Rama Krishna Pulla

Expression and stress tolerance of PR10 genes from Panax ginseng C. A. Meyer

Pathogenesis-related 10 protein families (PgPR10 proteins) from ginseng are reported to have ribonuclease activity, conferring defense-related resistance against various stresses. Homology-based PCR using PgPR10-2 specific primers allowed for the isolation of two additional PgPR10 genes. PgPR10-1 is identical to the previously reported ribonuclease 1, while PgPR10-3 is a newly-discovered protein, suggesting that the PgPR10s are a multi-gene family. Differential organ-specific transcripts of PgPR10-1 and PgPR10-2 in the flower bud and root, respectively, indicate that there are tissue-specific functional roles for this gene family. Overexpression of PgPR10-2 in Arabidopsis conferred longer root length and a tolerant growth phenotype on NaCl-supplemented media. Further changes in transcriptional levels against sets of abiotic stressors suggest similar functional roles of PgPR10-1 in the root and predominantly in the flower organ based on its higher expression levels. Overall, this suggests that the manipulation of PgPR10 genes in plants can be used as valuable tool to enhance its physiological status.

Isolation of a novel catalase (Cat1) gene from Panax ginseng and analysis of the response of this gene to various stresses.

A cDNA clone containing a catalase (CAT1) gene, designated PgCat1, was isolated from Panax ginseng C.A. Meyer (Korean ginseng). PgCat1 is predicted to encode a precursor protein of 492 amino acid residues, and its sequence shares high degrees of homology with a number of other CAT1s. Genomic DNA hybridization analysis indicated that PgCat1 represents a multi-gene family. Reverse transcriptase (RT)-PCR results showed that PgCat1 expressed at different levels in leaves, stem, roots of P. ginseng seedlings. Different stresses, heavy metals, plant hormones, osmotic agents, high light irradiance, abiotic stresses, triggered a significant induction of PgCat1. The positive responses of PgCat1 to the various stimuli suggested that P. ginseng PgCat1 may help to protect the plant against reactive oxidant related environmental stresses.

Isolation of S-adenosyl-L-methionine synthetase gene from Panax ginseng C.A. meyer and analysis of its response to abiotic stresses.

A cDNA clone containing a S-adenosyl-L-methionine synthetase (SAMS) gene, named as PgSAM, was isolated from a commercial medicinal plant Panax ginseng. PgSAM is predicted to encode a precursor protein of 307 amino acid residues, and its sequence shares high homology with a number of other plant SAMS. PgSAM is expressed at different levels in various organs of ginseng. The expression of PgSAM in adventitious roots and hairy roots of P. ginseng were analyzed using reverse transcriptase (RT)-PCR and real-time PCR under various abiotic stresses. Salt, salicylic acid, abscisic acid and chilling stresses induced PgSAM significantly at different time points within 2–72 h post-treatment. This study revealed that PgSAM may help to protect the plants against various abiotic stresses.

Brevibacillus panacihumi sp. nov., a β-glucosidase-producing bacterium

Two Gram-positive-staining, endospore-forming, rod-shaped, motile bacteria, strains DCY35(T) and C17, were isolated from soil of a ginseng field in South Korea and were characterized in order to determine their taxonomic positions. 16S rRNA gene sequence analysis revealed that the two strains belonged to the family Paenibacillaceae; strain DCY35(T) showed highest levels of similarity to strain C17 (99.9 %), Brevibacillus invocatus LMG 18962(T) (98.9 %), B. centrosporus DSM 8445(T) (98.0 %), B. borstelensis DSM 6347(T) (97.6 %), B. formosus DSM 9885(T) (97.4 %), B. agri DSM 6348(T) (97.3 %), B. brevis DSM 30(T) (97.3 %) and B. levickii LMG 22481(T) (97.0 %). Chemotaxonomic analyses revealed that strains DCY35(T) and C17 possess menaquinone MK-7, common to members of the genus Brevibacillus, and that the predominant fatty acids were iso-C(15 : 0) (37.3 % of the total), anteiso-C(15 : 0) (32.9 %), iso-C(14 : 0) (11.8 %) and iso-C(16 : 0) (6.5 %). The results of physiological and biochemical tests clearly demonstrated that strains DCY35(T) and C17 represent a distinct species. Based on these data, the two strains are considered to represent a novel species of the genus Brevibacillus, for which the name Brevibacillus panacihumi sp. nov. is proposed. The type strain is DCY35(T) (=KCTC 13206(T) =JCM 15085(T)).

Somatic embryogenesis of two new Panax ginseng cultivars, Yun-Poong and Chun-Poong

Somatic embryogenesis from single cells is important for normal plant regeneration of ginseng. Cotyledon explants from zygotic embryos of two new ginseng cultivars, Chun-Poong and Yun-Poong, produced somatic embryos on Murashige and Skoog (MS) basal medium and MS medium containing growth regulators. The highest frequency of single somatic embryo formation was obtained when cotyledon explants were excised from premature (cultured for 1 day) zygotic embryos (about 6 mm in length) of both cvs. Chun-Poong and Yun-Poong and then cultured on MS medium supplemented with 7% sucrose. The frequency of single somatic embryo formation was strongly enhanced when Chun-Poong cotyledons were subjected to plasmolysis with 0.1–0.5 M sucrose for 24 h and Yun-Poong cotyledons to plasmolysis with 1.0 M sucrose for 24 h and then cultured on MS medium with 2,4-D.

Isolation and Characterization of Cinnamoyl-CoA Reductase Gene from Panax ginseng C. A. Meyer

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) catalyses the reduction of cinnamic acid CoA esters into their corresponding aldehydes, the first step of the phenylpropanoid pathway specially dedicated to monolignol biosynthesis. A cDNA clones encoding CCR have been isolated from Panax ginseng C.A. Meyer and its expression was investigated in response to abiotic stresses. The cDNA, designated PgCCR which is 865 nucleotides long and has an open reading frame of 590 bp with a deduced amino acid sequence of 176 residues. The PgCCR encoded protein possesses substantial homology with CCRs isolated and cloned from other sources; the highest identity (51.8%) was observed with CCR from Tomato (Lycopersicon esculentum). Under various stress conditions, expression patterns of the PgCCR were highly induced in adventitious and hairy roots by several abiotic stresses. These results indicated that PgCCR plays protective role against diverse environmental stresses.

Isolation and Characterization of Calmodulin Gene from Panax ginseng C. A. Meyer

Ca 2+ and calmodulin (CaM), a key Ca 2+ sensor in all eukaryotes, have been implicated for defense responses of plants. Eukaryotic CaM contains four structurally and functionally similar Ca 2+ domains named Ⅰ, Ⅱ, Ⅲ and IV. Each Ca 2+ binding loop consists of 12 amino acid residues with ligands arranged spatially to satisfy the octahedral symmetry of Ca 2+ binding. To investigate the altered gene expression and the role of CaM in ginseng plant defense system, cDNA clone containing a CaM gene, designated PgCaM was isolated and sequenced from Panax ginseng. PgCaM, which has open reading frame of 450 nucleotides predicted to encode a precursor protein of 150 amino acid residues. Its sequence shows high homologies with a number of other CaMs, with more similarity to CaM of Daucus carota (AAQ63461). The expression of PgCaM in different P. ginseng organs was analyzed using real time PCR. The results showed that PgCaM expressed at different levels in young leaves, shoots, and roots of 3-week-old P. ginseng. In addition, the expressions of PgCaM under different abiotic stresses were analyzed at different time intervals.

Sanguibacter soli sp. nov., isolated from soil of a ginseng field.

A Gram-positive, non-spore-forming, rod-shaped, motile bacterium, designated strain DCY22(T), was isolated from soil of a ginseng field in South Korea and characterized in order to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that strain DCY22(T) belonged within the family Sanguibacteraceae, and highest levels of sequence similarity were found with Sanguibacter marinus 1-19(T) (96.8 %), Sanguibacter suarezii ST-26(T) (96.0 %), Sanguibacter inulinus ST-50(T) (95.9 %), Sanguibacter keddieii ST-74(T) (95.5 %), Terrabacter terrae PPLB(T) (94.0 %) and Terrabacter tumescens DSM 20308(T) (93.8 %). Chemotaxonomic investigations revealed that strain DCY22(T) possessed menaquinone MK-9, a common feature of members of the genus Sanguibacter. Predominant fatty acids were unknown ECL 13.961 (45.81 %), 17 : 0 anteiso (23.46 %), 18 : 0 iso (15.42 %) and unknown ECL 14.966 (8.70 %). The results of physiological and biochemical tests clearly demonstrated that strain DCY22(T) represents a novel species of the genus Sanguibacter, for which the name Sanguibacter soli sp. nov. is proposed. The type strain is DCY22(T) (=KCTC 13155(T)=JCM 14841(T)).

Isolation and Characterization of Glycolate Oxidase Gene from Panax ginseng C. A. Meyer

The oxidation of glycolate to glyoxylate, a key step in plant photorespiration, is carried out by the peroxisomal flavoprotein glycolate oxidase (EC 1.1.3.15). To investigate the altered gene expression and the role of GOX in ginseng plant defense system, a cDNA clone containing a GOX gene designated as PgGOX was isolated and sequenced from Panax ginseng. The cDNA was 692 nucleotides long and have an open reading frame of 552 bp with a deduced amino acid sequence of 183 residues. A GenBank BlastX search revealed that the deduced amino acid of PgGOX shares a high degree homology with the Glycine max (95% identity). In the present study we analyzed the expression of PgGOX under various environmental stresses at different times using real time-PCR. The results showed that the expressions of PgGOX increased after various treatments involving salt, light, cold, ABA, SA, and copper treatment.