Florence Bouvier

Oxidative Remodeling of Chromoplast Carotenoids: Identification of the Carotenoid Dioxygenase CsCCD and CsZCD Genes Involved in Crocus Secondary Metabolite Biogenesis

The accumulation of three major carotenoid derivatives—crocetin glycosides, picrocrocin, and safranal—is in large part responsible for the color, bitter taste, and aroma of saffron, which is obtained from the dried styles of Crocus. We have identified and functionally characterized the Crocus zeaxanthin 7,8(7′,8′)-cleavage dioxygenase gene (CsZCD), which codes for a chromoplast enzyme that initiates the biogenesis of these derivatives. The Crocus carotenoid 9,10(9′,10′)-cleavage dioxygenase gene (CsCCD) also has been cloned, and the comparison of substrate specificities between these two enzymes has shown that the CsCCD enzyme acts on a broader range of precursors. CsZCD expression is restricted to the style branch tissues and is enhanced under dehydration stress, whereas CsCCD is expressed constitutively in flower and leaf tissues irrespective of dehydration stress. Electron microscopy revealed that the accumulation of saffron metabolites is accompanied by the differentiation of amyloplasts and chromoplasts and by interactions between chromoplasts and the vacuole. Our data suggest that a stepwise sequence exists that involves the oxidative cleavage of zeaxanthin in chromoplasts followed by the sequestration of modified water-soluble derivatives into the central vacuole.

Xanthophyll Biosynthesis CLONING, EXPRESSION, FUNCTIONAL RECONSTITUTION, AND REGULATION OF β-CYCLOHEXENYL CAROTENOID EPOXIDASE FROM PEPPER (CAPSICUM ANNUUM)

Pepper (Capsicum annuum) β-cyclohexenyl xanthophyll epoxidase cDNA was cloned and the corresponding enzyme overexpressed and purified from Escherichia coli, for investigation of its catalytic activity. The recombinant protein did not directly accept NADPH for epoxidation of cyclohexenyl carotenoids, nor did it operate according to a peroxygenase-based mechanism. Instead, the reducing power of NADPH was transferred to the epoxidase via reduced ferredoxin as shown by reconstitution of epoxidase activity in the presence of NADPH, ferredoxin oxidoreductase, and ferredoxin. Bacterial rubredoxin could be substituted for ferredoxin. The pepper epoxidase acted specifically on the β-ring of xanthophylls such as β-cryptoxanthin, zeaxanthin, and antheraxanthin. The proposed reaction mechanism for epoxidation involves the formation of a transient carbocation. This characteristic allows selective inhibition of the epoxidase activity by different nucleophilic diethylamine derivatives, p-dimethylaminobenzenediazonium fluoroborate and N,N-dimethyl-2-phenylaziridinium. It was also shown that the epoxidase gene was up-regulated during oxidative stress and when chloroplasts undergo differentiation into chromoplasts in pepper fruit.

Biochemistry and Molecular Biology of Chromoplast Development

Plant cells contain a unique class of organelles, designated the plastids, which distinguish them from animal cells. According to the largely accepted endosymbiotic theory of evolution, plastids are descendants of prokaryotes. This process requires several adaptative changes which involve the maintenance and the expression of part of the plastid genome, as well as the integration of the plastid activity to the cellular metabolism. This is illustrated by the diversity of plastids encountered in plant cells. For instance, in tissues undergoing color changes, i.e., flowers and fruits, the chromoplasts produce and accumulate excess carotenoids. In this paper we attempt to review the basic aspects of chromoplast development.

Metabolic engineering of xanthophyll content in tomato fruits

Ripe tomato fruits accumulate significant amounts of the linear carotene lycopene, but only trace amounts of xanthophylls (oxygenated carotenoids). We overexpressed the lycopene β‐cyclase (b‐Lcy) and β‐carotene hydroxylase (b‐Chy) genes under the control of the fruit‐specific Pds promoter. Transgene and protein expression was followed through semi‐quantitative reverse transcription‐PCR, Western blotting, and enzyme assays. Fruits of the transformants showed a significant increase of β‐carotene, β‐cryptoxanthin and zeaxanthin. The carotenoid composition of leaves remained unaltered. The transgenes and the phenotype are inherited in a dominant Mendelian fashion. This is the first example of successful metabolic engineering of xanthophyll content in tomato fruits.

Xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper fruits (Capsicum annuum L.)

To dissect the mechanism by which carotenoid hydroxylases catalyze xanthophyll formation, we have cloned two pepper cDNAs encoding beta-cryptoxanthin and zeaxanthin biosynthetic enzymes. Using an in vitro system, we find that both enzymes are ferredoxin dependent and that their activity is strongly inhibited by iron chelators such as o-phenanthroline or 8-hydroxyquinoline. This suggests the transfer of a reducing equivalent from NADPH to the hydroxylase via ferredoxin and the involvement of an iron activated oxygen insertion process. Based on sequence analysis, the putative histidine clusters involved in the iron coordination were identified and their roles evaluated. Following site-directed mutagenesis of the identified histidine residues hydroxylase activity was totally inactivated. Collectively, our data indicate that carotenoid hydroxylases belong to a new class of diiron proteins structurally related to membrane fatty acid desaturases. Mechanistically, both types of enzymes exploit iron activated oxygen to break the C-H bond with concomitant formation of double bond or oxygen insertion. We propose that the same mechanism operates for beta-carotene ketolase and probably for other carotenoid oxygenases as well.

Developmental and stress regulation of gene expression for plastid and cytosolic isoprenoid pathways in pepper fruits.

Plant cells synthesize a myriad of isoprenoid compounds in different subcellular compartments, which include the plastid, the mitochondria, and the endoplasmic reticulum cytosol. To start the study of the regulation of these parallel pathways, we used pepper (Capsicum annuum) fruit as a model. Using different isoprenoid biosynthetic gene probes from cloned cDNAs, we showed that only genes encoding the plastid enzymes (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and capasanthin-capsorubin synthase) are specifically triggered during the normal period of development, at the ripening stage. This pattern of expression can be mimicked and precociously induced by a simple wounding stress. Concerning the cytosol-located enzymes, we observed that the expression of the gene encoding farnesyl pyrophosphate synthase is constitutive, whereas that of farnesyl pyrophosphate cyclase (5-epi-aristolochene synthase) is undetectable during the normal development of the fruit. The expression of these later genes are, however, only selectively triggered after elicitor treatment. The results provide evidence for developmental control of isoprenoid biosynthesis occurring in plastids and that cytoplasmic isoprenoid biosynthesis is regulated, in part, by environmental signals.

Arabidopsis SAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development

S-Adenosylmethionine (SAM) is formed exclusively in the cytosol but plays a major role in plastids; SAM can either act as a methyl donor for the biogenesis of small molecules such as prenyllipids and macromolecules or as a regulator of the synthesis of aspartate-derived amino acids. Because the biosynthesis of SAM is restricted to the cytosol, plastids require a SAM importer. However, this transporter has not yet been identified. Here, we report the molecular and functional characterization of an Arabidopsis thaliana gene designated SAM TRANSPORTER1 (SAMT1), which encodes a plastid metabolite transporter required for the import of SAM from the cytosol. Recombinant SAMT1 produced in yeast cells, when reconstituted into liposomes, mediated the counter-exchange of SAM with SAM and with S-adenosylhomocysteine, the by-product and inhibitor of transmethylation reactions using SAM. Insertional mutation in SAMT1 and virus-induced gene silencing of SAMT1 in Nicotiana benthamiana caused severe growth retardation in mutant plants. Impaired function of SAMT1 led to decreased accumulation of prenyllipids and mainly affected the chlorophyll pathway. Biochemical analysis suggests that the latter effect represents one prominent example of the multiple events triggered by undermethylation, when there is decreased SAM flux into plastids.

Light-Induced Acclimation of the Arabidopsis chlorina1 Mutant to Singlet Oxygen

This work shows that, similarly to green algae, vascular plants can acclimate to singlet oxygen (1O2). The Arabidopsis ch1 mutant is highly photosensitive due to increased release of 1O2 by photosystem II, but preexposure of ch1 plants to moderately elevated light intensities eliminated oxidative damage in high light without suppressing 1O2 formation. Regulation of the jasmonate biosynthesis pathway is a key factor in this acclimation process. Singlet oxygen (1O2) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, 1O2-induced transcriptomic changes result in acclimation to 1O2. Here, using a chlorophyll b–less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of 1O2 by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to 1O2 reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing 1O2 formation, indicating acclimation to 1O2. Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in 1O2-induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonate-treated ch1 plants and by the increased tolerance of the jasmonate-deficient mutant delayed-dehiscence2.

Molecular and Enzymatic Characterizations of Novel Bifunctional 3β-Hydroxysteroid Dehydrogenases/C-4 Decarboxylases from Arabidopsis thaliana

We have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3β-hydroxysteroid dehydrogenase/C-4 decarboxylases (3βHSD/D) involved in sterol synthesis, termed At3βHSD/D1 and At3βHSD/D2. Transformation of the yeast ergosterol auxotroph erg26 mutant, which lacks 3βHSD/D activity, with the At3βHSD/D1 isoform or with At3βHSD/D2 isoform containing a C-terminal At3βHSD/D1 endoplasmic reticulum-retrieval sequence restored growth and ergosterol synthesis in erg26. An in vitro enzymatic assay revealed high 3βHSD/D activity for both isoenzymes in the corresponding microsomal extracts. The two At3βHSD/D isoenzymes showed similar substrate specificities that required free 3β-hydroxyl and C-4-carboxyl groups but were quite tolerant in terms of variations of the sterol nucleus and side chain structures. Data obtained with 4α-carboxy-cholest-7-en-3β-ol and its 3α-deuterated analog revealed that 3α-hydrogen-carbon bond cleavage is not the rate-limiting step of the reaction. In planta reduction on the expression of the 3βHSD/D gene as a consequence of VIGS-mediated gene silencing in Nicotiana benthamiana led to a substantial accumulation of 3β-hydroxy-4β,14-dimethyl-5α-ergosta-9β,19-cyclo-24(241)-en-4α-carboxylic acid, consistent with a decrease in 3βHSD/D activity. These two novel oxidative decarboxylases constitute the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants.

Abscisic Acid Negatively Regulates Elicitor-Induced Synthesis of Capsidiol in Wild Tobacco

In the Solanaceae, biotic and abiotic elicitors induce de novo synthesis of sesquiterpenoid stress metabolites known as phytoalexins. Because plant hormones play critical roles in the induction of defense-responsive genes, we have explored the effect of abscisic acid (ABA) on the synthesis of capsidiol, the major wild tobacco (Nicotiana plumbaginifolia) sesquiterpenoid phytoalexin, using wild-type plants versus nonallelic mutants Npaba2 and Npaba1 that are deficient in ABA synthesis. Npaba2 and Npaba1 mutants exhibited a 2-fold higher synthesis of capsidiol than wild-type plants when elicited with either cellulase or arachidonic acid or when infected by Botrytis cinerea. The same trend was observed for the expression of the capsidiol biosynthetic genes 5-epi-aristolochene synthase and 5-epi-aristolochene hydroxylase. Treatment of wild-type plants with fluridone, an inhibitor of the upstream ABA pathway, recapitulated the behavior of Npaba2 and Npaba1 mutants, while the application of exogenous ABA reversed the enhanced synthesis of capsidiol in Npaba2 and Npaba1 mutants. Concomitant with the production of capsidiol, we observed the induction of ABA 8′-hydroxylase in elicited plants. In wild-type plants, the induction of ABA 8′-hydroxylase coincided with a decrease in ABA content and with the accumulation of ABA catabolic products such as phaseic acid and dihydrophaseic acid, suggesting a negative regulation exerted by ABA on capsidiol synthesis. Collectively, our data indicate that ABA is not required per se for the induction of capsidiol synthesis but is essentially implicated in a stress-response checkpoint to fine-tune the amplification of capsidiol synthesis in challenged plants.