Shadi Rahimi

Effect of salicylic acid and yeast extract on the accumulation of jasmonic acid and sesquiterpenoids in Panax ginseng adventitious roots

In different plant species, secondary metabolite biosynthesis is regulated by the phytohormone jasmonic acid (JA), which is derived by the action of lipoxygenase. In this study, we examined mono- and sesquiterpenoid accumulation and the related signal transduction pathways and biosynthetic genes in adventitious root cultures of Panax ginseng C.A. Meyer as induced by yeast extract (YE, 3 g/L), a biotic elicitor, and salicylic acid (SA, 200 μM), a signaling elicitor. The lipoxygenase (LOX) gene was highly expressed in 24 and 12 h after treatment with SA and YE. JA content was significantly increased in 24 h after SA treatment. The H2O2 content was the highest in 24 and 72 h after the onset of SA and YE treatment, respectively. RNA blot analysis showed that farnesyl diphosphate synthase (FPS) and isopentenyl pyrophosphate isomerase (IPPI) genes encoding enzymes of the biosynthesis of mono- and sesquiterpenoids were up-regulated by both elicitors. Farensol, isochiapin B sesquiterpenoids, champhor, and cineole monoterpenoids were highly accumulated after 24 h of SA treatment, while YE treatment induced bacchotricuneatin C, guaiazulene, isochiapin B, and p-benzoquinone sesquiterpenoid production. These results suggest that mono- and sesquiterpenoid accumulation induced by SA and YE occurs due to the IPPI and FPS expression and may be mediated by reactive oxygen species signaling and jasmonic acid signal transduction.

Grouping and characterization of putative glycosyltransferase genes from Panax ginseng Meyer

Glycosyltransferases are members of the multigene family of plants that can transfer single or multiple activated sugars to a range of plant molecules, resulting in the glycosylation of plant compounds. Although the activities of many glycosyltransferases and their products have been recognized for a long time, only in recent years were some glycosyltransferase genes identified and few have been functionally characterized in detail. Korean ginseng (Panax ginseng Meyer), belonging to Araliaceae, has been well known as a popular mysterious medicinal herb in East Asia for over 2,000 years. A total of 704 glycosyltransferase unique sequences have been found from a ginseng expressed sequence tag (EST) library, and these sequences encode enzymes responsible for the secondary metabolite biosynthesis. Finally, twelve UDP glycosyltransferases (UGTs) were selected as the candidates most likely to be involved in triterpenoid synthesis. In this study, we classified the candidate P. ginseng UGTs (PgUGTs) into proper families and groups, which resulted in eight UGT families and six UGT groups. We also investigated those gene candidates encoding for glycosyltransferases by analysis of gene expression in methyl jasmonate (MeJA)-treated ginseng adventitious roots and different tissues from four-year-old ginseng using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). For organ-specific expression, most of PgUGT transcription levels were higher in leaves and roots compared with flower buds and stems. The transcription of PgUGTs in adventitious roots treated with MeJA increased as compared with the control. PgUGT1 and PgUGT2, which belong to the UGT71 family genes expressed in MeJA-treated adventitious roots, were especially sensitive, showing 33.32 and 38.88-fold expression increases upon 24h post-treatments, respectively.

Molecular characterization of 5-chlorophyll a/b -binding protein genes from Panax ginseng Meyer and their expression analysis during abiotic stresses

The chlorophyll a/b-binding protein (CAB) serves in both photosystems (PS), I and II, as a coordinator of antenna pigments in the light-harvesting complex (LHC). The CABs constitute abundant and important proteins in the thylakoid membrane of higher plants. In our study, five CAB genes, which contained full-length cDNA sequences from the 4-year-old ginseng leaves (Panax ginseng Meyer), were isolated and named PgCAB. Phylogenetic comparison of the members of the subfamily between ginseng and higher plants, including Arabidopsis, revealed that the putative functions of these ginseng CAB proteins were clustered into the different family of Arabidopsis CABs; two PgCABs in LHCII family and three PgCABs in LHCI family. The expression analysis of PgCABs consistently showed dark-dependent inhibition in leaves. Expression analysis during abiotic stress identified that PgCAB genes responded to heavy metal, salinity, chilling, and UV stresses differently, suggesting their specific function during photosynthesis. This is the first comprehensive study of the CAB gene family in P. ginseng.

PgLOX6 encoding a lipoxygenase contributes to jasmonic acid biosynthesis and ginsenoside production in Panax ginseng

Highlight In ginseng, jasmonic acid promotes expression of the biosynthetic genes for ginsenosides. PgLOX6 encodes a lipoxygenase that is required for biosynthesis of jasmonic acid and its overexpression increases ginsenoside levels.

Characterization of squalene-induced PgCYP736B involved in salt tolerance by modulating key genes of abscisic acid biosynthesis

Cytochrome P450 genes as the one of the largest superfamily genes mediate a wide range of plant biochemical pathways. In this study, a full-length cytochrome P450 monooxygenase (CYP736B) cDNA was isolated and characterized from Panax ginseng. It was revealed that the deduced amino acid of PgCYP736B shares a high degree of sequence homology with CYP736A12 encoded by P. ginseng. Expression of PgCYP736B was differentially induced not only during a Pseudomonas syringae infection (7.7-fold) and wounding (47.3-fold) but also after exposure to salt (7.4-fold), cold (8.3-fold), and drought stress (3.24-fold). The gene transcription was highly affected by methyl jasmonate (476-fold) in the ginseng, suggesting that PgCYP736B was elicitor-responsive. Furthermore, we overexpressed the PgCYP736B gene in Arabidopsis and found that PgCYP736B is a transmembrane protein. Overexpression of PgCYP736B in Arabidopsis conferred enhanced resistance to salt stress via decreased H2O2 accumulation, increased carotenoid levels, and through abscisic acid biosynthesis gene expression. Our results suggest that the induction of ginsenoside biosynthetic pathway genes along with PgCYP736B by an exogenous supply of 10-100 μM of squalene most likely affects the metabolite profile of ginsenoside triterpenoid. Overall, our findings indicate that PgCYP736B protects ginseng from salt stress and may contribute to triterpenoid biosynthesis.

Molecular characterization of MYB transcription factor genes from Panax ginseng

Transcription factors (TFs) are essential for gene regulation in all living organisms, including plants, where among numerous other functions, they control temporal and spatial gene expression in response to environmental stimuli. The v-myb avian myeloblastosis viral oncogene homolog (MYB) family is the largest TF family in plants, with its members involved in developmental processes as well as secondary metabolism. Little is known about MYB genes in Panax ginseng Meyer, despite ginseng’s importance as a widely-used medicinal plant. In this study, we isolated nine MYB genes from P. ginseng (PgMYBs). Phylogenetic comparison of these genes with the MYB genes of other plant species revealed that the PgMYBs clustered into different families based on their putative functions including terpene regulation; five PgMYBs clustered in the R2R3 family, and four PgMYBs clustered in the MYB-related protein group. Further expression analysis of five PgMYBs showed consistently high expression in flower and leaf tissue, suggesting that these PgMYB genes may be involved in development of the above-mentioned tissues. Four PgMYBs were downregulated in response to methyl jasmonate (MJ) and salicylic acid (SA), whereas PgMYB3 was up-regulated, suggesting a role for all these genes in stress response. This is the first comprehensive study of the MYB gene family in P. ginseng, and the information provided here will facilitate further exploration of the functions of these TFs.

Molecular characterization and expression analysis of pathogenesis related protein 6 from Panax ginseng

Panax ginseng Meyer is one of the important medicinal plants in the world, particularly in Asian countries. Ginseng encounters many stress exposure during its long cultivation period. However, the molecular mechanism of stress resistance is still poorly understood in spite of its importance. In this study, pathogenesis-related protein 6 (PR6), also called proteinase inhibitor (PI), was isolated from ginseng embryogenic callus, named PgPR6. The small size of PR6, containing an open reading frame of 219 bp encoding 72 amino acids, the typical characteristic of PR6 protein, shares the highest sequence similarity to PR6 of Theobroma cacao (69% identity). Sequence and structural analysis indicated that PgPR6 belongs to class Kunitz-type PI family. This is the first report pertaining to the identification of PR6 gene from the P. ginseng genome. The high-level expression of PgPR6 was observed in root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR6 expression was highly up-regulated by signaling molecules, heavy metals, mechanical wounding, chilling, salt, sucrose, and mannitol stress, indicating that PgPR6 may play an important role in the molecular defense response of ginseng to a various range of environmental stresses.