Andrew D. Parry

The carotenoid and abscisic acid content of viviparous kernels and seedlings ofZea mays L.

Carotenoid and abscisic acid (ABA) levels were determined in endosperm, embryos and seedlings of wild-type and viviparous (vp) mutants ofZea mays L. Carotenoid concentrations were determined by absorption spectrometry following purification by high-performance liquid chromatography and ABA concentrations by combined gas chromatography-mass spectrometry. Lutein and zeaxanthin were the terminal carotenoids in wild-type tissue. The carotenoid profiles ofvp-1 andvp-8 tissue were similar to that of the wild type; invp-2, vp-5, vp-7 andvp-9 carotenogenesis was blocked at early stages so that xanthophylls were absent. Except forvp-1, where the ABA content was similar to the wild type, the ABA content ofvp embryos was substantially reduced, to 6–16% of the corresponding wild type. Thus, the absence of xanthophylls was associated with reduced ABA content, which was in turn correlated with vivipary. Kernels ofvp-8 had a reduced ABA content although xanthophylls were present. Seedlings of carotenoid-deficient mutants rescued from viviparous kernels contained less ABA than did wild-type seedlings grown in the same way. Furthermore, the ABA concentration of such seedlings did not increase in response to water deficit. Conversely,vp-1 seedlings contained normal levels of carotenoids and ABA. Carotenoid-deficient seedlings did not contain appreciable amounts of chlorophyll so that chloroplast development was not normal. Thus ABA-deficiency could be associated with abnormal plastid development rather than the absence of carotenoids per se.

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 Rts, 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.

Abscisic acid biosynthesis in roots. I: the identification of potential abscisic acid precursors, and other carotenoids

The pathway of water-stress-induced abscisic acid (ABA) biosynthesis in etiolated and light-grown leaves has been elucidated (see A.D. Parry and R. Horgan, 1991, Physiol. Plant. 82, 320–326). Roots also have the ability to synthesise ABA in response to stress and it was therefore of interest to examine root extracts for the presence of carotenoids, including those known to be ABA precursors in leaves. All-trans- and 9′-cis-neoxanthin, all-trans- and 9-cis-violaxanthin, antheraxanthin (all potential ABA precursors), lutein and β-carotene were identified on the basis of absorbance spectra, reactions with dilute acid, retention times upon high-performance liquid chromatography and by comparison with leaf carotenoids that had been analysed by mass spectrometry. The source of the extracted carotenoids was proved to be root tissue, and not contaminating compost or leaf material. The levels of total carotenoids in roots varied between 0.03–0.07% of the levels in light-grown leaves (Arabidopsis thaliana (L.) Heynh, Nicotiana plumbaginifolia Viv., Phaseolus vulgaris L. and Pisum sativum L.) up to 0.27% (Lycopersicon esculentum Mill.). The relative carotenoid composition was very different from that found in leaves, and varied much more between species. All-trans-neoxanthin and violaxanthin were the major carotenoids present (64–91 % of the total), but while Lycopersicon contained 67–80% all trans-neoxanthin, Phaseolus, Pisum and Zea mays L. contained 61–79% all-trans-violaxanthin. Carotenoid metabolism also varied between species, with most of the carotenoids in older roots of Phaseolus being esterified. Roots and leaves of the ABA-deficient aba mutant of Arabidopsis had reduced epoxy-xanthophyll levels compared to the wild-type.

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.

Abscisic acid biosynthesis in roots : II. The effects of water-stress in wild-type and abscisic-acid-deficient mutant (notabilis) plants of Lycopersicon esculentum Mill.

The ubiquity of the apo-carotenoid abscisic acid (ABA) biosynthetic pathway elucidated in water-stressed, etiolated leaves of Phaseolus vulgaris (see A.D. Parry and R. Horgan, 1991, Physiol. Plant. 82, 320–326), has been difficult to establish. Light-grown leaves contain very high carotenoid: ABA ratios, preventing correlative studies, and no etiolated leaves so far studied, other than those of Phaseolus, have been found capable of synthesising significant amounts of ABA in response to stress. Roots are known to synthesise ABA and contain low carotenoid levels; therefore ABA biosynthesis was investigated in soil- and hydroponically grown roots of Lycopersicon esculentum Mill. Hydroponically grown roots were stressed by immersion in 100 mM mannitol and soil-grown roots by withholding water. In both cases stress led to an increase in ABA levels and a decrease in the levels of specific xanthophylls, namely all-trans- and 9′-cis-neoxanthin and all-trans-violaxanthin. In hydroponically grown roots, and soil-grown roots stressed after removal of the shoot, ratios of xanthophyll cleaved:ABA synthesised of approx. 1∶1 were obtained. These findings are consistent with the operation of an apo-carotenoid pathway in roots, involving the conversion of all-trans-violaxanthin via all-trans-neoxanthin, to 9′-cis-neoxanthin, and the specific cleavage of 9′-cis-neoxanthin to yield the C15 ABA precursor xanthosin. Similar experiments with roots of the “leaky”, ABA-deficient mutant of Lycopersicon, notabilis, indicate that the mutation does not affect the perception or transduction of stress, or the ability of the plant to cleave carotenoids. Rather, it appears that notabilis possesses an enzyme with reduced substrate specificity which cleaves more all-trans-than 9′-cis-neoxanthin.

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.

Carotenoid metabolism and the biosynthesis of abscisic acid.

Abstract The conversion of all-trans-violaxanthin to 9′-cis-neoxanthin was shown to occur in fluridone-treated etiolated Lycopersicon and Phaseolus seedlings, following exposure to light. The results of deuterium oxide labelling experiments supported this precursor/product relationship, and provided further evidence for the origin of abscisic acid. Several apo-carotenoids, putative by-products of abscisic acid biosynthesis, were synthesised by chemical oxidation but were not detected in plant extracts. In vitro, lipoxygenase cleaved neoxanthin and violaxanthin down to small ( \ C13) fragments. It may be that in vivo any apo-carotenoids formed by the specific cleavage of 9′-cis-neoxanthin, during abscisic acid biosynthesis, are rapidly metabolized by lipoxygenase or similar enzymes.

A cytokinin-resistant mutant of Nicotiana plumbaginifolia is wilty

Selection for cytokinin resistance by incubating M2 seed of Nicotiana plumbaginifolia, after ethylmethanesulphonate mutagenesis, on 20 μM 6-benzylami-nopurine resulted in the isolation of a monogenic, recessive mutant, CKR1. Germination of the mutant is less sensitive to cytokinin inhibition than the wild type, and leaf development of the mutant occurs at cytokinin concentrations inhibitory to the wild type. Germination of the mutant is also resistant to auxin but not to abscisic acid. Three other traits jointly inherited with cytokinin resistance in the F2 are lack of root branching, precocious germination and wiltiness. The wilty phenotype is the consequence of the failure of stomatal closure during water stress.

Measurement of xanthoxin in higher plant tissues using 13C labelled internal standards.

Abstract A new procedure for the reliable measurement of xanthoxin from plant tissues has been developed. This accounts for both its breakdown and its isomerization to 2- trans -xanthoxin, and relies on the use of 12 C-labelled internal standards, HPLC purification and analysis by GC-MS SIM. The antioxidant tert -butylated hydroxyquinoline was found to reduce the amount of isomerization to 2- trans -xanthoxin during extraction. Levels of xanthoxin present in all tissues examined were much lower than previously thought, and 2- trans -xanthoxin: xanthoxin ratios much higher.

Jasmonic acid and abscisic acid in shoots, coleoptiles, and roots of wheat seedlings

The endogenous levels of abscisic acid (ABA) and jasmonic acid (JA) were analyzed in wheat seedlings grown in water, a system which in the past has been used to test the effects of these plant growth inhibitors. The levels in different plant parts and in the medium were measured by gas chromatography-mass spectrometry-selected ion monitoring, using [2H3]ABA and [2H6]JA as internal standards. In every plant part, JA levels were about 2 orders of magnitude greater than those of ABA. The exudation of JA from roots per seedling was about 14,000-fold greater than that of ABA, although the roots contained only about 170 times more JA than ABA. It is suggested that JA is a possible allelopathic compound.