Constitutive Cauliflower Mosaic Virus 35S Research Articles

Xylem-specific Overexpression of the GIBBERELLIN ACID 20 OXIDASE Gene (GA20-OXIDASE) from Pine in Hybrid Poplar (Populus tremula L. × P. alba L.) Revealed Reliable Increase in Growth and Biomass Production Just in a Single-copy-line

Overexpression of the GA20-OXIDASE gene under control of the constitutive cauliflower mosaic virus 35S promoter in poplar leads to increased shoot growth and biomass production, however, the trees suffer from unstable shoots and poor root growth. Transgenic hybrid poplar (Populus tremula L. × P. alba L.) plants overexpressing the GA20-OXIDASE gene from pine under control of a poplar-xylem-specific DX15-promoter also revealed a superior effect on growth and biomass production but without changing the overall phenotype. We tested seven DX15::GA20-OXIDASE-transgenic lines for growth and biomass production in the glasshouse in 2017, and repeated the experiment in 2018 with the “best-four” lines. Plants from one DX15::GA20-OXIDASE-transgenic line, N457‑4, turned out to be the tallest, with highest biomass, in both years under investigation. In contrast to the other lines tested in 2017 and 2018 carrying two or more copies of the transgene, N457‑4 carries only one copy. We suggest that transcriptional or post-transcriptional transgene silencing in the two- or more copies carrying lines might be responsible for lower GA20-OXIDASE transgene expression and that the single-copy-line N457‑4 has stable GA20-OXIDASE-gene expression.

Characterization of the bark storage protein gene (JcBSP) family in the perennial woody plant Jatropha curcas and the function of JcBSP1 in Arabidopsis thaliana.

BackgroundBark storage protein (BSP) plays an important role in seasonal nitrogen cycling in perennial deciduous trees. However, there is no report on the function of BSP in the perennial woody oil plant Jatropha curcas.MethodsIn this study, we identified six members of JcBSP gene family in J. curcas genome. The patterns, seasonal changes, and responses to nitrogen treatment in gene expression of JcBSPs were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Overexpression of JcBSP1 in transgenic Arabidopsis thaliana was driven by a constitutive cauliflower mosaic virus (CaMV) 35S RNA promoter.ResultsJcBSP members were found to be expressed in various tissues, except seeds. The seasonal changes in the total protein concentration and JcBSP1 expression in the stems of J. curcas were positively correlated, as both increased in autumn and winter and decreased in spring and summer. In addition, the JcBSP1 expression in J. curcas seedlings treated with different concentrations of an NH4NO3 solution was positively correlated with the NH4NO3 concentration and application duration. Furthermore, JcBSP1 overexpression in Arabidopsis resulted in a phenotype of enlarged rosette leaves, flowers, and seeds, and significantly increased the seed weight and yield in transgenic plants.

Expression of the Trichoderma reesei expansin-like protein, swollenin, in poplar results in biomass with improved sugar release by enzymatic hydrolysis

Ethanol production from lignocellulosic biomass has been considered an alternative to corn-based ethanol and fossil fuel. However, the secondary cell wall structure prevents efficient degradation of cellulose. Expression of cell wall degrading enzymes has been suggested as one solution to generate suitable feedstocks for lignocellulosic ethanol production. Swollenin (SWO), originating from Trichoderma reesei, disrupts cellulose structure with low-level hydrolytic activity to the glucan chain. Previously, characterization of T. reesei SWO has been carried out in multiple microorganisms and tobacco. This study heterologously overexpressed T. reesei SWO in hybrid poplar (Populus tremula × alba), a promising feedstock for biofuel production, under the control of either the constitutive Cauliflower Mosaic Virus 35S (CaMV 35S) promoter or the putative vascular tissue specific Subterranean Clover Stunt Virus S7 (SCSV) promoter. Transgenic poplar lines with high or moderate SWO expression level showed abnormal growth and reduced biomass production, along with increased glucose and reduced lignin contents. Increased cellulose crystallinity was observed in two transgenic events. This study also investigated the effect of in planta SWO expression on cellulose saccharification efficiency and found that transgenic events improved glucan conversion rates compared to wild type poplar. The results suggest SWO-overexpressing transgenic poplar lines could be a potential feedstock for ethanol production.

Increased Production of α-Linolenic Acid in Soybean Seeds by Overexpression of Lesquerella FAD3-1.

Soybean is a major crop that is used as a source of vegetable oil for human use. To develop transgenic soybean with high α-linolenic acid (ALA; 18:3) content, the FAD3-1 gene isolated from lesquerella (Physaria fendleri) was used to construct vectors with two different seed-specific promoters, soybean β-conglycinin (Pβ-con) and kidney bean phaseolin (Pphas), and one constitutive cauliflower mosaic virus 35S promoter (P35S). The corresponding vectors were used for Agrobacterium-mediated transformation of imbibed mature half seeds. The transformation efficiency was approximately 2%, 1%, and 3% and 21, 7, and 17 transgenic plants were produced, respectively. T-DNA insertion and expression of the transgene were confirmed from most of the transgenic plants by polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), reverse transcription PCR (RT-PCR), and Southern blot analysis. The fatty acid composition of soybean seeds was analyzed by gas chromatography. The 18:3 content in the transgenic generation T1 seeds was increased 7-fold in Pβ-con:PfFAD3-1, 4-fold in Pphas : PfFAD3-1, and 1.6-fold in P35S:PfFAD3-1 compared to the 18:3 content in soybean “Kwangankong”. The increased content of 18:3 in the Pβ-con:PfFAD3-1 soybean (T1) resulted in a 52.6% increase in total fatty acids, with a larger decrease in 18:1 content than 18:2 content. The increase in 18:3 content was also maintained and reached 42% in the Pphas : PfFAD3-1 transgenic generation T2. Investigations of the agronomic traits of 12 Pβ-con:PfFAD3-1 transgenic lines (T1) revealed that plant height, number of branches, nodes, pods, total seeds, and total seed weight were significantly higher in several transgenic lines than those in non-transgenic soybean. Especially, an increase in seed size was observed upon expression of the PfFAD3-1 gene with the β-conglycinin promoter, and 6%–14% higher seed lengths were measured from the transgenic lines.

Transgenic callus of Nicotiana glauca stably expressing a fungal laccase maintains its growth in presence of organic contaminants

Some plant species colonize mining-contaminated soils. They are adapted to harsh growth conditions and show high native phytoremediation abilities. For environmental cleanup, these species can be improved by genetic manipulation. Laccases are ligninolytic enzymes that oxidise a broad range of substrates by a radical-catalysed reaction mechanism using oxygen as the electron acceptor. Fungi of the genus Pycnoporus produce laccases with important biotechnological, industrial and environmental applications. Here we describe a successful attempt of Agrobacterium rhizogenes mediated stable transformation of Nicotiana glauca, a species naturally resistant to metal-contaminated soils. The coding region of a Pycnoporus sanguineus laccase gene was fused with the Nicotiana plumbaginifolia calreticulin apoplast targeting signal and driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter (2X35S). The obtained transgenic N. glauca callus cells secreted a recombinant fungal laccase into the growth medium and were capable to degrade an anthraquinone dye, Remazol Brilliant Blue R. Under phenol and cadmium stresses, the transgenic calli not only maintained their growth capacity but their relative growth rate was greater compared to callus transformed with empty vector. This work shows that stable expression of a fungal laccase in a plant species resistant to heavy metals presents a successful strategy that potentially can be used to combat combined organic and inorganic contamination. A fungal laccase was heterologously expressed in a heavy metal-tolerant plant species. The transgenic callus was capable of growth in medium supplemented with organic and inorganic contaminants.

The Potassium Transporter SlHAK10 Is Involved in Mycorrhizal Potassium Uptake.

Most terrestrial plants form a root symbiosis with arbuscular mycorrhizal (AM) fungi, which receive fixed carbon from the plant and enhance the plant's uptake of mineral nutrients. AM symbiosis improves the phosphorous and nitrogen nutrition of host plants; however, little is known about the role of AM symbiosis in potassium (K+) nutrition. Here, we report that inoculation with the AM fungus Rhizophagus irregularis improved tomato (Solanum lycopersicum) plant growth and K+ acquisition and that K+ deficiency has a negative effect on root growth and AM colonization. Based on its homology to a Lotus japonicus AM-induced K+ transporter, we identified a mycorrhiza-specific tomato K+ transporter, SlHAK10 (Solanum lycopersicum High-affinity Potassium Transporter10), that was exclusively expressed in arbuscule-containing cells. SlHAK10 could restore a yeast K+ uptake-defective mutant in the low-affinity concentration range. Loss of function of SlHAK10 led to a significant decrease in mycorrhizal K+ uptake and AM colonization rate under low-K+ conditions but did not affect arbuscule development. Overexpressing SlHAK10 from the constitutive cauliflower mosaic virus 35S promoter or the AM-specific Solanum melongena Phosphate Transporter4 not only improved plant growth and K+ uptake but also increased AM colonization efficiency and soluble sugar content in roots supplied with low K+ Our results indicate that tomato plants have a SlHAK10-mediated mycorrhizal K+ uptake pathway and that improved plant K+ nutrition could increase carbohydrate accumulation in roots, which facilitates AM fungal colonization.

Functional analysis of Arabidopsis and maize transgenic lines overexpressing the ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7.

The conserved poly(ADP-ribosyl)ation (PAR) pathway consists of three genetic components that are potential targets to modulate the plant's energy homeostasis upon stress with the aim to improve yield stability in crops and help secure food supply. We studied the role of the PAR pathway component ADP-ribose/NADH pyrophosphohydrolase (AtNUDX7) in yield and mild drought stress by using a transgenic approach in Arabidopsis thaliana and maize (Zea mays). Arabidopsis AtNUDX7 cDNA was overexpressed in Arabidopsis and maize by means of the constitutive Cauliflower Mosaic Virus 35S promoter and the strong constitutive Brachypodium distachyon pBdEF1α promoter, respectively. Overexpression of AtNUDX7 in Arabidopsis improved seed parameters that were measured by a novel, automated method, accelerated flowering and reduced inflorescence height. This combination of beneficial traits suggested that AtNUDX7 overexpression in Arabidopsis might enhance the ADP-ribose recycling step and maintain energy levels by supplying an ATP source in the poly(ADP-ribosyl)ation energy homeostasis pathway. Arabidopsis and maize lines with high, medium and low overexpression levels of the AtNUDX7 gene were analysed in automated platforms and the inhibition of several growth parameters was determined under mild drought stress conditions. The data showed that the constitutive overexpression of the Arabidopsis AtNUDX7 gene in Arabidopsis and maize at varying levels did not improve tolerance to mild drought stress, but knocking down AtNUDX7 expression did, however at the expense of general growth under normal conditions.

RNAi-mediated silencing of endogenous Vlnv gene confers stable reduction of cold-induced sweetening in potato (Solanum tuberosum L. cv. Désirée)

Potato tubers must be cold-stored to extend their shelf life and maintain an uninterrupted supply chain for food processors. However, a side-effect of low-temperature storage is manifested in terms of cold-induced sweetening (CIS) of potato tubers, which reduces the processing quality and the commercial value of the end-products. RNA interference (RNAi) technology, whereby transgene-derived small interfering RNAs can trigger the homology-based knockdown of cognate host genes and can initiate gene silencing, has been successfully applied in crop improvement through targeted gene knockout in host plants. In the current study, transgenic potato plants (Solanum tuberosum cv. Desiree) were generated, expressing a 300 bp hairpin loop nucleotide sequence targeting the potato vacuolar invertase gene (VInv), under the constitutive Cauliflower mosaic virus 35S promoter. Tubers collected from transgenic lines showed a significant reduction in reducing sugar content after 180 days of cold storage, without showing any measurable off-target effects on plant morphology and tuberization compared to non-transformed control plants. The cold-stored tubers were further assayed for chip color, which showed a fairly light colored quality in the samples originating from RNAi lines. Together with similar effects seen in previously published experiments involving other potato varieties, the Desiree results described here establish the efficacy of using RNAi for the successful reduction of CIS in potato tubers.

Production of Recombinant Human Interleukin-11 (IL-11) in Transgenic Tobacco (Nicotiana tabacum) Plants

Interleukin-11 (IL-11) is a cytokine that plays a key regulatory role in the immune system. Recombinant human IL-11 (rhIL-11) exerts a preventative effect against apoptotic cell death and inhibits preadipocyte differentiation. IL-11 also is used to stimulate the bone marrow to produce platelets in order to prevent low platelets that may be caused by chemotherapy. Unfortunately, the high production cost of IL-11 associated. In this study, we investigated the feasibility of transgenic plants for the cost-effective production of rhIL-11. Production of rhIL-11 proteins in whole-plant expression system will be more economical when compared to the current E. coli based expression system. The human rhIL-11 gene was codon optimized to maximize plant host system expression. IL-11 expression vector under the control of a constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into tobacco by Agrobacteriummediated transformation. The 5′-leader sequence (called O) of tobacco mosaic virus (TMV) as a translational enhancer was added to construct. Transgenic tobacco plants expressing various levels of rhIL-11 protein were generated. Western blotting of the stably transformed lines demonstrated accumulation of the appropriately sized rhIL-11 protein in leaves. This research demonstrated the efficacy of using tobacco as an expression system for the production of rhIL-11. © Korean Society for Plant Biotechnology.

115 - Plant-Based Production, Purification, and Characterization of Oxidation-Resistant Alpha-1 Antitrypsin

Alpha-1 antitrypsin (AAT) is an acute phase protease inhibitor. Currently, the only available treatment for patients with COPD caused by AAT deficiency is replacement therapy, requiring weekly infusions of human plasma-derived AAT. While AAT has shown potential when inhaled to treat both emphysema and cystic fibrosis, the oxidation susceptibility of methionine residues at positions 351 and 358 in the reactive site loop have led to difficulties in maintaining AAT activity and stability when aerosolized. Using a novel cucumber mosaic virus-based inducible transient expression system, we have produced a biobetter plant recombinant AAT (prAAT) in Nicotiana benthamiana . This variant replaces the oxidation-susceptible methionine residue at position 358 with a valine residue to increase resistance to oxidation. N. benthamiana plants were vacuum infiltrated as detached leaves with two strains of Agrobacterium tumefaciens : one carrying the biobetter AAT gene and one carrying a gene for the tomato bushy stunt virus p19 viral RNA silencing suppressor driven by a constitutive cauliflower mosaic virus 35S promoter. Leaves were incubated in humidity chambers at 20°C in the dark for 6 days before flash freezing in liquid nitrogen, grinding, and protein extraction in a 20 mM Tris, 150 mM NaCl, and 0.01% (v/v) Tween-80 AAT stability buffer. Purification was achieved using Alpha-1 Antitrypsin Select Affinity Chromatography (GE Healthcare) with a 58.1±8.1% yield of active AAT. Activity of purified AAT was confirmed via a band shift in a Western blot after incubation with excess porcine pancreatic elastase, however, degradation of prAAT was also observed in all purified extracts. Purified prAAT was tested against an analytical standard (Calbiochem) and Prolastin-C (Griffols), a common therapeutic formulation of human AAT, to determine its activity and oxidation resistance. 41±11% of purified prAAT and 71.9±5.5% of Prolastin-C showed activity against porcine pancreatic elastase as determined by a residual elastase activity inhibitory assay. Under oxidation with 48.9 mM H 2 O 2 for 60 minutes at room temperature, prAAT was found to retain 102.8±20.4% of its original anti-elastase activity, while Prolastin-C retained 13.8±5.0% anti-elastase activity and analytical standard AAT retained 11.6±7.5% anti-elastase activity. Future work will include determinations of oxidation resistance to OCl – /HOCl and development of more optimal isolation and purification processes.

Integration of a hairpin RNA-encoding gene derived from Tobacco streak virus confers resistance in groundnut (Arachis hypogaea L.) against peanut stem necrosis disease

The feasibility of controlling peanut stem necrosis disease caused by Tobacco streak virus (TSV) in groundnut (Arachis hypogaea L.) was explored by expressing double-stranded RNA of the replicase (Rep) gene of TSV in groundnut through genetic engineering. A hairpin (hp) RNAi construct containing 535-bp sense and antisense TSV-Rep sequences flanking a 742-bp spacer sequence (Pdk intron) under the control of the constitutive Cauliflower mosaic virus 35S promoter was made in the binary vector pART27. This chimeric gene construct was then mobilized into Agrobacterium tumefaciens strain LBA4404 via triparental mating using pRK2013 as a helper. Cotyledon explants of groundnut cultivar TMV-7 were transformed with A. tumefaciens harboring the hpRNA cassette. The presence of the transgene in the transgenic plants was confirmed up to T3 generation by PCR amplification of the 535-bp fragment of TSV-Rep gene. The bioassay results indicated that necrotic lesions were observed on the leaves of the wild-type plants 7–9 days after inoculation with TSV and stem necrosis appeared 16–20 days after inoculation, whereas the transgenic plants did not develop symptoms until harvest. ELISA results indicated that the wild-type plants inoculated with TSV recorded the highest virus concentration as compared to the transgenic lines.

Genetic complementation analysis of rice sucrose transporter genes in Arabidopsis SUC2 mutant atsuc2

Sucrose transporters (SUTs) play a critical role on the phloem plasma membrane in loading sucrose into the phloem of source leaves for long-distance transport to sink organs. Rice has a small gene family of five SUTs, Oryza sativa SUT1 (OsSUT1) to OsSUT5. To identify rice SUTs that function as phloem loaders, we adopted a growth restoration assay of the severe growth retardation phenotype of atsuc2, a mutant of the best-characterized Arabidopsis phloem loader AtSUC2, by introducing OsSUTs. The rice SUT genes were expressed by two different promoters, the native phloem-specific promoter of AtSUC2 (pAtSUC2) and the constitutive Cauliflower Mosaic Virus 35S (pCaMV35S) promoter. Of all the transgenic atsuc2 plants, only pAtSUC2: OsSUT1 complemented the atsuc2 mutant phenotype in a comparable manner to wild type (WT), and consistent levels of soluble sugars and starch were recovered compared to those of WT. This suggests that OsSUT1 is a functional ortholog of the Arabidopsis AtSUC2 and functions as an apoplastic phloem loader. In addition, ossut1 mutants were produced via anther culture and their primary carbohydrate levels and growth phenotypes were indistinguishable from those of WT. This suggests that the rice phloem loader OsSUT1 function may not be essential for rice vegetative growth under normal conditions.

Expression of cadR Enhances its Specific Activity for Cd Detoxification and Accumulation in Arabidopsis.

Cadmium (Cd) is a transition metal that is highly toxic in biological systems. Anthropogenic emissions of Cd have increased biogeochemical cycling and the amount of Cd in the biosphere. Here we studied the utility of a bacterial Cd-binding protein, CadR, for the remediation of Cd contamination. CadR was successfully targeted to chloroplasts using a constitutive Cauliflower mosaic virus (CaMV) 35S promoter or a shoot-specific Chl a/b-binding protein 2 gene (CAB2) promoter and an RbcS (small subunit of the Rubisco complex) transit peptide. Under short-term (2 d) exposure to Cd, the cadR transgenic plants showed up to a 2.9-fold Cd accumulation in roots compared with untransformed plants. Under medium term (7 d) exposure to Cd, the concentrations of Cd in leaves began to increase but there were no differences between the wild type and the cadR transgenic plants. Under long-term (16 d) exposure to Cd, the cadR transgenic plants accumulated greater amounts of Cd in leaves than the untransformed plants. Total Cd accumulation (µg per plant) in shoots and roots of the plants expressing cadR were significantly higher (up to 3.5-fold in shoots and 5.2-fold in roots) than those of the untransformed plants. We also found that targeting CadR to chloroplasts facilitated chloroplastic metal homeostasis and Chl b accumulation. Our results demonstrate that manipulating chelating capacity in chloroplasts or in the cytoplasm may be effective in modifying both the accumulation of and resistance to Cd.

Transgenic Arabidopsis thaliana plants expressing a β-1,3-glucanase from sweet sorghum (Sorghum bicolor L.) show reduced callose deposition and increased tolerance to aluminium toxicity.

Seventy-one cultivars of sweet sorghum (Sorghum bicolor L.) were screened for aluminium (Al) tolerance by measuring relative root growth (RRG). Two contrasting cultivars, ROMA (Al tolerant) and POTCHETSTRM (Al sensitive), were selected to study shorter term responses to Al stress. POTCHETSTRM had higher callose synthase activity, lower β-1,3-glucanase activity and more callose deposition in the root apices during Al treatment compared with ROMA. We monitored the expression of 12 genes involved in callose synthesis and degradation and found that one of these, SbGlu1 (Sb03g045630.1), which encodes a β-1,3-glucanase enzyme, best explained the contrasting deposition of callose in ROMA and POTCHETSTRM during Al treatment. Full-length cDNAs of SbGlu1 was prepared from ROMA and POTCHETSTRM and expressed in Arabidopsis thaliana using the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Independent transgenic lines displayed significantly greater Al tolerance than wild-type plants and vector-only controls. This phenotype was associated with greater total β-1,3-glucanase activity, less Al accumulation and reduced callose deposition in the roots. These results suggest that callose production is not just an early indicator of Al stress in plants but likely to be part of the toxicity pathway that leads to the inhibition of root growth.

Overexpression of the rice phosphate transporter gene OsPT6 enhances tolerance to low phosphorus stress in vegetable soybean

Low phosphorus (P) availability is a major constraint in the growth and production of vegetable soybean. To improve P efficiency in vegetable soybean, a rice (Oryza sativa L.) phosphate transporter gene, OsPT6, was transferred into the vegetable soybean cultivar ‘NY-1001’ using an Agrobacterium-mediated cotyledonary-node transformation system. OsPT6 was driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter in the binary vector pCAMBIA3301-OsPT6. Two independent fertile T0 transgenic plants (12PT6-1 and 12PT6-2) were obtained and confirmed by PCR and Southern blotting analyses. The overexpression of the OsPT6 gene of the T2 transgenic plants was detected by quantitative RT-PCR analysis. The tolerance of the T2 transgenic lines overexpressing OsPT6 to low concentrations of phosphate (low-Pi; 10μMPi) was evaluated by hydroponic culture using a modified Hoagland's nutrient solution. The P contents in leaves, stems, and roots of the transgenic plants were significantly higher than those of the non-transformed (NT) plants under the concentrations of both low- and normal-Pi of standard Hoagland's nutrient solution. Under low-Pi stress, the transgenic plants grew better and exhibited significant increases in plant height, root length, root weight, number of pods and seeds, and seed weight per plant compared with the NT plants. Our data indicate that the overexpression of OsPT6 in transgenic vegetable soybean lines improves P accumulation and growth performance and thereby represents a promising approach for the improvement of P efficiency in vegetable soybean.

Inducible and constitutive expression of an elicitor gene Hrip1 from Alternaria tenuissima enhances stress tolerance in Arabidopsis.

Hrip1 is a novel hypersensitive response-inducing protein secreted by Alternaria tenuissima that activates defense responses and systemic acquired resistance in tobacco. This study investigates the role that Hrip1 plays in responses to abiotic and biotic stress using transgenic Arabidopsis thaliana expressing the Hrip1 gene under the control of the stress-inducible rd29A promoter or constitutive cauliflower mosaic virus 35S promoter. Bioassays showed that inducible Hrip1 expression in rd29A∷Hrip1 transgenic lines had a significantly higher effect on plant height, silique length, plant dry weight, seed germination and root length under salt and drought stress compared to expression in 35S∷Hrip1 lines and wild type plants. The level of enhancement of resistance to Botrytis cinerea by the 35S∷Hrip1 lines was higher than in the rd29A∷Hrip1 lines. Moreover, stress-related gene expression in the transgenic Arabidopsis lines was significantly increased by 200mM NaCl and 200mM mannitol treatments, and defense genes in the jasmonic acid and ethylene signaling pathway were significantly up-regulated after Botrytis inoculation in the Hrip1 transgenic plants. Furthermore, the activity of some antioxidant enzymes, such as peroxidase and catalase increased after salt and drought stress and Botrytis infection. These results suggested that the Hrip1 protein contributes to abiotic and biotic resistance in transgenic Arabidopsis and may be used as a useful gene for resistance breeding in crops. Although the constitutive expression of Hrip1 is suitable for biotic resistance, inducible Hrip1 expression is more responsive for abiotic resistance.

Isolation and functional characterization of JcFT, a FLOWERING LOCUS T (FT) homologous gene from the biofuel plant Jatropha curcas.

BackgroundPhysic nut (Jatropha curcas L.) is a potential feedstock for biofuel production because Jatropha oil is highly suitable for the production of the biodiesel and bio-jet fuels. However, Jatropha exhibits low seed yield as a result of unreliable and poor flowering. FLOWERING LOCUS T (FT) –like genes are important flowering regulators in higher plants. To date, the flowering genes in Jatropha have not yet been identified or characterized.ResultsTo better understand the genetic control of flowering in Jatropha, an FT homolog was isolated from Jatropha and designated as JcFT. Sequence analysis and phylogenetic relationship of JcFT revealed a high sequence similarity with the FT genes of Litchi chinensis, Populus nigra and other perennial plants. JcFT was expressed in all tissues of adult plants except young leaves, with the highest expression level in female flowers. Overexpression of JcFT in Arabidopsis and Jatropha using the constitutive promoter cauliflower mosaic virus 35S or the phloem-specific promoter Arabidopsis SUCROSE TRANSPORTER 2 promoter resulted in an extremely early flowering phenotype. Furthermore, several flowering genes downstream of JcFT were up-regulated in the JcFT-overexpression transgenic plant lines.ConclusionsJcFT may encode a florigen that acts as a key regulator in flowering pathway. This study is the first to functionally characterize a flowering gene, namely, JcFT, in the biofuel plant Jatropha.

Sucrose transporter NtSUT1 confers aluminum tolerance on cultured cells of tobacco (Nicotiana tabacum L.)

The role of plasma membrane-localized sucrose transporter (NtSUT1) was investigated using cultured tobacco cell (Nicotiana tabacum L.) line BY-2. The wild type (WT) cells were first transformed with the NtSUT1 gene or its fragments cloned from tobacco cell line SL to form the over-expression (OX) and suppression (RNAi) cell lines, respectively. Using OX and RNAi transgenics, the role of NtSUT1 in growth capacity of actively growing cells and in aluminum (Al)-treated cells was examined. During the logarithmic phase of growth in nutrient medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), both the rate of sucrose uptake measured with radio-tracer and the content of soluble sugars were higher in OX and lower in RNAi cell lines compared to WT. Overall, the content of soluble sugars negatively correlated with the time necessary for doubling mass (fresh weight). When cells were treated without (control) or with Al in a simple medium containing calcium, sucrose and 2-(N-morpholino)ethanesulfonic acid (MES; pH 5.0) for up to 18 h, the expression of NtSUT1 under its native promoter, or under the control of strong constitutive cauliflower mosaic virus (CaMV) 35S promoter, was strongly dependent on the presence of 2,4-D. Thereafter, the cells were preferentially treated in the presence of 2,4-D. During 6 h after a start of the control treatment, sucrose uptake rates were, compared to WT, slightly higher and lower in OX and RNAi lines respectively. The addition of Al reduced the sucrose uptake rates of OX and WT to the level of RNAi line, indicating that Al inhibits sucrose uptake via NtSUT1. During the post-Al culture of control and Al-treated cells in a nutrient medium, sucrose uptake rates were much higher in OX compared to WT and RNAi lines, which closely and positively correlated with the growth capacity of the cells. Judging from the growth capacity of Al-treated cells relative to that of control cells, OX cells were more tolerant to Al than WT and RNAi. In summary, we conclude that over-expression of NtSUT1 confers higher growth capacity in actively growing cells as well as in Al-treated cells.

OsSUV3 dual helicase functions in salinity stress tolerance by maintaining photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. IR64)

To overcome the salinity-induced loss of crop yield, a salinity-tolerant trait is required. The SUV3 helicase is involved in the regulation of RNA surveillance and turnover in mitochondria, but the helicase activity of plant SUV3 and its role in abiotic stress tolerance have not been reported so far. Here we report that the Oryza sativa (rice) SUV3 protein exhibits DNA and RNA helicase, and ATPase activities. Furthermore, we report that SUV3 is induced in rice seedlings in response to high levels of salt. Its expression, driven by a constitutive cauliflower mosaic virus 35S promoter in IR64 transgenic rice plants, confers salinity tolerance. The T1 and T2 sense transgenic lines showed tolerance to high salinity and fully matured without any loss in yields. The T2 transgenic lines also showed tolerance to drought stress. These results suggest that the introduced trait is functional and stable in transgenic rice plants. The rice SUV3 sense transgenic lines showed lesser lipid peroxidation, electrolyte leakage and H2 O2 production, along with higher activities of antioxidant enzymes under salinity stress, as compared with wild type, vector control and antisense transgenic lines. These results suggest the existence of an efficient antioxidant defence system to cope with salinity-induced oxidative damage. Overall, this study reports that plant SUV3 exhibits DNA and RNA helicase and ATPase activities, and provides direct evidence of its function in imparting salinity stress tolerance without yield loss. The possible mechanism could be that OsSUV3 helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in transgenic rice.

Fast-Forward Genetics Identifies Plant CPL Phosphatases as Regulators of miRNA Processing Factor HYL1

MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE). We have combined a luciferase-based genetic screen with whole-genome sequencing for rapid identification of new regulators of miRNA biogenesis and action. Among the first six mutants analyzed were three alleles of C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1)/FIERY2 (FRY2). In the miRNA processing complex, SE functions as a scaffold to mediate CPL1 interaction with HYL1, which needs to be dephosphorylated for optimal activity. In the absence of CPL1, HYL1 dephosphorylation and hence accurate processing and strand selection from miRNA duplexes are compromised. Our findings thus define a new regulatory step in plant miRNA biogenesis.