Functional integration of non‐native carotenoids into chloroplasts by viral‐derived expression of capsanthin–capsorubin synthase in <i>Nicotiana benthamiana</i>

Kumagai, Monto H.; Keller, Yves; Bouvier, Florence; Clary, David C.; Camara, Bilal

doi:10.1046/j.1365-313x.1998.00128.x

Cited by 54 publications

(42 citation statements)

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“…For example, total pigment extracts from the algae from Euglena gracilis (containing neoxanthin and diadinoxanthin (5,6-epoxy-7Ј,8Ј-didehydro-5,6-dihydro-␤,␤-carotene-3,3Ј-diol)) and Chlamydomonas reinhardtii (containing loroxanthin) have been used in heterologous reconstitution of spinach light-harvesting complexes (39). More recently, capsanthin (which supports in vitro reassembly of LHCIIb; see above) has been shown to be a functional component (at ϳ36% of total carotenoid) of transformed plants of Nicotiana benthamiana (25).…”

Section: Discussionmentioning

confidence: 99%

The Binding of Xanthophylls to the Bulk Light-harvesting Complex of Photosystem II of Higher Plants

Phillip¹,

Hobe²,

Paulsen³

et al. 2002

Journal of Biological Chemistry

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The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single ␤-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-transneoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3 diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6 -C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.

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Section: Discussionmentioning

confidence: 99%

The Binding of Xanthophylls to the Bulk Light-harvesting Complex of Photosystem II of Higher Plants

Phillip¹,

Hobe²,

Paulsen³

et al. 2002

Journal of Biological Chemistry

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“…For TTO-CrtB, the forward 59-GCCTCGAGATTACGCCCCGCATGGCGCTC-GATCAC-39 and reverse 59-TCCCTAGGGCGGCGATCCCAGGCGCT-GCCTCCCGC-39 primers containing XhoI and AvrII restriction sites, respectively, were used. The PCR and N. benthamiana transfection were performed as described previously (Kumagai et al, 1995(Kumagai et al, , 1998. In plants transfected with TTO-Nb SAMT1, the detection of endogenous Nb SAMT1 transcripts was performed by RT-PCR .…”

Section: Virus-induced Gene Silencingmentioning

confidence: 99%

ArabidopsisSAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development

et al. 2006

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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.

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“…A 500-bp fragment of the N. benthamiana 3␤-hydroxysteroid-dehydrogenase-C-4 decarboxylase geneamino acid residues from the C terminus was (Nb3␤HSD/D) (AM236597) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (DXR) (AM236596), which was used as a control, were PCR-amplified from RNAs extracted from leaves of N. benthamiana using the following forward primers, respectively: 5Ј-GCCTCGAGGCTGATTTGGGTCCATC-CATTAAACTTGAG-3Ј, 5Ј-GCCTCGAGGGAGTGGCTA-TCAAAAGAAAGGAG-3Ј, and the following reverse primers: 5Ј-TCCCTAGGTCCTGCCTTTGCAGCTGCAACTA-ATGAAGGAAC-3Ј, 5Ј-TCCCTAGGCTTTATTGGCCAA-GGCAATGTCCTTTC-3Ј. These products were cloned into the XhoI-AvrII restricted TTO viral vector (16) to generate TTO-Nb3␤HSD/D and TTO-NbDXR, respectively.…”

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confidence: 99%

“…Virus-induced Gene Silencing in N. benthamiana-TTONb3␤HSD/D and TTO-NbDXR constructs were used to inoculate young N. benthamiana plants as described previously (16). To measure silencing of Nb3␤HSD/D, semi-quantitative reverse transcription was performed also as described previously (8,17).…”

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confidence: 99%

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

Rahier

Darnet

Bouvier

et al. 2006

Journal of Biological Chemistry

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We have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3␤-hydroxysteroid dehydrogenase/C-4 decarboxylases (3␤HSD/D) involved in sterol synthesis, termed At3␤HSD/D1 and At3␤HSD/D2. Transformation of the yeast ergosterol auxotroph erg26 mutant, which lacks 3␤HSD/D activity, with the At3␤HSD/D1 isoform or with At3␤HSD/D2 isoform containing a C-terminal At3␤HSD/D1 endoplasmic reticulum-retrieval sequence restored growth and ergosterol synthesis in erg26. An in vitro enzymatic assay revealed high 3␤HSD/D activity for both isoenzymes in the corresponding microsomal extracts. The two At3␤HSD/D isoenzymes showed similar substrate specificities that required free 3␤-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 4␣-carboxy-cholest-7-en-3␤-ol and its 3␣-deuterated analog revealed that 3␣-hydrogen-carbon bond cleavage is not the rate-limiting step of the reaction. In planta reduction on the expression of the 3␤HSD/D gene as a consequence of VIGS-mediated gene silencing in Nicotiana benthamiana led to a substantial accumulation of 3␤-hydroxy-4␤,14-dimethyl-5␣-ergosta-9␤,19-cyclo-24(24 1 )-en-4␣-carboxylic acid, consistent with a decrease in 3␤HSD/D activity. These two novel oxidative decarboxylases constitute the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants.Sterols are essential components of all eukaryotic cell membranes, and the biosynthetic pathways differ significantly. The sterol molecule becomes functional only after removal of the two methyl groups at C-4. Both methyl groups at C-4 are removed early and successively in animals and yeast, whereas in higher plants one methyl group is initially removed from a 4,4-dimethyl-9␤,19-cyclopropylsterol precursor, and the second is eliminated several steps later (1-3). In plants, we have characterized the activities of a sterol C-4 methyl oxidase (SMO), 3 a 4␣-carboxysterol-3␤-hydroxysteroid dehydrogenase/C-4 decarboxylase (3␤HSD/D), and an NADPH-dependent 3-oxosteroid reductase from partially purified preparations (4 -6) in order to define the steps involved in C-4 demethylation in plants. The first step is initiated by the SMO, whereby this enzyme converts the C-4␣ methyl group to produce a 4␣-carboxysterol derivative that is subsequently oxidatively decarboxylated by the 3␤HSD/D to produce a C-4-monodemethylated 3-oxosteroid, which is then stereospecifically reduced by the 3-ketoreductase. In contrast to animals and yeast where the SMO is encoded by a single gene (7), we biochemically characterized two distinct microsomal SMOs in Zea mays (4), and we identified two distinct families of SMO genes in Arabidopsis thaliana (8). Until now, the gene(s) coding the single bifunctional protein 3␤HSD/D (Fig. 1) have not been characterized.Furthermore, little is known about plant hydroxysteroid dehydrogenases that are either members of the short chain dehydrogenases/reductases (SDR) (9 -11) or the a...

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Functional integration of non‐native carotenoids into chloroplasts by viral‐derived expression of capsanthin–capsorubin synthase in Nicotiana benthamiana

Cited by 54 publications

References 53 publications

The Binding of Xanthophylls to the Bulk Light-harvesting Complex of Photosystem II of Higher Plants

The Binding of Xanthophylls to the Bulk Light-harvesting Complex of Photosystem II of Higher Plants

ArabidopsisSAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development

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

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