Genome-wide analysis of terpene synthases in soybean: functional characterization of GmTPS3

Terpenes (terpenoids or isoprenoids) constitute a large class of plant natural products and play numerous functional roles in primary and secondary metabolism as well as inecological interactions. This study presents a genomic analysis of 23 putative soybean (Glycine max) terpene synthase genes (GmTPSs) distributed over 10 of 20 chromosomes. The GmTPSs are grouped into six types based on gene architecture and sequence identity. Sequence alignment indicates that most GmTPSs contain the conserved aspartate-rich DDX₂D motif, and two clades encoded by TPS-a and TPS-b contain variations of an arginine-rich RRX₈W motif. Quantitative real-time PCR analysis demonstrated that GmTPSs were predominantly expressed in reproductive organs. Heterologous expression followed by enzymatic assay suggested that GmTPS3 functions as a geraniol synthase. We also generated transgenic tobacco plants ectopically expressing GmTPS3. In dual-choice feeding-preference and force-feeding assays, the transgenic tobacco lines expressing GmTPS3 exhibited enhanced resistance to cotton leafworms and an increased level of geraniol. Taken together, these data provide a comprehensive understanding of the TPS family in soybeans and suggest a promising approach to engineering transgenic plants with enhanced insect resistance.     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta.     

Cotton PRP5 gene encoding a proline-rich protein is involved in fiber development

Proline-rich proteins contribute to cell wall structure of specific cell types and are involved in plant growth and development. In this study, a fiber-specific gene, GhPRP5, encoding a proline-rich protein was functionally characterized in cotton. GhPRP5 promoter directed GUS expression only in trichomes of both transgenic Arabidopsis and tobacco plants. The transgenic Arabidopsis plants with overexpressing GhPRP5 displayed reduced cell growth, resulting in smaller cell size and consequently plant dwarfs, in comparison with wild type plants. In contrast, knock-down of GhPRP5 expression by RNA interference in cotton enhanced fiber development. The fiber length of transgenic cotton plants was longer than that of wild type. In addition, some genes involved in fiber elongation and wall biosynthesis of cotton were up-regulated or down-regulated in the transgenic cotton plants owing to suppression of GhPRP5. Collectively, these data suggested that GhPRP5 protein as a negative regulator participates in modulating fiber development of cotton.     

Differential Expression of Endogenous Ferritin Genes and Iron Homeostasis Alteration in Transgenic Tobacco Overexpressing Soybean Ferritin Gene

For studying the effects of endogenous ferritin gene expressions (NtFer1, GenBank accession number ay083924; and NtFer2, GenBank accession number ay141105) on the iron homeostasis in transgenic tobacco (Nicotiana tabacum L.) plants expressing soybean (Glycine max Merr) ferritin gene (SoyFer1, GenBank accession number m64337), the transgenic tobacco has been produced by placing soybean ferritin cDNA cassette under the control of the CaMV 35S promoter. The exogenous gene expression was examined by both Northern- and Western-blot analyses. Comparison of endogenous ferritin gene expressions between nontransformant and transgenic tobacco plants showed that the expression of NtFer1 was increased in the leaves of transgenic tobacco plants, whereas the NtFer2 expression was unchanged. The iron concentration in the leaves of transgenic tobacco plants was about 1.5-folds higher than that in nontransformant. Enhanced growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weights significantly greater than those in the nontransformant. These results demonstrated that exogenous ferritin expression induced increased expression of at least one of the endogenous ferritin genes in transgenic tobacco plants by enhancing the ferric chelate reductase activity and iron transport ability of the root, and improved the rate of photosynthesis.     

GmHKT1;4, a novel soybean gene regulating Na+/K+ ratio in roots enhances salt tolerance in transgenic plants

Salt is an important factor affecting the growth and development of soybean in saline or alkaline soil. The aims of the present study were to identify and functionally analyse the soybean GmHKTs gene family, and to explore their roles under NaHCO₃ and NaCl stresses. The GmHKTs gene family were isolated from soybean using genome sequence information. The GmHKTs gene family were further analysed for the structure and phylogenetic relationship. The expression patterns of soybean GmHKTs genes under NaHCO₃ and NaCl stresses were analysed via quantitative real-time PCR. As a result, the expression level of GmHKT1;4 was extremely up regulated in root under each treatment. Overexpression of GmHKT1;4 significantly enhanced the tolerance of transgenic tobacco plants to NaHCO₃ and NaCl stresses, compared with null plants. The overexpressed transgenic plants of this gene accomulated more K⁺ and less Na⁺ under salt stress, compaired with null plants. Our findings suggest that GmHKT1;4 plays an important role for regulation Na⁺/K⁺ ratio in roots under alkaline (NaHCO₃) and saline (NaCl) stresses.     

Differential expression of limonene synthase gene affects production and composition of essential oils in leaf and floret of transgenic lavandin (Lavandula × intermedia Emeric ex Loisel.)

Transgenic lavandin (Lavandula × intermedia) expressing the limonene synthase gene (LIMS) cDNA of true lavender (L. angustifolia) driven by a constitutive 35S promoter was generated from leaf-derived callus inoculated with disarmed Agrobacterium tumefaciens. Only three LIMS regenerants could be acclimatized and their transgenes confirmed. Of them, LM-3, showed closed internodes and short stalks with small spikes, similar to the morphological characters of dwarfism. These results might be attributable to depleted geranyl diphosphate, the monoterpene precursor, which is required for the production of gibberellin. LIMS was differentially expressed in leaves and florets of the transgenic plant, which affected the production of several monoterpenes and therefore essential oil production. In vegetative leaves, overexpressed LIMS increased not only limonene but also total essential oil production, although no alteration in fragrance was observed. Conversely, suppression of LIMS expression in florets of the reproductive stage reduced their total essential oil production, including the dramatic decrease of limonene, linalool, and linalyl acetate. These results suggest that the constitutive promoter acts as a suppressor in tissues in which endogenous targeted gene expression is strong. Consequently, a slight change in fragrance at weak intensity in florets of the transgenic plants, producing a camphoraceous odor, was apparent when compared with non-transgenic plants, because the relative proportions of camphoraceous compounds such as 1,8-cineole, camphor, and borneol were increased in the transgenic plants. These results suggest that the suppression of terpene synthase gene expression is an effective way to alter fragrance, despite a dramatic reduction in total essential oil production.     

Overexpression of sense and antisense ced-9 in tobacco plants confers resistance to Meloidogyne incognita

Transgenic tobacco plants expressing the Caenorhabditis elegans programmed cell death gene ced-9, in both sense and antisense orientations, were produced using Agrobacterium tumefaciens-mediated transformation. The generated transgenic tobacco plants were tested for resistance to the root-knot nematode Meloidogyne incognita by measuring gall formation, size of galls generated, and the ability of juvenile-2 (J2) to hatch. Results showed that expression of ced-9 gene in either sense (ced-9F) or antisense (ced-9R) orientation in hemizygous transgenic tobacco plants induced prevention of M. incognita proliferation (as measured by gall number reduction) and J2 hatching. Furthermore, the results also showed that ced-9R in homozygous transgenic tobacco plants prevented J2 hatching, whereas ced-9F homozygous transgenic tobacco plants lost nematicidal function. Although our study demonstrates that expression of either ced-9R or ced-9F genes in tobacco plants significantly reduces infection by M. incognita, further investigation is required to understand the specific mechanisms involved for this control. It is possible that the nematode resistance seen with both sense (ced-9F) and antisense (ced-9R) sequences is the result of two independent mechanisms, one acting on invading nematodes and the other acting during embryogenesis of M. incognita, ultimately resulting in plant protection.     

Heat-inducible hygromycin resistance in transgenic tobacco

We have constructed a chimaeric gene consisting of the promoter of the soybean heat shock (hs) gene Gmhsp17,6-L, the coding region of a hygromycin phosphotransferase (hpt) gene, and the termination sequence of the nopaline synthase (nos) gene. This gene fusion was introduced into tobacco by Agrobacterium-mediated gene transfer. Heat-inducible synthesis of mRNA was shown by northern hybridization, and translation of this RNA into a functional protein was indicated by plant growth on hygromycin-containing media in a temperature-dependent fashion. One hour incubation at 40 °C per day, applied for several weeks, was sufficient to express the resistant phenotype in transgenic plants containing the chimaeric hs-hpt gene. These data suggest that the hygromycin resistance gene is functional and faithfully controlled by the soybean hs promoter. The suitability of these transgenic plants for selection of mutations that alter the hs response is discussed.     

Ectopically expressed leaf and bulb lectins from garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) protect transgenic tobacco plants against cotton leafworm (<Emphasis Type="Italic">Spodoptera littoralis</Emphasis>)

The insecticidal activity of the leaf (ASAL) and bulb (ASAII) agglutinins from Allium sativum L. (garlic) against the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied using transgenic tobacco plants expressing the lectins under the control of the constitutive CaMV35S promoter. PCR analysis confirmed that the garlic lectin genes were integrated into the plant genome. Western blots and semi-quantitative agglutination assays revealed lectin expression at various levels in the transgenic lines. Biochemical analyses indicated that the recombinant ASAL and ASAII are indistinguishable from the native garlic lectins. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed ASAL and ASAII significantly (P < 0.05) reduced the weight gain of 4th instar larvae of S. littoralis. Further on, the lectins retarded the development of the larvae and their metamorphosis, and were detrimental to the pupal stage resulting in weight reduction and lethal abnormalities. Total mortality was scored with ASAL compared to 60% mortality with ASAII. These findings suggest that garlic lectins are suitable candidate insect resistance proteins for the control of S. littoralis through a transgenic approach.     

The Sesquiterpenes(E)-?-Farnesene and (E)-α-Bergamotene Quench Ozone but Fail to Protect the Wild Tobacco Nicotiana attenuata from Ozone,UVB, and Drought Stresses

Among the terpenes, isoprene (C₅) and monoterpene hydrocarbons (C₁₀) have been shown to ameliorate abiotic stress in a number of plant species via two proposed mechanisms: membrane stabilization and direct antioxidant effects. Sesquiterpene hydrocarbons (C₁₅) not only share the structural properties thought to lend protective qualities to isoprene and monoterpene hydrocarbons, but also react rapidly with ozone, suggesting that sesquiterpenes may similarly enhance tolerance of abiotic stresses. To test whether sesquiterpenes protect plants against ozone, UVB light, or drought, we used transgenic lines of the wild tobacco Nicotiana attenuata. The transgenic plants expressed a maize terpene synthase gene (ZmTPS10) which produced a blend of (E)-ß-farnesene and (E)-α-bergamotene, or a point mutant of the same gene (ZmTPS10M) which produced (E)-ß-farnesene alone,. (E)-ß-farnesene exerted a local, external, and transient ozone-quenching effect in ozone-fumigated chambers, but we found no evidence that enhanced sesquiterpene production by the plant inhibited oxidative damage, or maintained photosynthetic function or plant fitness under acute or chronic stress. Although the sesquiterpenes (E)-ß-farnesene and (E)-α-bergamotene might confer benefits under intermittent heat stress, which was not tested, any roles in relieving abiotic stress may be secondary to their previously demonstrated functions in biotic interactions.     

Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene

Cotton leaf curl virus (CLCuV) is a serious pathogen causing leaf curl disease and affecting the cotton production in major growing areas. The transgenic cotton (Gossypium hirsutum cv. Coker 310) plants were developed by using βC1 gene in antisense orientation gene driven by Cauliflower mosaic virus-35S promoter and nos (nopaline synthase) terminator and mediated by Agrobacterium tumefaciens transformation and somatic embryogenesis system. Molecular confirmation of the transformants was carried out by polymerase chain reaction (PCR) and Southern blot hybridization. The developed transgenic and inoculated plants remained symptomless till their growth period. In conclusion, the plants were observed as resistant to CLCuV.