Metal tolerance proteins (MTPs) play an important role in the transport of metals at the cellular, tissue and whole plant levels. In the present study, 11 MTP genes were identified and these clustered in three major sub-families Fe/Zn-MTP, Zn-MTP, and Mn-MTP, and seven groups, which are similar to the grouping of MTP genes in both Arabidopsis and rice. Vitis vinifera metal tolerance proteins (VvMTP) ranged from 366 to 1092 amino acids, were predicted to be located in the cell vacuole, and had four to six putative TMDs, except for VvtMTP12 and VvMTP1. The VvMTPs had putative cation diffusion facilitator (CDF) domains and the putative Mn-MTPs also had zinc transporter dimerization domains (ZD-domains). V. vinifera Mn-MTPs had gene structures and motif distributions similar to those of the Fe/Zn-MTP and Zn-MTP sub-families. The upstream regions of VvMTP genes had variable frequencies of cis-regulatory elements that could indicate regulation at different developmental stages and/or differential regulation in response to stress. Comparison of the VvMTP coding sequences with known miRNAs found in various plant species indicated the presence of 13 putative miRNAs, with 7 of these associated with VvMTPs. Temporal and spatial expression profiling indicates a potential role for VvMTP genes during growth and development in grape plants, as well as the involvement of these genes in plant responses to environmental stress, especially osmotic stress. The data generated from this study provides a basis for further investigation of the roles of MTP genes in grapes.

Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoid and phenolic families. The aim of this study is to evaluate the effect of salinity stress on the expression of key genes involved in the biosynthetic pathway of triterpenoids such as glycyrrhizin, betulinic acid, soyasaponins, and phytosterols in licorice root, as well as providing a phonemic platform to characterize antioxidant properties, glycyrrhizin, and total phenolic content. This study also includes measuring the gene expression level and glycyrrhizin content in leaves and roots of control plants. The studied genes included squalene synthase (SQS1 and SQS2), β-amyrin synthase (bAS), lupeol synthase (LUS), cycloartenol synthase (CAS), β-amyrin 11-oxidase (CYP88D6), and β-amyrin 24-hydroxylase (CYP93E6). Our results revealed that all of the mentioned genes were upregulated following the stress condition with different transcription rates. The highest increase (12-fold) was observed for the expression of the LUS gene, which is related to the betulinic acid pathway. Also, the highest content of glycyrrhizin was observed at 72 h posttreatment, which was consistent with the upregulated transcription levels of the glycyrrhizin pathway genes especially SQS1 and CYP88D6 at the same time. Correlation and stepwise regression analysis proved the key role of SQS1 gene in the biosynthetic pathway of glycyrrhizin. Antioxidant activity and phenolic content also were increased following stress condition. A comparison between the expression levels of SQS1 and other genes involved in the production of glycyrrhizin, phytosterols, and soyasaponins revealed a similar transcription trend, which shows the gene expression in the roots was significantly higher than the leaves. In contrast, SQS2 and LUS genes displayed a higher expression in leaf tissues. The genes related to betulinic acid biosynthetic pathway exhibited an expression rate different from other triterpenoid pathway genes, which could be observed in the leaves and roots of control plants and the roots of salt-treated plants. Furthermore, results showed that these two SQS genes have different expression rates due to different plant tissues (roots and leaves) and stress conditions. Importantly, in contrast to previous reports, we detected the glycyrrhizin in leaf tissues. This result may indicate the presence of a different genetic background in native Iranian licorice germplasm. Keywords Gene expression . Glycyrrhiza glabra . Glycyrrhizin . Quantitative real-time PCR . Secondary metabolites Abbreviations bAS β-Amyrin synthase CAS Cycloartenol synthase CYP88D6 β-Amyrin 11-oxidase CYP93E6 β-Amyrin 24-hydroxylase FDP Farnesyl diphosphate GA3 Gibberellic acid HPLC High-performance liquid chromatography LUS Lupeol synthase MeJA Methyl jasmonate OSCs Oxidosqualene cyclases QRT-PCR Quantitative reverse transcription PCR SQS Squalene synthase

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Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces various triterpene saponins such as glycyrrhizin. Beta-amyrin 11-oxidase (CYP88D6) plays a key role in engineering pathway of glycyrrhizin production and converts an intermediated beta-amyrin compound to glycyrrhizin. In this study, pBI121:CYP88D6 construct was transferred to G. glabra using Agrobacterium rhizogene ATCC 15834. The quantitation of transgene was measured in putative transgenic hairy roots using qRT-PCR. The amount of glycyrrhizin production was measured by HPLC in transgenic hairy root lines. Gene expression analysis demonstrated that CYP88D6 was over-expressed only in one of transgenic hairy root lines and was reduced in two others. Beta-amyrin 24-hydroxylase (CYP93E6) was significantly expressed in one of the control hairy root lines. The amount of glycyrrhizin metabolite in over-expressed line was more than or similar to that of control hairy root lines. According to the obtained results, it would be recommended that multi-genes of glycyrrhizin biosynthetic pathway be transferred simultaneously to the hairy root in order to increase glycyrrhizin content.

Licorice is the roots and stolons of Glycyrrhiza uralensis which have several chemical compounds. Triterpene saponins such as glycyrrhizin and glycyrrhetinic acid and flavonoids like liquiritin, isoliquiritigenin and glabron are main compounds detected in liquorice root. The plant's major constituent is a glycyrrhizin. The β-amyrin 11-oxidase catalyzes the sequential two-step oxidation of β-amyrin in C-11 to produce 11-oxo-β-amyrin, a possible biosynthetic intermediate between β-amyrin and glycyrrhizin. In this study, the total RNA was extracted from licorice roots and cDNA synthesis, then PCR products were cloned into pTZ57R/T vector. Sequencing confirmed piece length of 1482 bp that encodes a protein of 493 amino acid residues. The results of alignment showe d 99% similarity to β-amyrin sequence of Glycyrrhiza uralensis. Subcellular studies using Softberry and Psort software showed that the activity of this protein is in endoplasmic reticulum. Moreover the protein has a signal peptide and is targeted to the secretory pathway. The results of phylogenetic tree determined most similar amino acid sequence to the CYP88D subfamily of cytochrome P450. These findings can be used for nucleotide or protein manipulation and transformation.

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Shirazi et al.