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Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

ABSTRACT: Cinnamate 4-hydroxylase [CA4H; trans-cinnamate,NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11] is a cytochrome P450 that catalyzes the first oxygenation step of the general phenylpropanoid metabolism in higher plants. The compounds formed are essential for lignification and defense against predators and pathogens. We recently reported the purification of this enzyme from Mn(2+)-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. Highly selective polyclonal antibodies raised against the purified protein were used to screen a lambda gt11 cDNA expression library from wound-induced Jerusalem artichoke, allowing isolation of a 1130-base-pair insert. Typical P450 domains were identified in this incomplete sequence, which was used as a probe for the isolation of a 1.7-kilobase clone in a lambda gt10 library. A full-length open reading frame of 1515 base pairs, encoding a P450 protein of 505 residues (M(r) = 57,927), was sequenced. The N terminus, essentially composed of hydrophobic residues, matches perfectly the microsequenced N terminus of the purified protein. The calculated pI is 9.78, in agreement with the chromatographic behavior and two-dimensional electrophoretic analysis of CA4H. Synthesis of the corresponding mRNA is induced in wounded plant tissues, in correlation with CA4H enzymatic activity. This P450 protein exhibits the most similarity (28% amino acid identity) with avocado CYP71, but also good similarity with CYP17 and CYP21, or with CYP1 and CYP2 families. According to current criteria, it qualifies as a member of a new P450 family.

SUBMITTER: Teutsch HG

PROVIDER: S-EPMC46454 | biostudies-other | 1993 May

REPOSITORIES: biostudies-other

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Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

Teutsch H G HG Hasenfratz M P MP Lesot A A Stoltz C C Garnier J M JM Jeltsch J M JM Durst F F Werck-Reichhart D D

Proceedings of the National Academy of Sciences of the United States of America 19930501 9

Cinnamate 4-hydroxylase [CA4H; trans-cinnamate,NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11] is a cytochrome P450 that catalyzes the first oxygenation step of the general phenylpropanoid metabolism in higher plants. The compounds formed are essential for lignification and defense against predators and pathogens. We recently reported the purification of this enzyme from Mn(2+)-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. Highly selective polyclonal antibodi ...[more]

PMID: 8097885

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Project description:Helianthus tuberosus L., known as the Jerusalem Artichoke, is a hexaploid plant species, adapted to low nutrient soils, that accumulates high levels of inulin in its tubers. Inulin is a fructose-based polysaccharide used either as dietary fiber or for the production of bioethanol. Key enzymes involved in inulin biosynthesis are well known. However, the gene networks underpinning tuber development and inulin accumulation in H. tuberous remain elusive. To fill this gap, we selected 6,365 ESTs from a H. tuberosus library to set up a microarray platform and record their expression across three tuber developmental stages, when rhizomes start enlarging (T0), at maximum tuber elongation rate (T3) and at tuber physiological maturity (Tm), in “VR” and “K8-HS142”clones. The former was selected as an early tuberizing, the latter as a late-tuberizing clone. We quantified inulin and starch levels, and q-RT PCR confirmed the expression of key genes accounting for inulin biosynthesis. The microarray analysis revealed that the differences in morphological and physiological traits between tubers of the two clones are genetically determined since T0 and that is relatively low the number of differentially expressed ESTs across the stages shared between the clones (93). The expression of ESTs for sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT), the two critical genes for fructans polymerization, resulted to be temporarily synchronized and mirror the progress of inulin accumulation and stretching. The expression of ESTs for starch biosynthesis was insignificant throughout the developmental stages of the clones in line with the negligible level of starch into their mature tubers, where inulin was the dominant polysaccharide. Overall, our study disclosed candidate genes underpinning the development and storage of carbohydrates in the tubers of two H. tuberosus clones. A model according to which the steady state levels of 1-SST and 1-FFT transcripts are developmentally controlled and might represent a limiting factor for inulin accumulation has been provided. Our finding may have significant repercussions for breeding clones with improved levels of inulin for food and chemical industry.

Dataset Information

Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

Publications

Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

Similar Datasets

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