type and notabilis converted xanthoxin at similar rates, while extracts from sitiens and flacca showed little or no activity. We also observed no activity when extracts of sitiens and flacca were mixed. Similar results were obtained when ABA aldehyde was used as a substrate, in that extracts from wild type and notabilis were equally active, but extracts from flacca and sitiens showed little activity. None of the tomato extracts showed significant activity with xanthoxin acid, xanthoxin alcohol, or ABA-1',4-'Trans-diol as substrates. Extracts from bean leaves (Phaseolus vulgaris L. cv Blue Lake) were similar to the wild-type tomato extracts in their ability to convert the various substrates to ABA, although excised bean leaves did convert ABA-1',4'-trans-diol and xanthoxin alcohol to ABA when these substances were taken up through the petiole. These results are consistent with a role for xanthoxin as a normal intermediate on the ABA biosynthetic pathway, and they suggest that ABA aldehyde is the final ABA precursor.