Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

A soybean plastidic ATP/ADP transporter gene, <Emphasis Type="Italic">GmAATP</Emphasis>, is involved in carbohydrate metabolism in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic ATP/ADP transporter (AATP) imports adenosine triphosphate (ATP) from the cytosol into plastids, resulting in the increase of the ATP supply to facilitate anabolic synthesis in heterotrophic plastids of dicotyledonous plants. The regulatory role of GmAATP from soybean in increasing starch accumulation has not been investigated. In this study, a gene encoding the AATP protein, named GmAATP, was successfully isolated from soybean. Transient expression of GmAATP in Arabidopsis protoplasts and Nicotiana benthamiana leaf epidermal cells revealed the plastidic localization of GmAATP. Its expression was induced by exogenous sucrose treatment in soybean. The coding region of GmAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of GmAATP significantly increased the sucrose and starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III, and AtSSS IV), and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS, and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild type (WT). These findings suggest that GmAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis. All these results suggest that GmAATP could be used as a candidate gene for developing high starch-accumulating plants as alternative energy crops.     

Overexpression of <Emphasis Type="Italic">MeDREB1D</Emphasis> confers tolerance to both drought and cold stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cassava (Manihot esculenta) is an important tropical crop with extraordinary tolerance to drought stress but few reports on it. In this study, MeDREB1D was significantly and positively induced by drought stress. Two allelic variants of the gene named MeDREB1D(R-2) and MeDREB1D(Y-3) were identified. Overexpressing MeDREB1D(R-2) and MeDREB1D(Y-3) in Arabidopsis resulted in stronger tolerance to drought and cold stresses. Under drought stress, transgenic plants had more biomass, higher survival rates and less MDA content than wild-type plants. Under cold stress, transgenic plants also had higher survival rates than wild-type plants. To further characterize the molecular function of MeDREB1D, we conducted an RNA-Seq analysis of transgenic and wild-type Arabidopsis plants. The results showed that the Arabidopsis plants overexpressing MeDREB1D led to changes in downstream genes. Several POD genes, which may play a vital role in drought and cold tolerance, were up-regulated in transgenic plants. In brief, these results suggest that MeDREB1D can simultaneously improve plant tolerance to drought and cold stresses.     

Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, <Emphasis Type="Italic">AtSRK2C</Emphasis> and <Emphasis Type="Italic">AtGolS2</Emphasis>

Recent environmental issues have increased the demand for woody biomass as a renewable resource for industry and energy. For a stable supply of woody biomass, it is critical to decrease the effects of abiotic stresses, such as drought and salinity, which hinder plant growth. For the goal to develop practical stress-tolerant trees, we generated transgenic poplar plants (P. tremula × tremuloides), in which a key Arabidopsis regulatory factor involved in stress responses, SNF1-related protein kinase 2C (AtSRK2C), or galactinol synthase 2 (AtGolS2), was overexpressed. Both types of transgenic poplar plants displayed higher tolerance to abiotic stresses, in comparison with nontransgenic plants, indicating that AtSRK2C and AtGolS2 can function in the abiotic stress response pathway of poplar. We also examined the expression profiles of ten poplar genes putatively homologous to well-known Arabidopsis stress response genes and found that several of the poplar genes showed different responses to abiotic stress from their Arabidopsis counterparts. Whereas the overexpression of AtSRK2C in transgenic Arabidopsis plants was reported to upregulate the expression of endogenous genes, the overexpression of AtSRK2C or AtGolS2 in transgenic poplar did not. Taken together, our findings suggest that the details of the underlying molecular mechanisms of the abiotic stress response may differ, but that the key regulatory factors in Arabidopsis and poplar have common features and are effective molecular targets for further breeding to enhance abiotic stress tolerance in poplar.     

<Emphasis Type="Italic">LMI1</Emphasis>-like genes involved in leaf margin development of <Emphasis Type="Italic">Brassica napus</Emphasis>

In rapeseed (Brassica napus L.), leaf margins are variable and can be entire, serrate, or lobed. In our previous study, the lobed-leaf gene (LOBED-LEAF 1, BnLL1) was mapped to a 32.1 kb section of B. napus A10. Two LMI1-like genes, BnaA10g26320D and BnaA10g26330D, were considered the potential genes that controlled the lobed-leaf trait in rapeseed. In the present study, these two genes and another homologous gene (BnaC04g00850D) were transformed into Arabidopsis thaliana (L.) Heynh. plants to identify their functions. All three LMI1-like genes of B. napus produced serrate leaf margins. The expression analysis indicated that the expression level of BnaA10g26320D determined the difference between lobed- and entire-leaved lines in rapeseed. Therefore, it is likely that BnaA10g26320D corresponds to BnLL1.     

Co-transformation of canola by chimeric chitinase and <Emphasis Type="Italic">tlp</Emphasis> genes towards improving resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T₂ generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.     

Overexpression of <Emphasis Type="Italic">IrlVHA</Emphasis>-<Emphasis Type="Italic">c</Emphasis>, A Vacuolar-Type H<Superscript>+</Superscript>-ATPase c Subunit Gene from <Emphasis Type="Italic">Iris lactea</Emphasis>, Enhances Salt Tolerance in Tobacco

Vacuolar-type H⁺-ATPase (V-ATPase), a multi-subunit endomembrane proton pump, plays an important role in plant growth and response to environmental stresses. In the present study, transgenic tobacco that overexpressed the V-ATPase c subunit gene from Iris lactea (IrlVHA-c) was used to determine the function of IrlVHA-c. Quantitative PCR analysis showed that IrlVHA-c expression was induced by salt stress in I. lactea roots and leaves. Subcellular localization of green fluorescent protein (GFP) as marker combined with FM4-64 staining showed that the IrlVHA-c-GFP was localized to the endosomal compartment in tobacco cells. Compared with the wild-type, the IrlVHA-c transgenic tobacco plants exhibited greater seed germination rates, root length, fresh weight, and higher relative water content (RWC) of leaves under salt stress. Furthermore, the IrlVHA-c transgenic tobacco leaves have lower stomatal densities and larger stomatal apertures than wild-type. Under salt stress, superoxide dismutase (SOD) activity in the transgenic tobacco was significantly enhanced. Moreover, the level of malondialdehyde (MDA) in the transgenic tobacco was significantly lower than that in wild-type plants under salt stress. Taken together, these results suggested that the IrlVHA-c plays an important role in salt tolerance in transgenic tobacco by influencing stomatal movement and physiological changes.