Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence‐related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full‐length cDNAs of GmSAMT1 from a SCN‐resistant soybean line and from a SCN‐susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli‐expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μm . To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN‐susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.     

Isolation and characterization of novel defence-related genes induced by copper, salicylic acid, methyl jasmonate, abscisic acid and pathogen infection in Brassica carinata

To examine the defence response in Brassica carinata we differentially screened a cDNA library made from CuCl₂-treated (Cu) leaves. The sequence of 17 of the 27 cDNA clones examined that showed Cu-induction had a high similarity to defence genes from other plant species. Among other clones that showed higher expression in the Cu leaves were two cDNAs encoding polypeptides of 351 and 250 amino acids, designated BcCJS1 and BcCJAS1. BcCJS1 had similarity to S-adenosyl-l -methionine: salicylic acid carboxyl methyltransferase from Clarkia breweri. However, the enzyme activity was not found in extracts from E. coli expressing BcCJS1. BcCJAS1 did not show extensive similarity to any genes with known function in the databases but it did contain three regions of amino acid sequence that are frequently found in amidotransferases. A third Cu-induced mRNA, Bcp6PGL, showed very high (86%) similarity to a putative 6-phosphogluconolactonase (6PGL) from Arabidopsis thaliana. In addition to Cu induction, BcCJS1 expression was induced by methyl jasmonate (MeJA) and salicylic acid (SA), BcCJAS1 expression by MeJA, SA and abscisic acid and Bcp6PGL expression by MeJA. The expression of all three genes increased after Alternaria brassicae infection. BcCJS1 and BcCJAS1 were induced within 1 h after MeJA- but not until 3 h after SA-treatment. The expression of both genes was systemically induced after infection with a compatible or incompatible fungal pathogen. SA systemically induced only BcCJAS1. The effects of various inhibitors of signalling pathways on expression of the three genes were studied.     

Enzymatic Kolbe-Schmitt reaction to form salicylic acid from phenol: Enzymatic characterization and gene identification of a novel enzyme, Trichosporon moniliiforme salicylic acid decarboxylase

Salicylic acid decarboxylase (Sdc) can produce salicylic acid from phenol; it was found in the yeast Trichosporon moniliiforme WU-0401 and was for the first time enzymatically characterized, with the sdc gene heterologously expressed. Sdc catalyzed both reactions: decarboxylation of salicylic acid to phenol and the carboxylation of phenol to form salicylic acid without any byproducts. Both reactions were detected without the addition of any cofactors and occurred even in the presence of oxygen, suggesting that this Sdc is reversible, nonoxidative, and oxygen insensitive. Therefore, it is readily applicable in the selective production of salicylic acid from phenol, the enzymatic Kolbe-Schmitt reaction. The deduced amino acid sequence of the gene, sdc, encoding Sdc comprises 350 amino acid residues corresponding to a 40-kDa protein. The recombinant Escherichia coli BL21(DE3) expressing sdc converted phenol to salicylic acid with a 27% (mol/mol) yield at 30 °C for 9 h.     

Molecular characterization of a novel soybean gene encoding a neutral PR-5 protein induced by high-salt stress

In this study, we characterized a novel soybean gene encoding a neutral PR-5 protein and compared it to two acidic isoforms of soybean PR-5 protein. This gene, designated as Glycine max osmotin-like protein, b isoform (GmOLPb, accession no. AB370233), encoded a putative protein having the greatest similarity to chickpea PR-5b (89% identity). Unlike the two acidic PR-5, GmOLPa and P21, the protein had a C-terminal elongation responsible for possible vacuolar targeting and after maturation showed a calculated molecular mass of 21.9 kDa with pI 6.0. The 3D models, predicted by the homology modeling, contained four α-helixes and 16 β-strands and formed three characteristic domains. The two acidic PR-5 proteins also showed a 3D structure very similar to GmOLPb, although the electrostatic potential on molecular surface of each PR-5 was significantly different. In the study of the gene expression under conditions of high-salt stress, GmOLPb was highly induced in the leaves of the soybean, particularly in the lower part of a leaf. The expression started at 2 h after initiation of the stress and was highly induced between 18–72 h. Gene expression of P21e (protein homologous to P21) was transiently induced by high-salt stress, but took place earlier than the gene expressions of GmOLPa and GmOLPb. Such differential expression was observed also under investigation with methyl jasmonate and salicylic acid. These results suggested that each soybean PR-5 might play a distinctive role in the defensive system protecting the soybean plant against high-salt stress, particularly in the leaves of the soybean.     

Isolation of a novel protein kinase-encoding gene from yeast by oligodeoxyribonucleotide probing

We have identified a novel protein kinase-encoding gene, KIN3, in the genome of the budding yeast Saccharomyces cerevisiae. The gene was isolated from a library of cloned genomic fragments by probing with an oligodeoxyribonucleotide mixture corresponding to part of a highly-conserved region in the catalytic domain of protein serine-threonine kinases. KIN3 is unique in the yeast genome, maps to chromosome VI and is actively expressed in mitotically dividing cells to produce a 1400 nucleotide (nt) message. The nt sequence of KIN3 predicts a protein product of 43.4 kDa which contains all of the conserved elements found in known protein serine-threonine kinases, although the organisation of these elements in the KIN3 gene product differs significantly from the consensus. The function of the KIN3-encoded protein kinase is unclear although it appears not to be essential for growth, conjugation or sporulation.     

Short-day flowering of Lemna gibba G3 induced by salicylic acid

Salicylic acid, probably as a chelating agent of the EDTA-salicylaldoximetype, can eliminate the light requirement during the inductivephase of Lemna gibba G3, and thus is able to induce short-dayflowering of this long-day plant. (Received September 4, 1975; )     

Isolation and characterization of a novel dehydrin gene from Capsella bursa-pastoris

A novel dehydrin gene designated as Cbcor29 was cloned from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE) and genome walker technique. The full-length cDNA of Cbcor29 was 1101 bp long with a 783 bp open reading frame (ORF), encoding a putative protein of 261 amino acids. Like other dehydrin proteins, CbCOR29 contained a high percentage of charged and polar amino acids, in which Cys and Trp amino acids were absent. In addition, the predicted CbCOR29 protein possesses three conserved repeats of the characterized Lys-rich domains (K-segments), and a Ser-rich domain (S-segment) prior to the first Lys-rich domain, which presented a typical SK3 structure of dehydrins. Analysis of Cbcor29 genomic DNA revealed that it contains 2 exons and 1 intron, which is a typical character of dehydrin genes. Subsequent bioinformatic analysis also showed that the sequence of CbCOR29 has high homology with other dehydrin proteins, especially with cor47 from Arabidopsis thaliana. Moreover, semi-quantitative RT-PCR revealed that the expression of Cbcor29 can be induced by exposure to drought, low temperature, NaCl, and exogenous ABA treatment. Our study led to the conclusion that the Cbcor29 gene is a new member of the dehydrin gene family and might exert functions in responsiveness to drought, cold, and salt in Capsella bursa-pastoris. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 1, pp. 52–60. The article was submitted by the authors in English.     

Isolation and characterization of a novel dehydrin gene from Capsella bursa-pastoris

A novel dehydrin gene designated as Cbcor29 was cloned from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE) and genome walker technique. The full-length cDNA of Cbcor29 was 1101 bp long with a 783 bp open reading frame (ORF), encoding a putative protein of 261 amino acids. Like other dehydrin proteins, CbCOR29 contained a high percentage of charged and polar amino acids, in which Cys and Trp amino acids were absent. Besides, predicted CbCOR29 protein possesses three conserved repeats of the characterized Lys-rich domains (K-segments), and a Ser-rich domain (S-segment) prior to the first Lys-rich domain, which presented a typical SK3 structure of dehydrins. Analysis of Cbcor29 genomic DNA revealed that it contained 2 exons and 1 intron, which was a typical character of dehydrin genes. Subsequent bioinformatic analysis also showed that the sequence of CbCOR29 had high homology with other dehydrin proteins, especially with cor47 from Arabidopsis thaliana. Moreover, semi-quantitative RT-PCR revealed that the expression of Cbcor29 could be induced by exposure to drought, low-temperature, NaCl and exogenous ABA treatment respectively. Our study implied that the Cbcor29 gene was a new member of the dehydrin gene family and might exert functions in drought-, cold- and salt- responsiveness in Capsella bursa-pastoris.