Promoter of Vegetable Soybean GmTIP1;6 Responds to Diverse Abiotic Stresses and Hormone Signals in Transgenic Arabidopsis

Tonoplast intrinsic proteins (TIPs), a sub-family of aquaporins (AQPs), are known to play important roles in plant abiotic stress responses. However, evidence for the promoters of TIPs involvement in abiotic stress processes remains scarce. In this study, the promoter of the vegetable soybean GmTIP1;6 gene, which had the highest similarity to TIP1-type AQPs from other plants, was cloned. Expression pattern analyses indicated that the GmTIP1;6 gene was dramatically induced by drought, salt, abscisic acid (ABA), and methyl jasmonate (MeJA) stimuli. Promoter analyses revealed that the GmTIP1;6 promoter contained drought, ABA, and MeJA cis-acting elements. Histochemical staining of the GmTIP1;6 promoter in transgenic Arabidopsis corroborated that it was strongly expressed in the vascular bundles of leaves, stems, and roots. Beta-glucuronidase (GUS) activity assays showed that the activities of the GmTIP1;6 promoter were enhanced by different concentrations of polyethylene glycol 6000 (PEG 6000), NaCl, ABA, and MEJA treatments. Integrating these results revealed that the GmTIP1;6 promoter could be applied for improving the tolerance to abiotic stresses of the transgenic plants by promoting the expression of vegetable soybean AQPs.     

Identification,Functional Study,and Promoter Analysis of HbMFT1, a Homolog of MFT from Rubber Tree (Hevea brasiliensis)

A homolog of MOTHER OF FT AND TFL1 (MFT) was isolated from Hevea brasiliensis and its biological function was investigated. Protein multiple sequence alignment and phylogenetic analysis revealed that HbMFT1 conserved critical amino acid residues to distinguish MFT, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1)-like proteins and showed a closer genetic relationship to the MFT-like group. The accumulation of HbMFT1 was generally detected in various tissues except pericarps, with the highest expression in embryos and relatively higher expression in roots and stems of seedlings, flowering inflorescences, and male and female flowers. HbMFT1 putative promoter analysis showed that tissue-specific, environmental change responsive and hormone-signaling responsive elements were generally present. HbMFT1 was strongly induced under a short-day condition at 28 °C, with the highest expression after the onset of a day. Overexpression of HbMFT1 inhibited seed germination, seedling growth, and flowering in transgenic Arabidopsis. The qRT-PCR further confirmed that APETALA1 (AP1) and FRUITFULL (FUL) were drastically down-regulated in 35S::HbMFT1 plants. A histochemical β-glucuronidase (GUS) assay showed that HbMFT1::GUS activity was mainly detected in stamens and mature seeds coinciding with its original expression and notably induced in rosette leaves and seedlings of transgenic Arabidopsis by exogenous abscisic acid (ABA) due to the presence of ABA cis-elements in HbMFT1 promoter. These results suggested that HbMFT1 was mainly involved in maintenance of seed maturation and stamen development, but negatively controlled germination, growth and development of seedlings and flowering. In addition, the HbMFT1 promoter can be utilized in controlling transgene expression in stamens and seeds of rubber tree or other plant species.     

Comparison and Characterization of a Cell Wall Invertase Promoter from Cu-Tolerant and Non-Tolerant Populations of Elsholtzia haichowensis

Cell wall invertase (CWIN) activity and the expression of the corresponding gene were previously observed to be significantly elevated in a Cu-tolerant population of Elsholtzia haichowensis relative to a non-tolerant population under copper stress. To understand the differences in CWIN gene regulation between the two populations, their CWIN promoter β-glucuronidase (GUS) reporter vectors were constructed. GUS activity was measured in transgenic Arabidopsis in response to copper, sugar, and phytohormone treatments. Under the copper treatment, only the activity of the CWIN promoter from the Cu-tolerant population was slightly increased. Glucose and fructose significantly induced the activity of CWIN promoters from both populations. Among the phytohormone treatments, only salicylic acid induced significantly higher (p < 0.05) activity of the Cu-tolerant CWIN promoter relative to the non-tolerant promoters. Analysis of 5′-deletion constructs revealed that a 270-bp promoter fragment was required for SA induction of the promoter from the Cu-tolerant population. Comparison of this region in the two CWIN promoters revealed that it had 10 mutation sites and contained CAAT-box and W-box cis-elements in the Cu-tolerant promoter only. This work provides insights into the regulatory role of SA in CWIN gene expression and offers an explanation for differences in CWIN expression between E. haichowensis populations.     

The Metallothionein Gene,TaMT3, from Tamarix androssowii Confers Cd2+ Tolerance in Tobacco

Cadmium (Cd) is a nonessential microelement and low concentration Cd²⁺ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd²⁺ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd²⁺ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd²⁺ stress.     

Seed-Specific Expression of a Lysine-Rich Protein Gene,GhLRP, from Cotton Significantly Increases the Lysine Content in Maize Seeds

Maize seed storage proteins are a major source of human and livestock consumption. However, these proteins have poor nutritional value, because they are deficient in lysine and tryptophan. Much research has been done to elevate the lysine content by reducing zein content or regulating the activities of key enzymes in lysine metabolism. Using the naturally lysine-rich protein genes, sb401 and SBgLR, from potato, we previously increased the lysine and protein contents of maize seeds. Here, we examined another natural lysine-rich protein gene, GhLRP, from cotton, which increased the lysine content of transgenic maize seeds at levels varying from 16.2% to 65.0% relative to the wild-type. The total protein content was not distinctly different, except in the six transgenic lines. The lipid and starch levels did not differ substantially in Gossypium hirsutum L. lysine-rich protein (GhLRP) transgenic kernels when compared to wild-type. The agronomic characteristics of all the transgenic maize were also normal. GhLRP is a high-lysine protein candidate gene for increasing the lysine content of maize. This study provided a valuable model system for improving maize lysine content.     

Full-Length cDNA Cloning,Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries

Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5′-untranslated region (UTR) of 24 bp, a 3′-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future.     

Phenolic Compounds from Halimodendron halodendron (Pall.) Voss and Their Antimicrobial and Antioxidant Activities

Halimodendron halodendron has been used as forage in northwestern China for a long time. Its young leaves and flowers are edible and favored by indigenous people. In this study, eleven phenolic compounds were bioassay-guided and isolated from the aerial parts of H. halodendron for the first time. They were identified by means of physicochemical and spectrometric analysis as quercetin (1), 3,5,7,8,4′-pentahydroxy-3′-methoxy flavone (2), 3-O-methylquercetin (3), 3,3′-di-O-methylquercetin (4), 3,3′-di-O-methylquercetin-7-O-β-d-glucopyranoside (5), isorhamentin-3-O-β-d-rutinoside (6), 8-O-methylretusin (7), 8-O-methylretusin-7-O-β-d-glucopyranoside (8), salicylic acid (9), p-hydroxybenzoic acid (ferulic acid) (10), and 4-hydroxy-3-methoxy cinnamic acid (11). They were sorted as flavonols (1–6), soflavones (7 and 8), and phenolic acids (9–11). Among the compounds, flanools 1–4 revealed a strong antibacterial activity with minimum inhibitory concentration (MIC) values of 50–150 μg/mL, and median inhibitory concentration (IC₅₀) values of 26.8–125.1 μg/mL. The two isoflavones (7 and 8) showed moderate inhibitory activity on the test bacteria. Three phenolic acids (9, 10 and 11) showed strong antibacterial activity with IC₅₀ values of 28.1–149.7 μg/mL. Antifungal activities of the compounds were similar to their antibacterial activities. All these phenolic compounds showed significant antimicrobial activity with a broad spectrum as well as antioxidant activity based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and β-carotene-linoleic acid bleaching assays. In general, the flavonol aglycones with relatively low polarity exhibited stronger activities than the glycosides. The results suggest the potential of this plant as a source of functional food ingredients and provide support data for its utilization as forage as well.     

Bio-Guided Isolation of the Cytotoxic Terpenoids from the Roots of Euphorbia kansui against Human Normal Cell Lines L-O2 and GES-1

The dried roots of Euphorbia kansui (kansui) have been used for centuries in China as a herbal medicine for edema, ascites, and asthma. The 95% ethanol extract showed a significant inhibition of cell proliferation against human normal cell lines L-O2 and GES-1. Bioassay-guided separation of the 95% ethanol extract from the roots of E. kansui led to the isolation of 12 diverse terpenoids whose structures were identified by ¹H, ¹³C NMR spectroscopy and ESI-MS as kansuinine A (1), kansuinine B (2), kansuinine C (3), kansuiphorin C (4), 3-O-(2′E,4′Z-decadienoyl)-20-O-acetylingenol (5), 3-O-(2′E,4′Edecadienoyl)-20-O-acetylingenol (6), 3-O-(2′E,4′Z-decadienoyl)-20-deoxyingenol (7), 3-O-benzoyl-20-deoxyingenol (8), 5-O-benzoyl-20-deoxyingenol (9), kansenone (10), epi-kansenone (11), euphol (12). All these 12 terpernoids were evaluated in vitro for cytotoxicity on L-O2 and GES-1 cell lines. Most ingenane-type diterpenoids and 8-ene-7-one triterpenoids (5–11) exhibited a relatively lower IC₅₀ value; therefore, these compounds had stronger cytotoxicity against human normal cell lines L-O2 and GES-1 with dose-dependent relationships. These results will be significantly helpful to reveal the mechanism of toxicity of kansui and to effectively guide safer clinical application of this herb.     

A Novel Thylakoid Ascorbate Peroxidase from Jatrophacurcas Enhances Salt Tolerance in Transgenic Tobacco

Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide in higher plants. In the present study, a novel APX gene (JctAPX) was cloned from Jatropha curcas L. The deduced amino acid sequence was similar to that of APX of some other plant species. JctAPX has a chloroplast transit peptide and was localized to the chloroplasts by analysis with a JctAPX-green fluorescent protein (GFP) fusion protein. Quantitative polymerase chain reaction (qPCR) analysis showed that JctAPX was constitutively expressed in different tissues from J. curcas and was upregulated by NaCl stress. To characterize its function in salt tolerance, the construct p35S: JctAPX was created and successfully introduced into tobacco by Agrobacterium-mediated transformation. Compared with wild type (WT), the transgenic plants exhibited no morphological abnormalities in the no-stress condition. However, under 200 mM NaCl treatment, JctAPX over-expressing plants showed increased tolerance to salt during seedling establishment and growth. In addition, the transgenic lines showed higher chlorophyll content and APX activity, which resulted in lower H₂O₂ content than WT when subjected to 400 mM NaCl stress. These results suggest that the increased APX activity in the chloroplasts from transformed plants increased salt tolerance by enhancing reactive oxygen species (ROS)-scavenging capacity under short-term NaCl stress conditions.