Structure and expression of a cDNA encoding a putative neoxanthin cleavage enzyme (NCE), isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Oxidative cleavage of a 9-cis xanthophyll probably representsthe key regulatory step in abscisic acid (ABA) biosynthesis.A transposon tagged maize (Zea mays) mutant vp14, provided theoriginal DNA sequence data needed to design a VP14 fusion proteincapable of catalysing this reaction in vitro. A cDNA encodinga similar protein has now been isolated from a wilt-relatedtomato (Lycopersicon esculentum Mill.) library. The tomato cDNAand derived amino acid sequence have been compared to thoseof maize and of other enzymes catalysing broadly similar oxidativecleavage reactions. The results of Northern analysis in tomatoindicated that mRNA levels of this vp14 homologue increaseddramatically in response to water stress. Key words: ABA biosynthesis, oxidative cleavage step, tomato     

The 33 kDa Protein of the Oxygen-Evolving Complex: a Multi-Gene Family in Tomato

A cDNA was isolated by chance from tomato which had a high similarityto a cDNA clone from potato known to code for the 33 kDa proteinof the oxygen-evolving complex [van Spanje et al. (1991) PlantMol. Biol. 17: 157]. The sequence of a previously describedpartial cDNA clone from tomato [Ko et al. (1990) Plant Mol.Biol. 14: 217] which has also a high similarity but is not identicalto the sequence described here indicates that tomato containsat least two genes coding for 33 kDa proteins per haploid genome.This conclusion is supported by Southern blot analysis. Thetissue specific expression of the corresponding genes is described. (Received September 28, 1992; Accepted February 15, 1993)     

Abscisic-acid metabolism in a wilty mutant of Nicotiana plumbaginifolia

A mutant of Nicotiana plumbaginifolia, CKR1, isolated on the basis of its enhanced resistance to cytokinins was found to have a greater tendency to wilt than the wild type (Blonstein et al., 1991, Planta 183, 244–250). Further characterisation has shown that the wiltiness in the mutant is not caused by an insensitivity to abscisic acid (ABA) because the external application of ABA leads to stomatal closure and phenotypic reversion. The basal ABA level in the mutant is < 20% of that in the wild type. Following stress, the ABA level in wild-type leaves increases by approx 9-to 10-fold while the mutant shows only a slight increase. This deficiency in ABA is unlikely to be the consequence of accelerated catabolism as the levels of two major metabolites of ABA, phaseic and dihydrophaseic acid, are also much reduced in the mutant. The qualitative and quantitative distributions of carotenoids, the presumed presursors of ABA, are the same for the leaves of both wild type and mutant. Biosynthesis of ABA at the C15 level was investigated by feeding xanthoxin (Xan) to detached leaves. Wild-type leaves convert between 9–19% of applied Xan to ABA while the mutant converts less than 1%. The basal level of trans-ABA-alcohol (t-ABA-alc) is 3-to 10-fold greater in the mutant and increases by a further 2.5-to 6.0-fold after stress. This indicates that the lesion in the wilty mutant of N. plumbaginifolia affects the conversion of ABA-aldehyde to ABA, as in the flacca and sitiens mutants of tomato and the droopy mutant of potato (Taylor et al., 1988, Plant Cell Environ. 11, 739–745; Duckham et al., 1989, J. Exp. Bot. 217, 901–905). Wild-type tomato and N. plumbaginifolia leaves can convert trans-Xan into t-ABA-alc, and Xan into ABA, while those of flacca and the wilty N. plumbaginifolia mutant convert both Xan and t-Xan to t-ABA-alc.     

The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

C-ABI3, the Carrot Homologue of the Arabidopsis ABI3, is Expressed during Both Zygotic and Somatic Embryogenesis and Functions in the Regulation of Embryo-Specific ABA-Inducible Genes

A carrot gene homologous to the ABI3 gene of Arabidopsis wasisolated from a carrot somatic embryo cDNA library and designatedC-ABI3. The sequence of C-ABI3 was very similar to those ofABI3 of Arabidopsis and VP1 of maize in certain conserved regions.The expression of C-ABI3 was detected specifically in embryogeniccells, somatic embryos and developing seeds. Thus, expressionof C-ABI3 was limited to tissues that acquired desiccation tolerancein response to endogenous or exogenous abscisic acid (ABA).Endogenous levels of ABA in seeds increased transiently andthen desiccation of seeds started. The expression of C-ABI3in developing seeds was observed prior to the increase in levelsof endogenous ABA that was followed by desiccation of seeds.In transgenic mature leaves in which C-ABI3 was ectopicallyexpressed, expression of ECP31, ECP63 and ECP40 was inducedby treatment with ABA, which indicates that the expression ofECP genes was controlled by the pathway(s) that involved C-ABI3and ABA. This suggests that C-ABI3 has the same function asVP1/ABI3 factor in carrot somatic embryos. (Received March 4, 1998; Accepted September 4, 1998)     

Apical Dominance in the Tomato: Some Further Observations on Isogenic Lines Showing Varying Degrees of Side-shoot Development

A study has been made of the distribution of substances resemblingindol-3-ylacetic acid (IAA), abscisic acid (ABA) and cytokinin-likesubstances in the stem tissue of Craigella tomato plants ascompared with that found in two isogenic lines of this variety,Craigella Blind (blbl) and Craigella Lateral Suppressor (lsls),in both of which side shoot growth is suppressed to varyingdegrees. There was no evidence to suggest that the distributionof these hormones in the stem had any association with the differentpatterns of side shoot development of the three types, thoughsome of the lateral suppressor plants which exhibited only partialbud inhibition did show a relation between high auxin and abscisicacid levels and lack of side shoot development from the centralnodes of the shoot. Decapitation led to a stimulation of bud outgrowth from allnodes of the Craigella plants but the lateral suppressor plantsremained inhibited. The blind plants were found to initiatebud primordia at the cotyledonary nodes only when the severedapex was replaced by exogenous IAA. The results are discussed in relation to our knowledge of themechanisms controlling apical dominance in the tomato. Lycopersicon esculentumL, tomato, apical dominance, growth regulation, indol-3-ylacetic acid, abscisic acid, cytokinins     

Characterization of the ABA-deficient tomato mutant notabilis and its relationship with maize Vp14

The notabilis (not) mutant of tomato has a wilty phenotype due to a deficiency in the levels of the plant hormone abscisic acid (ABA). The mutant appears to have a defect in a key control step in ABA biosynthesis--the oxidative cleavage of a 9-cis xanthophyll precursor to form the C15 intermediate, xanthoxin. A maize mutant, viviparous 14 (vp14) was recently obtained by transposon mutagenesis. This maize genetic lesion also affects the oxidative cleavage step in ABA synthesis. Degenerate primers for PCR, based on the VP14 predicted amino acid sequence, have been used to provide probes for screening a wilt-related tomato cDNA library. A full-length cDNA clone was identified which is specific to the not gene locus. The ORFs of the tomato cDNA and maize Vp14 are very similar, apart from parts of their N-terminal sequences. The not mutation has been characterized at the DNA level. A specific A/T base pair deletion of the coding sequence has resulted in a frameshift mutation, indicating that not is a null mutant. This observation is discussed in connection with the relatively mild phenotype exhibited by not mutant homozygotes.     

Structure and induction pattern of a novel proteinase inhibitor class II gene of tobacco

A cDNA and a corresponding genomic clone encoding a protein with partial identity to type II proteinase inhibitors from potato, tomato and Nicotiana alata, were isolated from tobacco libraries. The protein of 197 amino acids contains a putative signal peptide of 24 residues and three homologous domains, each with a different reactive site. The tobacco PI-II gene is not expressed in leaves of healthy plants, but is locally induced in leaves subjected to different types of stress (TMV infection, wounding, UV irradiation) and upon ethephon treatment. As opposed to the analogous PI-II genes of potato and tomato, the tobacco gene is not systemically induced by wounding or pathogenic infection. A far-upstream region in the PI-II promoter, containing various direct and indirect repeats, shares considerable sequence similarity to a similar region in the stress-inducible Cu/Zn-superoxide dismutase gene of N. plumbaginifolia.     

A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes.     

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     

Characterization and expression of an mRNA encoding a wound-induced (Win) protein from ethylene-treated tomato leaf abscission zone tissue

A cDNA clone (TAB7) encoding a putative woundinduced (Win) proteinhas been isolated from a tomato (Lycopersicon esculentum Mill.cv. Ailsa Craig) leaf abscission zone cDNA library using a differentialscreening strategy. The clone has a high degree of homologyat the amino acid level to both the potato win1 and 2 genes,Hevea brasiliensis hevein and Nicotiana tabacum PR-4a and PR-4bproteins. The mRNA encoded by TAB7 is up-regulated within 12h of exposure to ethylene (10µl l^–1) and its expressionincreases steadily within the cells comprising the leaf abscissionzone and to a lesser extent in the adjacent non-zone tissue.This rise precedes the onset of cell separation. Southern analysisindicates that the mRNA is encoded by either a single gene ora small gene family. The role of the protein during abscissionis discussed. Key words: Lycopersicon esculentum, abscission zone, ethylene, tomato, wound-induced proteins     

Isolation and Characterization of a Water Stress-Specific Genomic Gene, pwsi 18, from Rice

One of the water stress-specific cDNA clones of rice characterisedpreviously, wsi18, was selected for further study. The wsi18gene can be induced by water stress conditions such as mannitol,NaCl, and dryness, but not by ABA, cold, or heat. A genomicclone for wsi18, pwsi18, contained about 1.7 kbp of the 5' upstreamsequence, two introns, and the full coding sequence. The 5'-upstreamsequence of pwsi18 contained putative cis-acting elements, namelyan ABA-responsive element (ABRE), three G-boxes, three E-boxes,a MEF-2 sequence, four direct and two inverted repeats, andfour sequences similar to DRE, which is involved in the dehydrationresponse of Arabi-dopsis genes. The gusA reporter gene underthe control of the pwsi18 promoter showed transient expressionin response to water stress. Deletion of the downstream DRE-likesequence between the distal G-boxes-2 and -3 resulted in ratherlow GUS expression. (Received March 27, 1997; Accepted November 5, 1997)     

Molecular Cloning and Sequence of a Complementary DNA Encoding 1-Aminocyclopropane-l-carboxylate Synthase Induced by Tissue Wounding

1-Aminocyclopropane-l-carboxylate (ACC) synthase [EC 4.4.1.14 [EC] ]is the key enzyme regulating ethylene biosynthesis in higherplants. A complementary DNA encoding wound-induced ACC synthasefrom mesocarp of winter squash (Cucurbita maxima Duch.) fruitswas cloned, and its complete nucleotide sequence determined.The cloned cDNA contained an open reading frame of 1479 basepairs encoding a sequence of 493 amino acids. Identificationof the cDNA was accomplished by expression of active enzymein Escherichia coli harboring the cDNA and by the presence ofa partial amino acid sequence identical to that found in thepurified enzyme. A putative pyridoxal phosphate binding siteof the enzyme is suggested. Northern blot analysis showed thatthe ACC synthase gene was activated by tissue wounding, andits expression was repressed by ethylene. Genomic Southern analysisindicates the presence of at least another sequence which weaklyhybridizes with the cDNA. (Received June 26, 1990; Accepted August 7, 1990)     

Identification of the tomato ABA-deficient mutant <Emphasis Type="Italic">sitiens</Emphasis> as a member of the ABA-aldehyde oxidase gene family using genetic and genomic analysis

The sitiens (sit) wilty mutant of tomato (Solanum lycopersicum L.) is deficient in functional enzyme activity at the final step in abscisic acid (ABA) biosynthesis. The biochemical lesion is believed to be an impaired aldehyde oxidase (AO). Molecular mapping using various interspecies crosses has previously shown sit to co-map with a cluster of unresolved RFLP markers on the short arm of chromosome 1. Here, the utilisation of bridging lines to produce interspecies mapping populations involving a self-compatible S. peruvianum accession (LA2157) allowed the fine mapping of sit within this cluster. Identification of a novel AO gene, within the region now known to contain the sit locus, was confirmed by analysis of the tomato whole genome shotgun sequence assembly. This novel AO protein shares 76-78% identity at the amino acid level with the previously characterised tomato AO proteins. The DNA sequence of this putative sit gene was characterised in wild type and in two allelic sit mutants (sit and sit ^w): changes in DNA sequence were identified in these mutant alleles that cause a truncation of exon 2 and the deletion of exon 7, respectively. These results establish the identity of the tomato sit gene and are consistent with its proposed function of encoding the ABA aldehyde oxidase apoenzyme.     

The Expression of an Abscisic Acid-Responsive Glycine-Rich Protein Coincides with the Level of Seed Dormancy in Fagus sylvatica

By differential screening of a cDNA library constructed frompoly (A⁺) RNA of ABA-treated seeds of Fagus sylvatica L., wehave isolated an ABA-responsive clone that is present in dormantseeds and under conditions that maintain dormancy, but it tendsto disappear under conditions breaking seed dormancy. A searchof the sequence data bases showed that the clone codes for aGlycine-Rich Protein and has sequence similarity to RNA-bindingproteins. The clone, which exibits the characteristics of lea-genes,is up-regulated by ABA and down-regulated by GA3. Paclobutrazolabolishes the effect of GA³, which is restored upon additionof GA3. The possible relationship of this Glycine-Rich Proteinto seed dormancy in F. sylvatica is discussed. (Received May 23, 1997; Accepted September 22, 1997)     

cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.