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.     

Isolation of a poplar and anArabidopsis thaliana dihydrodipicolinate synthase cDNA clone

A poplar DHDPS cDNA clone has been isolated by functional rescue of thedapA-deficient AT997 mutant ofEscherichia coli. By sequence comparison between the poplar and maize DHDPS cDNAs, two oligonucleotides were designed to perform polymerase chain reaction (PCR) onArabidopsis thaliana genomic DNA. The PCR fragment was subsequently used to isolate anArabidopsis DHDPS genomic and cDNA clone.     

Molecular and biochemical characterization of the<Emphasis Type="Italic"> Capsicum annuum calcium-dependent protein kinase 3</Emphasis> (<Emphasis Type="Italic">CaCDPK3</Emphasis>) gene induced by abiotic and biotic stresses

The isolated full-length Capsicum annuum calcium-dependent protein kinase 3 (CaCDPK3) cDNA clone was selected from the chili pepper expressed sequence tag database (). Phylogenetic analysis based on the deduced amino acid sequence of CaCDPK3 cDNA revealed significant sequence similarity to the winter squash (Cucurbita maxima) CmCPK2 gene (81% identity). Genomic gel blot analysis disclosed that CaCDPK3 belongs to a multigene family in the pepper genome. CaCDPK3 expression was root tissue-specific, as shown by Northern blot data. The gene was rapidly induced in response to various osmotic stress factors and exogenous abscisic acid application in pepper leaves. Moreover, CaCDPK3 RNA expression was induced by an incompatible pathogen and by plant defense-related chemicals such as ethephon, salicylic acid and jasmonic acid. The biochemical properties of CaCDPK3 were investigated using a CaCDPK3 and glutathione S-transferase (GST) fusion protein. The recombinant proteins retained calcium-binding ability, and displayed autophosphorylation activity in vitro in a calcium-dependent manner. Further transient-expression studies showed that CaCDPK3 fused with soluble modified green fluorescent protein (smGFP) localized to the cytosol in chili pepper protoplasts. We propose that CaCDPK3 is implicated in biotic and abiotic stresses in pepper plants.     

Functional expression of an Arabidopsis cDNA clone encoding a flavonol 3'-O-methyltransferase and characterization of the gene product

We report that the cDNA clone (Accession No. U70424), previously isolated from Arabidopsis thaliana as encoding a caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT) (1), has now been overexpressed in Escherichia coli BL21 and its recombinant protein identified as a novel flavonol 3'-OMT. It is, therefore, renamed AtOMT1. This cDNA clone has previously been identified on the basis of its 88% amino acid sequence similarity and 80% identity to the aspen bispecific lignin OMT (2), the type member of the group involved in lignin biosynthesis. Our data indicate that this novel OMT uses the flavonol quercetin as the preferred substrate, but neither of the hydroxycinnamic acids, caffeic or 5-hydroxyferulic, to any significant extent. This indicates that the high sequence similarity/identity of AtOMT1 to that of the aspen lignin OMT (2) is not sufficient to assign the function of this gene product.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

Molecular cloning and characterization of a novel human kinase gene,PDIK1L

We isolated a 4301-bp cDNA from a human foetal brain cDNA library by high-throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the putative kinase may interact with PDZ and LIM domain proteins. Therefore the protein and its cDNA were named ’PDLIM1 interacting kinase 1 like’ (PDIK1L; nomenclature approved by the HUGO Gene Nomenclature Committee). Ensembl Genome Browser locatedPDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be localized to the nucleus.     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.     

Detection and characterization of anS-adenosyl-l-methionine: Flavonoid 7-O-methyltransferase from leaves ofPrunus x yedoensis matsum. with special reference to prunetrin formation

An enzyme,S-adenosyl-l-methionine: flavonoid 7-O-methyltransferase (F7OMT), catalyzing the transfer of the methyl group fromS-adenosyl-l-methionine (SAM) to the 7 position of sophoricoside (5, 7, 4′-trihydroxyisoflavone 4′-O-glucoside) and some of the other flavonoids, was detected in extracts from leaves ofPrunus x yedoensis, and it was partially purified (about 203-fold) by a combination of gel filtration and ion-exchange column chromatographies. F7OMT was isolated as a soluble enzyme with a pH optimun of 7.5 in K-phosphate buffer. The molecular mass of F7OMT, which had an isoelectric point at pH 4.1, was estimated by elution from a column of Sephadex G-100 to be about 36 kDa. The activity of F7OMT was stimulated by 14 mM 2-Co²⁺ and reagents that react with sulfhydryl groups. The apparentKm values for sophoricoside, its aglycone genistein (5, 7, 4′-trihydroxyisoflavone) and quercetin were 1.49, 2.19 and 1.89 μM, respectively. The apparentKm value for SAM as methyl donor was 2.08 mM. The specificity of F7OMT for methyl acceptors was not strict; flavonols, flavanones and flavanonols in addition to isoflavones served as methyl acceptor. An examination ofP. x yedoensis leaves during spring and autumn showed variations in the activities of F7OMT and UDP-glucose: isoflavone 4′-O-glucosyltransferase (I4′ GT). The activities of F7OMT and I4′GT increased in enlarging leaf tissues and then markedly declined when the leaves approached maturation. In autumn leaves F7OMT activity was scarcely detected, but a small peak of I4′GT activity was observed during autumnal reddening.     

Isolation and characterization of the cytoplasmic male sterility-associated <Emphasis Type="Italic">orf456</Emphasis> gene of chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T₁ transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T₁ plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper. GenBank accession number DQ116040 (orf456 genomic sequence), DQ126683 (pepper coxII genomic sequence)     

Isolation,sequencing and analysis of the expression of Bryonia calmodulin after mechanical perturbation

A cDNA clone (Bc329) encoding calmodulin was isolated from a Bryonia cDNA library by screening with cloned Arabidopsis calmodulin cDNA. The cDNA Bc329 was 899 bp full-length clone. The predicted amino acid sequence consists of 149 residues and reveals a high homology with other known plant calmodulins (91 to 99% identity). Genomic southern blot suggests that Bryonia calmodulin is encoded by a single-copy gene. The Bc329 clone was used as a probe to study the expression of calmodulin mRNA after a mechanical stimulus applied on young Bryonia internodes. The steady-state of calmodulin mRNA reached a maximum 30 min after the treatment before it progressively decreased. The role of calcium and calmodulin as second messengers is discussed with regard to environmental changes.     

Members of the<Emphasis Type="Italic"> S</Emphasis>-receptor kinase multigene family in<Emphasis Type="Italic"> Senecio squalidus</Emphasis> L. (Asteraceae), a species with sporophytic self-incompatibility

While the molecular basis of sporophytic self-incompatibility (SSI) has been investigated extensively in the Brassicaceae, almost nothing is known about the molecular regulation of SSI in other families, such as the Asteraceae. In species of Brassica and in Arabidopsis lyrata, a stigma-specific serine-threonine receptor kinase (SRK) and its cognate ligand, a pollen coating-borne cysteine-rich protein (SCR/SP11), determine the female and male sides of the SSI response, respectively. Here we have used RT-PCR with degenerate primers to conserved regions of SRK to amplify three SRK-like gene fragments expressed in stigmas of Senecio squalidus (Asteraceae). The Senecio S-receptor-like kinase (SSRLK) sequences share ~43% amino acid sequence identity with Brassica SRK₃ but higher amino acid sequence identity (~50%) with two Solanum bulbocastanum receptor-like kinase genes of unknown function. Despite expression in stigmas, all three SSRLKs were expressed at varying levels in floral and vegetative tissues. No allelic polymorphism was detected for the three SSRLKs in two S homozygous lines of S. squalidus or three other lines of S. squalidus carrying different S alleles. A full-length cDNA clone was obtained for SSRLK1 and its predicted amino acid sequence revealed significant structural differences to Brassica SRKs, most notably a major N-terminal truncation of 169 amino acids and the presence of just 8 conserved cysteine residues within the putative receptor domain instead of 12. Together, the sequence characteristics and expression characteristics of SSRLKs suggest that they are unlikely to be directly involved in the SSI response of S. squalidus. These findings are discussed in terms of the evolution of the SRK multigene family and the molecular basis of SSI in S. squalidus and the Asteraceae.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.