Xanthoxin levels and metabolism in the wild-type and wilty mutants of tomato

Using ¹³C-labelled internal standards and gas chromatography-mass spectrometry/multiple-ion monitoring the levels of xanthoxin (Xan) and 2-trans-xanthoxin (t-Xan) have been determined in stressed and non-stressed leaves of wildtype tomato (Lycopersicon esculentum Mill cv. Ailsa Craig), and the wilty mutants, notabilis (not), flacca (flc) and sitiens (sit). Levels of Xan were very low in all tissues. Ratios of t-Xan: Xan ranged from 10:1 to <500:1. In the wild-type and flc, t-Xan levels increased following stress. The results from feeding experiments using [¹³C]Xan and t-Xan demonstrated that whilst wild-type and not plants readily converted Xan into abscisic acid (ABA), flc and sit plants converted only a small amount of applied Xan into ABA. In all plants t-Xan was not converted into ABA. These results indicate that the flc and sit mutants are impaired in ABA biosynthesis because they are unable to convert Xan into ABA, whereas the not mutant is blocked at a metabolic step prior to Xan. Another possible ABA precursor, ABA-1,4-trans-diol (ABA-t-diol) was found to occur in wild-type and mutant tissue. All four tissues could convert [²H]ABA-t-diol to ABA. Incubation of stressed leaves in the presence of ¹⁸O₂ provided evidence consistent with Xan and ABA originating via oxidative cleavage of a xanthophyll such as violaxanthin.Abbreviations ABA abscisic acid - ABA-t-diol abscisic acid-1,4-trans-diol - DDC sodium diethyldithiocarbamate - FW fresh weight - GC-MS gas chromatography-mass spectrometry - i.d. internal diameter - MIM multiple-ion monitoring - PA phaseic acid - Xan xanthoxin - flc flacca - not notabilis - sit sitiens     

Abscisic Acid accumulation is not required for proline accumulation in wilted leaves

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.     

Xanthoxin Metabolism in Cell-free Preparations from Wild Type and Wilty Mutants of Tomato

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.     

Dynamic Properties of Endogenous Phytochrome A in Arabidopsis Seedlings

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.     

2-trans-ABA alcohol accumulation in the wilty tomato mutants flacca and sitiens

Abstract Two wilty tomato mutants, flacca and sitiens, fail to increase their endogenous ABA concentration in response to water stress. Instead, a compound accumulates which has been identified as 2-trans-ABA alcohol. Levels of this compound have been estimated for three wilty mutants and the control; both before and after water stress. When the compound was biosynthesized in the presence of ¹⁸O₂, one atom appeared to be incorporated into the primary alcohol group. The possible implications of this for the ABA biosynthetic pathway are discussed.     

Wild barley<Emphasis Type="Italic"> eibi1</Emphasis> mutation identifies a gene essential for leaf water conservation

Drought is a major abiotic stress that limits plant growth and crop productivity. A spontaneous wilty mutant (eibi1) hypersensitive to drought was identified from wild barley (Hordeum spontaneum Koch). eibi1 showed the highest relative water loss rate among the known wilty mutants, which indicates that eibi1 is one of the most drought-sensitive mutants. eibi1 had the same abscisic acid (ABA) level, the same ability to accumulate stress-induced ABA, and the same stomatal movement in response to light, dark, drought, and exogenous ABA as the wild type, revealing that eibi1 was neither an ABA-deficient nor an ABA-insensitive mutant. The eibi1 leaves had a larger chlorophyll efflux rate in 80% ethanol than the wild-type leaves; and the transpiration rate of eibi1 was more closely related to chlorophyll efflux rate than to stomatal density, demonstrating that the cuticle of eibi1 was defective. eibi1 will be a promising candidate to study the actual barrier layer in the cuticle that limits water loss of the plant. Exogenous ABA reduced leaf length growth in eibi1 more than in the wild type, implying an interaction on plant growth of ABA signal transduction and the eibi1 product. One may infer that the eibi1 product may reverse the growth inhibition induced by ABA.Abbreviation ABA Abscisic acid     

Phenotypic expression of wild-type tomato and three wilty mutants in relation to abscisic Acid accumulation in roots and leaflets of reciprocal grafts

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.     

The role of cis-carotenoids in abscisic acid biosynthesis

Evidence has been obtained which is consistent with 9-cis-neoxanthin being a major precursor of abscisic acid (ABA) in higher plants. A mild, rapid procedure was developed for the extraction and analysis of carotenoids from a range of tissues. Once purified the carotenoids were identified from their light-absorbance properties, reactions with dilute acid, high-performance liquid chromatography R_ts, mass spectra and the quasiequilibria resulting from iodine-catalysed or chlorophyllsensitised photoisomerisation. Two possible ABA precursors, 9-cis-neoxanthin and 9-cis-violaxanthin, were identified in extracts of light-grown and etiolated leaves (of Lycopersicon esculentum, Phaseolus vulgaris, Vicia faba, Pisum sativum, Cicer arietinum, Zea mays, Nicotiana plumbaginifolia, Plantago lanceolata and Digitalis purpurea), and roots of light-grown and etiolated plants (Lycopersicon, Phaseolus and Zea). The 9,9-di-cisisomer of violaxanthin was synthesised but its presence was not detected in any extracts. Levels of 9-cis-neoxanthin and all-trans-violaxanthin were between 20- to 100-fold greater than those of ABA in light-grown leaves. The levels of 9-cis-violaxanthin were similar to those of ABA but unaffected by water stress. Etiolated Phaseolus leaves contained reduced amounts of carotenoids (15–20% compared with light-grown leaves) but retained the ability to synthesise large amounts of ABA. The amounts of ABA synthesised, measured as increases in ABA and its metabolites phaseic acid and dihydrophaseic acid, were closely matched by decreases in the levels of 9-cis-neoxanthin and all-trans-violaxanthin. In etiolated seedlings grown on 50% D₂O, deuterium incorporation into ABA was similar to that into the xanthophylls. Relative levels of carotenoids in roots and light-grown and etiolated leaves of the ABA-deficient mutants, notabilis, flacca and sitiens were the same as those found in wild-type tomato tissues.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PA phaseic acid - t trans - Xan xanthoxin - flc flacca - not notabilis - sit sitiens The authors would like to thank the following for their help and advice: G. Britton (Department of Biochemistry, University of Liverpool, UK), B.H. Davies (Department of Biochemistry, University of Wales, Aberystwyth), P. Molnar, J. Szabolcs, D.C. Walton (Department of Biology, Suny, Syracuse, N.Y., USA), and Mr. J.K. Heald for his expert operation of the mass spectrometer. A.D.P. was supported initially by a Science and Engineering Research Council CASE award with Shell Biosciences, Sittingbourne, Kent, UK, and later by a Agricultural and Food Research Council (AFRC) grant. M.J.B. received a NATO fellowship. The mass spectrometer and HPLC-photodiode-array detector were purchased with funds provided by the AFRC.     

Wild-type levels of abscisic Acid are not required for heat shock protein accumulation in tomato

Levels of endogenous abscisic acid (ABA) in wild type were not required for the synthesis of heat shock proteins in detached leaves of tomato (Lycopersicon esculentum Mill., cv Ailsa Craig). Heat-induced alterations in gene expression were the same in the ABA-deficient mutant of tomato, flacca, and the wild type. Heat tolerance of the mutant was marginally less that the wild type, and in contrast, ABA applications significantly reduced the heat tolerance of wild-type leaves. It was concluded that elevated levels of endogenous ABA are not involved in the tomato heat shock response.     

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.     

Relationship between Mineral Nitrogen Influx and Transpiration in Radish and Tomato

Net ammonium and nitrate influx were independent of transpiration rate for intact seedlings of both a wild species of radish (Raphanus raphanistrum) and a wilty tomato mutant (Lycopersicon esculentum Mill. cv RR flacca).     

Abscisic Acid and C10 Dicarboxylic Acids in Wilty Tomato Mutants

Linforth, R. S. T., Taylor, I. B. and Hedden, P. 1987. Abscisicacid and C10 dicarboxylic acids in wilty tomato mutants.—J.exp. Bot. 38: 1734–1740. The concentration of C₁₀ dicarboxylic acids in wilty tomatomutants was investigated. Three of the genotypes studied (flacca,sitiens and the double mutant homozygote flacca/sitiens) werefound to have higher concentrations of 2,7-dimethyl-2,4-octadienedioicacid (ODA) than the isogenic normal form. In contrast, the othergenotypes (notabilisand the double mutant homozygotes notabilis/flaccaand notabilis/sitiens) were found to have lower concentrationsof ODA than the isogenic normal form. The concentration of ODAin flacca plants was increased by water stress and reduced byexogenously applied abscisic acid (ABA). A second structurallyrelated compound, 2,7-dimethyl-4-octenedioic acid (OEA) wasalso quantified, but it showed no clear genotype-related pattern. The concentration of ABA in the wilty tomato mutants was alsoinvestigated. As expected in the light of previously publishedresults, it was reduced in the single mutants relative to theisogenic control plants. In the double mutant flacca/sitiensABA levels were similar to those of the single mutant sitiens.However, in the two double mutants notabilis/flacca and notabilis/sitiensABA was substantially lower than those in any other genotypeinvestigated. Key words: Abscisic acid, 2,7-dimethyl-2,4-octadienedioic acid, 2,7-dimethyl-4-octenedioc acid, tomato, wilty mutants     

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO ₃ ^- absorption increased two-to five fold within 2 d of being deprived of NO ₃ ^- supply. Nitrogen-starved barley roots continued to maintain a high potential for NO ₃ ^- absorption, whereas NO ₃ ^- absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO ₃ ^- solution, roots of both species transported more NO ₃ ^- and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO ₃ ^- stores took precedence over NO ₃ ^- transport to the xylem. Consequently, as N stress became more severe, transport of NO ₃ ^- and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L _p) and exudate volume (J _v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L _p and J _v were increased, but NO ₃ ^- absorption and NO ₃ ^- flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO ₃ ^- absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO ₃ ^- absorption and transport and L _p had been observed (wild-type tomato); (2) changes in NO ₃ ^- absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO ₃ ^- transport to those observed in the wild type.Abbreviation and symbols ABA abscisic acid - J_v exudate volume - L_p root hydraulic conductance     

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     

The genetic relationship between the tomato mutants,flacca and lateral suppressor,with reference to abscisic acid accumulation

Two tomato mutants, Lycopersicon esculentum flacca and lateral suppressor, are assigned to map position 59 of chromosome 7. The tight linkage between these two gene loci was detected as a result of attempts to establish whether they would exhibit phenotypic interaction. The possibility that both mutants result in abnormalities of abscisic acid (ABA) accumulation is considered. ABA analysis supports the suggestion that plants homozygous for flacca have a substantially lower concentration but indicates that lateral suppressor homozygotes do not differ from normal in ABA content. An attempt is made to reconcile the results with those of Tucker (1976, New. Phytol. 77, 561–568) by suggesting that lateral suppressor plants may accumulate high levels of an ABA metabolite which is indistinguishable from ABA using the Commelina epidermal strip bioassay.Abbreviations ABA abscisic acid - flc flacca - ls lateral suppressor - La Lanceolate     

Maternal synthesis of abscisic acid controls seed development and yield in<Emphasis Type="Italic"> Nicotiana plumbaginifolia</Emphasis>

The role of maternally derived abscisic acid (ABA) during seed development has been studied using ABA-deficient mutants of Nicotiana plumbaginifolia Viviani. ABA deficiency induced seed abortion, resulting in reduced seed yield, and delayed growth of the remaining embryos. Mutant grafting onto wild-type stocks and reciprocal crosses indicated that maternal ABA, synthesized in maternal vegetative tissues and translocated to the seed, promoted early seed development and growth. Moreover ABA deficiency delayed both seed coat pigmentation and capsule dehiscence. Mutant grafting did not restore these phenotypes, indicating that ABA synthesized in the seed coat and capsule envelope may have a positive effect on capsule and testa maturation. Together these results shed light on the positive role of maternal ABA during N. plumbaginifolia seed development.Abbreviations ABA Abscisic acid - DAP Days after pollination - g Grafted - Wt Wild-type     

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.