Identification, purification, and characterization of S-adenosyl-L-methionine: isoliquiritigenin 2'-O-methyltransferase from alfalfa (Medicago sativa L.).

An O-methyltransferase (OMT) which methylates the 2'-hydroxyl of isoliquiritigenin (2',4,4'-trihydroxychalcone) was identified in alfalfa (Medicago sativa L.) seedlings and cell cultures. The OMT activity increased during early stages of seedling development and was predominantly located in roots. Treatment of alfalfa cell cultures with an elicitor from yeast resulted in a fivefold increase in chalcone OMT activity, whereas treatment of seedlings with CuCl2 caused a reduction in activity. The chalcone OMT was purified to near homogeneity from elicited alfalfa cell cultures. Only one form of the enzyme was found. It consisted of an active monomer of subunit Mr 43,000 which could be photoaffinity labeled with S-adenosyl-L-[methyl-3H]methionine. The purified OMT had a pH optimum of 9.0, pI of 4.7, and was highly specific for the 2'-hydroxyl of 2',4,4'-trihydroxychalcone, with essentially no activity toward narigenin chalcone, caffeic acid, or daidzein. Kinetic analysis indicated a sequential bi bi mechanism with Km values of 2.2 and 17.7 microM for 2',4,4'-trihydroxychalcone and S-adenosyl-L-methionine, respectively. S-Adenosyl-L-homocysteine was a potent inhibitor. The chalcone OMT represents the third distinct OMT isolated from alfalfa cell cultures.     

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.     

Localization of flavonoid enzymes in Arabidopsis roots

Immunofluorescence and immuno-electron microscopy have been used to test the hypothesis that flavonoid metabolism is organized as a membrane-associated enzyme complex. The cellular and subcellular locations of chalcone synthase (CHS) and chalcone isomerase (CHI), the first two enzymes of this pathway, were examined in Arabidopsis roots. High levels of both enzymes were found in the epidermal and cortex cells of the elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. Co-localization of CHS and CHI was observed at the endoplasmic reticulum and tonoplast in these cells, and also in electron-dense regions that are, as yet, unidentified. In addition, a striking asymmetric distribution was observed for these enzymes in cortex cells of the elongation zone, which may provide clues about the physiological function of flavonoids in roots. The accumulation of CHS and CHI was also examined in tt7(88), a mutant in the gene for flavonoid 3'-hydroxylase (F3'H), which has been postulated to serve as a membrane anchor for the flavonoid enzyme complex. CHS and CHI accumulated to lower levels in cortex cells and higher levels in epidermal cells in the roots of this mutant as compared with wild-type plants. Moreover, the electron-dense regions containing these two enzymes were not observed. However, localization of CHS and CHI to the ER and tonoplast did not appear to be affected, suggesting that other proteins may function in recruiting the "soluble" flavonoid enzymes to membranes. Staining of flavonoid endproducts with DPBA was consistent with expression of CHS and CHI in these seedlings.     

Expression and Localization of Plant Protein Disulfide Isomerase

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules.     

The alfalfa (Medicago sativa) TDY1 gene encodes a mitogen-activated protein kinase homolog.

Development of root nodules, specifically induction of cortical cell division for nodule initiation, requires expression of specific genes in the host and microsymbiont. A full-length cDNA clone and the corresponding genomic clone encoding a MAP (mitogen-activated protein) kinase homolog were isolated from alfalfa (Medicago sativa). The genomic clone, TDY1, encodes a 68.9-kDa protein with 47.7% identity to MMK4, a previously characterized MAP kinase homolog from alfalfa. TDY1 is unique among the known plant MAP kinases, primarily due to a 230 amino acid C-terminal domain. The putative activation motif, Thr-Asp-Tyr (TDY), also differs from the previously reported Thr-Glu-Tyr (TEY) motif in plant MAP kinases. TDY1 messages were found predominantly in root nodules, roots, and root tips. Transgenic alfalfa and Medicago truncatula containing a chimeric gene consisting of 1.8 kbp of 5' flanking sequence of the TDY1 gene fused to the beta-glucuronidase (GUS) coding sequence exhibited GUS expression primarily in the nodule parenchyma, meristem, and vascular bundles, root tips, and root vascular bundles. Stem internodes stained intensely in cortical parenchyma, cambial cells, and primary xylem. GUS activity was observed in leaf mesophyll surrounding areas of mechanical wounding and pathogen invasion. The promoter was also active in root tips and apical meristems of transgenic tobacco. Expression patterns suggest a possible role for TDY1 in initiation and development of nodules and roots, and in localized responses to wounding.     

Expression of a functional type-I chalcone isomerase gene is localized to the infected cells of root nodules of Elaeagnus umbellata

A putative type-I chalcone isomerase (CHI) cDNA clone EuNOD-CHI was previously isolated from the root nodule of Elaeagnus umbellata [Kim et al. (2003)]. To see if it encodes a functional CHI, we ectopically overexpressed it in the Arabidopsis (Arabidopsis thaliana) transparent testa 5 (tt5) mutant, which is defective in naringenin production and has yellow seeds due to proanthocyanidin deficiency. Ectopic overexpression of EuNOD-CHI resulted in recovery of normal seed coat color. Naringenin produced by CHI from naringenin chalcone was detected in the transgenic lines like in the wild-type, whereas it was absent from the tt5 mutant. We conclude that EuNOD-CHI encodes a functional type-I CHI. In situ hybridization revealed that EuNOD-CHI expression is localized to the infected cells of the fixation zone in root nodules.     

Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase

Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcone synthesized by chalcone synthase (CHS) into (2S)-naringenin, an essential compound in the biosynthesis of anthocyanin pigments, inducers of Rhizobium nodulation genes, and antimicrobial phytoalexins. The 1.85 A resolution crystal structure of alfalfa CHI in complex with (2S)-naringenin reveals a novel open-faced beta-sandwich fold. Currently, proteins with homologous primary sequences are found only in higher plants. The topology of the active site cleft defines the stereochemistry of the cyclization reaction. The structure and mutational analysis suggest a mechanism in which shape complementarity of the binding cleft locks the substrate into a constrained conformation that allows the reaction to proceed with a second-order rate constant approaching the diffusion controlled limit. This structure raises questions about the evolutionary history of this structurally unique plant enzyme.     

A Vesicular Arbuscular Mycorrhizal Fungus (Glomus intraradix) Induces a Defense Response in Alfalfa Roots

Flavonoid accumulation and activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and chitinase were followed during early colonization of alfalfa roots (Medicago sativa L. cv Gilboa) by vesicular arbuscular (VA) fungi (Glomus intraradix). Formononetin was the only flavonoid detected that showed a consistent increase in the inoculated roots. This increase depended only on the presence of the fungus in the plant rhizosphere; no colonization of the root tissue was required. CHI and chitinase activities increased in inoculated roots prior to colonization, whereas the increase in PAL activity coincided with colonization. After reaching a maximum, activities of all enzymes declined to below those of uninoculated roots. PAL inactivation was not caused by a soluble inhibitor. Our results indicate that VA fungi initiate a host defense response in alfalfa roots, which is subsequently suppressed.     

Isolation and characterization ofo-diphenol-O-methyltransferase cDNA clone in hot pepper (Capsicum annuum L.)

A cDNA clone,CaOMTl encoding ano-diphenol-O-methyltransferase (OMT), which is involved in capsaicin biosynthesis, was isolated by screening of a cDNA library prepared from the mRNA of pepper (Capsicum annuum L.) pericarp. Nucleotide sequence analysis ofCaOMTl revealed that it had an open reading frame of 1080 bp which encodes a polypeptide with a predicted molecular weight of 39,430 D, corresponding well with the size of the known OMT’s of tobacco, poplar, aspen, alfalfa, and cabbage. It also had five conserved boxes which appear in all known OMT’s. The nucleotide sequence ofCaOMTl had 89–74% identity with the OMT cDNA’s of tobacco, aspen, alfalfa, and poplar, but a relatively lower identity of 59% with the OMT cDNA of maize. Amino acid sequence analysis also revealed that CaOMT1 has high identity with the known OMT’s which have a substrate ofo-diphenolic compounds, especially 5-hydroxyferulic acid and caffeic acid. It supportsCaOMTl which encodes an OMT. Southern blot analysis suggested thatCaOMTl might exist in the form of multiple copies in the pepper genome.CaOMTl is expressed preferentially in pepper fruit and its expression levels increased during pepper fruit development, but decreased during fruit ripening, suggesting that theCaOMTl gene is fruit development-related.CaOMTl is the first reported cDNA clone for enzymes related to the phenlypropanoid pathway in pepper.