Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

Steviol glycosides (SGs) are extracted from Stevia leaves for use as a natural sweetener. Among SGs, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP‐glycosyltransferase 76G1 (SrUGT76G1) that is known to convert stevioside to Reb A through 1,3‐β‐d ‐glucosylation in vitro. Interestingly, by overexpressing SrUGT76G1, the Reb A to stevioside ratio was drastically increased from 0.30 in wild‐type (WT) plants up to 1.55 in transgenic lines without any significant changes in total SGs content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S‐transferase‐tagged SrUGT76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by SrUGT76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of SrUGT76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.     

甜菊糖苷积累与其生物合成基因表达的关系

为探究甜叶菊叶片中甜菊糖苷积累与其合成途径上关键基因表达的关系,本研究分别检测了鑫丰3号苗期不同冠层和3个不同品种甜叶菊(守田3号、江甜3号、谱星1号)收获期混合叶片样品中多种糖苷的含量,同时定量检测对应样品中甜菊糖苷合成关键基因的表达水平。结果显示,总糖苷在鑫丰3号顶叶中最高,底叶中最低,而多数检测基因(6/8)表达水平也在顶叶最高底叶中最低;单一糖苷甜菊苷在顶叶中积累最高,而其催化酶编码基因Sr UGT74G1表达也在顶叶中最高;莱鲍迪苷A则在底叶中积累最多,其催化酶编码基因Sr UGT76G1表达水平也在底叶中表达最高。3个品种相比,总糖苷和莱鲍迪苷A的积累在江甜3号中最高,谱星1号中最低;甜菊苷的积累在守田3号中最高,江甜3号中最低,但基因表达水平与糖苷积累趋势一致的类似结果并未在不同品种间出现。由此可知,甜菊糖苷合成基因的表达水平可以影响总糖苷的积累,且在同一甜叶菊品种中单一糖苷合成调控基因的表达水平可以反映其调控的单糖苷的积累量。     

A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni)

Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA₃) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.     

A DESD-box helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. PB1)

The exact mechanism of helicase-mediated salinity tolerance is not yet understood. We have isolated a DESD-box containing cDNA from Pisum sativum (Pea) and named it as PDH45. It is a unique member of DEAD-box helicase family; containing DESD instead of DEAD/H. PDH45 overexpression driven by constitutive cauliflower mosaic virus-35S promoter in rice transgenic [Oryza sativa L. cv. Pusa Basmati 1 (PB1)] plants confers salinity tolerance by improving the photosynthesis and antioxidant machinery. The Na⁺ ion concentration and oxidative stress parameters in leaves of the NaCl (0, 100 or 200 mM) treated PDH45 overexpressing T₁ transgenic lines were lower as compared to wild type (WT) rice plants under similar conditions. The 200 mM NaCl significantly reduced the leaf area, plant dry mass, net photosynthetic rate (P_N), stomatal conductance (gs), intercellular CO₂ (Ci), chlorophyll (Chl) content in WT plants as compared to the transgenics. The T₁ transgenics exhibited higher glutathione (GSH) and ascorbate (AsA) contents under salinity stress. The activities of antioxidant enzymes viz. superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) were significantly higher in transgenics; suggesting the existence of an efficient antioxidant defence system to cope with salinity induced-oxidative damage. Yeast two-hybrid assay indicated that the PDH45 protein interacts with Cu/Zn SOD, adenosine-5′-phosphosulfate-kinase, cysteine proteinase and eIF(4G), thus confirming the involvement of ROS scavenging machinery in the transgenic plants to provide salt tolerance. Furthermore, the T₂ transgenics were also able to grow, flower, and set viable seeds under continuous salinity stress of 200 mM NaCl. This study provides insights into the mechanism of PDH45 mediated salinity stress tolerance by controlling the generation of stress induced reactive oxygen species (ROS) and also by protecting the photosynthetic machinery through a strengthened antioxidant system.     

Overexpression of SaRBP1 enhances tolerance of Arabidopsis to salt stress

Abiotic stresses are the major concern in recent years as their effect on world food production is constantly increasing. We have obtained salt tolerant Arabidopsis lines overexpressing SaRBP1 (Suaeda asparagoides RNA binding protein 1) of a Korean halophyte, S. asparagoides. Homozygous T3 Arabidopsis transgenic lines were developed and used for salt stress tolerance studies. The transgenic seedlings displayed tolerance to salt and mannitol compared to the wild type (WT) seedlings. Transgenic lines produced longer primary roots, more fresh weight, and higher number of lateral roots than WT. In planta stress tolerance assay results showed that the survival rates of transgenic plants were significantly higher than WT plants. Transgenic lines showed delayed germination under 200 mM NaCl stress. In addition, the transgenics showed higher water retention ability than WT. Subcellular localization results revealed that SaRBP1 was targeted to the cytoplasm. Northwestern blot analysis results confirmed the RNA binding property of SaRBP1. Quantitative Real-Time Polymerase Chain Reaction results revealed that many stress marker genes were upregulated by SaRBP1 overexpression. Thus, our data demonstrate that SaRBP1 overexpression lines are tolerant to salt stress. Hence, this is the first report for the functional characterization of SaRBP1, a novel RBP gene isolated from S. asparagoides cDNA library.     

Antioxidant potential in <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> callus in response to polyethylene glycol,paclobutrazol and gibberellin treatments

The study on Stevia callus has the potential to advance the knowledge of antioxidant mechanisms involved in unorganized cells response to drought stress. The effects of polyethylene glycol (PEG; 0 and 4% w/v) in combination with paclobutrazol (PBZ; 0 and 2 mg l^?1) and gibberellin (GA; 0 and 2 mg l^?1) were studied on Stevia rebaudiana callus. PEG treatment led to an oxidative stress, as indicated by increased H₂O₂ content whose accumulation was prevented with PBZ and GA treatments. All treatments of PEG, PBZ and GA increased the total antioxidant capacity, with the highest antioxidant power in PBZ and GA treatments without PEG. The activity of superoxide dismutase, catalase and ascorbate peroxidase significantly increased in PEG treatment alone or in combination with PBZ and GA. All treatments of PEG, PBZ and GA significantly increased proteins, amino acids and proline contents, with the highest increase in presence of PBZ in medium culture. In contrary to proline, the activity of pyrroline-5-carboxylate synthetase and proline dehydrogenase did not change in response to any of the treatments. Collectively, our results demonstrated that PBZ and GA increased reactive oxygen species scavenging and osmolytes in PEG-treated calli more than PEG treatment alone to alleviate negative effects of PEG on Stevia calli. These findings will enable us to design effective genetic engineering strategies in callus culture to generate some somaclonal variation that may be useful in enhancing drought resistance in Stevia.     

Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) JAZ3 and SLR1 function in jasmonate and gibberellin mediated epidermal cell differentiation and elongation

Jasmonate ZIM-domain (JAZ) proteins and DELLA proteins are key negative regulators of jasmonates (JAs) and gibberellin (GA) signaling, respectively. In this study, we found JA and GA synergistically promote fiber cell initiation. We characterized the cellular function of a JAZ protein (GhJAZ3), and a DELLA protein (GhSLR1) of cotton (Gossypium hirsutum). GhJAZ3 is specifically expressed in elongating fibers, while GhSLR1 is expressed in different tissues and at a relatively higher level in 3 DPA ovules. GhSLR1 and GhJAZ3 proteins are localized in the cell nucleus. Yeast two-hybrid analysis indicated that GhSLR1, GhJAZ3 and GhDEL65 could interact with each other, and GhSLR1 could also interact with GhBZR1. Overexpression of GhJAZ3 in Arabidopsis increased hypocotyl and root length, leaf trichome length, and plant height, but decreased the number of leaf trichome, while overexpression of GhSLR1 in Arabidopsis decreased hypocotyl length, leaf trichome length and density. Expression of several leaf trichome initiation determinators (GL3, GL2, TTG2 and MYB23) was down-regulated in GhJAZ3 or GhSLR1 transgenic Arabidopsis, while expression of the cell elongation related genes (EXP1, EXP8, EXPL2 and XTH4) was altered in the GhJAZ3 and GhSLR1 transgenic Arabidopsis. Taken together, these results demonstrate that GhJAZ3 and GhSLR1 function in jasmonate and gibberellin mediated epidermal cell differentiation and elongation.     

A <Emphasis Type="Italic">Vitis vinifera</Emphasis> xanthine dehydrogenase gene, <Emphasis Type="Italic">VvXDH</Emphasis>, enhances salinity tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.     

Cloning, characterization and impact of up- and down-regulating subabul cinnamyl alcohol dehydrogenase (CAD) gene on plant growth and lignin profiles in transgenic tobacco

Both cDNA including 5′UTR and 3′UTR and genomic clones of cinnamyl alcohol dehydrogenase (CAD) were isolated and characterized from a pulp-yielding leguminous tree Leucaena leucocephala (LlCAD1). The deduced amino acid sequence shared high identity with orthologous sequences of Acacia mangium?×?Acacia auriculiformis (83%), Medicago sativa (83%), Nicotiana tabaccum (83%) and Aralia cordata (81%). Full length cDNA contained 78 bases of 5′UTR and 283 bases of 3′UTR, while the genomic clone contained 5 exons and 4 introns. Western blot analysis revealed elevated expression of LlCAD1 in seedling roots and shoots compared to leaves. Sense and antisense CAD tobacco transgenics showed increased and reduced CAD activity accompanied by a change in monomeric lignin composition. Histochemical staining of lignin in down-regulated plants suggested an increase in aldehyde units and a decrease in S/G ratio. Down-regulation of CAD resulted in accumulation of syringic, ferulic, p-coumaric and sinapic acids compared to untransformed controls. These observations were validated by anatomical studies of down-regulated transgenic stems which showed thin walled, elongated phloem and xylem fibres, accompanied by a reduction in the density of vessel elements and amount of secondary xylem when compared to untransformed plants. Furthermore, Klason lignin analysis of CAD antisense transgenics showed 7–32% reduced lignin and normal phenotype as compared to untransformed plants. Such a reduction was not noticed in up-regulated transgenics. These results demonstrate a unique opportunity to explore the significant role that down-regulation of CAD gene plays in reducing lignin content thereby offering potential benefits to the pulp and paper industry.     

Cloning,antibody production,expression and cellular localization of universal stress protein gene (<Emphasis Type="Italic">USP1</Emphasis>-GFP) in transgenic cotton

This study was aimed to clone the universal stress protein (GUSP1) gene isolated from Gossypium arboreum in E. coli expression vector pET30(a) and to raise the specific antibody in rabbit to devise a system that could be used for localization and expression of this gene under drought stress. The amplification of GUSP1 transgene revealed a fragment of 500 bp via PCR in genomic DNA of transgenic cotton plants and expression was confirmed through ELISA and Western blot by using the GUSP1 specific polyclonal antibodies. ELISA showed the expression of GUSP1 protein in roots, stem and leaves of transgenic plants at seedling, vegetative and mature plant developmental stages. Total protein isolated from drought stressed transgenic plants revealed a fragment of 47 kDa (GUSP1-GFP fusion protein) in Western blot which confirmed the expression of transgene. Confocal microscopy detected the GFP fluorescence as localization of GUSP1 in the midrib, guard cells of stomata, trichome and globular trichome of intact leaf of transgenic plants. The co-localization was observed within cytoplasm, palisade, spongy mesophyll, guard cells of stomata, vascular bundle, trichome and globular trichome of transgenic plants by using the GUSP1 specific primary antibodies and Alexa fluor conjugated secondary antibodies. This study of GUSP1 gene will advance the mechanism of abiotic stress tolerance in plants.     

Expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in Escherichia coli and Arabidopsis enhances heavy metal(loid)s accumulation

Phytochelatin synthase (PCS) gene encoding key enzyme for heavy metal detoxification and accumulation has been characterised from different sources and used to develop a technology for bioremediation. Past efforts provided limited success and contradictory results. Therefore, functional characterisation of PCS gene from new sources into different target systems is considered as an important task in the area of bioremediation. Earlier, we isolated and functionally characterised PCS gene from an aquatic macrophyte Ceratophyllum demersum L., a metal accumulator aquatic plant. Expression of this gene, CdPCS1, in tobacco enhanced PC synthesis and metal accumulation of transgenic tobacco plants. In the present study, we have expressed CdPCS1 in more diverse systems, Escherichia coli and Arabidopsis, and studied growth and metal accumulation of transgenic organisms. The expression of CdPCS1 in E. coli offered tolerance against cadmium as well as higher accumulation accompanied with PCS1 activity. The expression of CdPCS1 in Arabidopsis showed a significant enhanced accumulation of heavy metal(loid)s in aerial parts without significant difference in growth parameters in comparison to wild-type Arabidopsis plants. Our study suggests that CdPCS1 can be utilised for enhancing bioremediation potential of different organisms using biotechnological approaches.     

Overexpression of glucosyltransferase UGT85A1 influences trans-zeatin homeostasis and trans-zeatin responses likely through O-glucosylation

Trans-zeatin is a kind of cytokinins that plays a crucial role in plant growth and development. The master trans-zeatin O-glucosyltransferase of Arabidopsis thaliana, UGT85A1, has been previously identified through biochemical approach. To determine the in planta role of UGT85A1 gene, the characterization of transgenic Arabidopsis plants overexpressing UGT85A1 was carried out. Under normal conditions, transgenic Arabidopsis did not display clearly altered phenotypes. A remarkable alteration is that the accumulation level of the trans-zeatin O-glucosides was significantly increased in UGT85A1 overexpressing transgenic Arabidopsis, while other forms of cytokinins kept the similar concentrations compared to the wild type. When treated with exogenously applied trans-zeatin, UGT85A1 overexpressing Arabidopsis showed much less sensitivity to trans-zeatin in primary root elongation and lateral root formation. Meanwhile, the chlorophyll content of detached leaves of transgenic Arabidopsis was much lower than wild type. Studies of spatial–temporal expression patterns showed that UGT85A1 was mainly expressed in the early seedlings and developing seeds. Analysis of subcellular localization suggested that UGT85A1 was localized to cytoplasm and nucleus. Taken together, our data suggest that overexpression of Arabidopsis glucosyltransferase UGT85A1 influences trans-zeatin homeostasis and trans-zeatin responses likely through O-glucosylation in planta.