OsMSR3, a Small Heat Shock Protein, Confers Enhanced Tolerance to Copper Stress in Arabidopsis thaliana.

Copper is a mineral element essential for the normal growth and development of plants; however, excessive levels can severely affect plant growth and development. Oryza sativa L. multiple stress-responsive gene 3 (OsMSR3) is a small, low-molecular-weight heat shock protein (HSP) gene. A previous study has shown that OsMSR3 expression improves the tolerance of Arabidopsis to cadmium stress. However, the role of OsMSR3 in the Cu stress response of plants remains unclear, and, thus, this study aimed to elucidate this phenomenon in Arabidopsis thaliana, to further understand the role of small HSPs (sHSPs) in heavy metal resistance in plants. Under Cu stress, transgenic A. thaliana expressing OsMSR3 showed higher tolerance to Cu, longer roots, higher survival rates, biomass, and relative water content, and accumulated more Cu, abscisic acid (ABA), hydrogen peroxide, chlorophyll, carotenoid, superoxide dismutase, and peroxidase than wild-type plants did. Moreover, OsMSR3 expression in A. thaliana increased the expression of antioxidant-related and ABA-responsive genes. Collectively, our findings suggest that OsMSR3 played an important role in regulating Cu tolerance in plants and improved their tolerance to Cu stress through enhanced activation of antioxidative defense mechanisms and positive regulation of ABA-responsive gene expression.

Cloning, expression, and characterization of a porcine pancreatic α-amylase in Pichia pastoris.

Pancreatic α-amylase (α-1, 4-glucan-4-glucanohydrolase, EC.3.2.1.1) plays a primary role in the intestinal digestion of feed starch and is often deficient in weanling pigs. The objective of this study was to clone, express, and characterize porcine pancreatic α-amylase (PPA). The full-length cDNA encoding the PPA was isolated from pig pancreas by RT-PCR and cloned into the pPICZαA vector. After the resultant pPICZαΑ-PPA plasmid was transferred into Pichia pastoris, Ni Sepharose affinity column was used to purify the over-expressed extracellular recombinant PPA protein (rePPA) that contains a His-tag to the C terminus and was characterized against the natural enzyme (α-amylase from porcine pancreas). The rePPA exhibited a molecular mass of approximately 58 kDa and showed optimal temperature (50 °C), optimal pH (7.5), Km (47.8 mg/mL), and Vmax (2,783 U/mg) similar to those of the natural enzyme. The recombinant enzyme was stable at 40 °C but lost 60% to 90% (P < 0.05) after exposure to heating at ≥50 °C for 30 min. The enzyme activity was little affected by Cu2+ or Fe3+, but might be inhibited (40% to 50%) by Zn2+ at concentrations in pig digesta. However, Ca2+ exhibited a dose-dependent stimulation of the enzyme activity. In conclusion, the present study successfully cloned the porcine pancreatic α-amylase gene and over-expressed the gene in P.pastoris as an extracellular, functional enzyme. The biochemical characterization of the over-produced enzyme depicts its potential and future improvement as an animal feed additive.

OsDSSR1, a novel small peptide, enhances drought tolerance in transgenic rice.

Small signaling peptides play important roles in plant development and responses to abiotic and biotic stresses. We have identified a novel small peptide gene in rice, OsDSSR1, which is expressed mainly in the root, stem, node, leaf, and panicle. OsDSSR1 expression is also induced by drought, salinity, ABA, and H2O2 treatment. OsDSSR1 is localized in the nucleus and cytoplasm. Transgenic plants overexpressing OsDSSR1 exhibited enhanced drought stress tolerance and decreased ABA sensitivity as compared to the wild type. Overexpression of OsDSSR1 promoted the accumulation of compatible osmolytes, such as free proline and soluble sugars. OsDSSR1-overexpressing plants displayed enhanced OsSodCc2 and OscAPX expression and superoxide dismutase and ascorbate peroxidase activities under drought stress. RNA-sequencing data revealed that the expression of 72 abiotic stress-responsive genes was significantly altered in homozygous transgenic plants. These stress-responsive candidate genes will aid in expanding our understanding of the mechanisms by which small peptides mediate tolerance in crop species.

Overexpression of a novel MYB-related transcription factor, OsMYBR1, confers improved drought tolerance and decreased ABA sensitivity in rice.

The MYB proteins play important roles in regulating plant responses to environmental stresses. We cloned and functionally characterized a novel MYB-related gene, OsMYBR1, from rice. Our microarray and qRT-PCR analyses showed that its expression was induced by drought and cold in different tissues at various developmental stages. This gene encodes a putative MYB-related protein of 463 amino acid residues. Compared with wild-type (WT) plants, transgenic plants over-expressing OsMYBR1 exhibited much greater tolerance to drought stress and decreased sensitivity to abscisic acid (ABA). Under drought treatment, levels of free proline and soluble sugar were higher in transgenic plants than in the WT. Furthermore, transcriptional expression of four stress-related genes -- OsP5CS1, OsProt, OsLEA3, and OsRab16 -- was significantly increased in transgenic plants under drought stressed conditions and ABA. Our results provide evidence that OsMYBR1 is involved in mediating plant responses to ABA and drought.

Deciphering the physiological and molecular mechanisms for copper tolerance in autotetraploid Arabidopsis.

KEY MESSAGE: Autotetraploid Arabidopsis line esd and 4COL exhibit enhanced tolerance to Cu stress by enhancing activation of antioxidative defenses, altering expression of genes related to Cu transport, chelation, and ABA-responsive. Autopolyploidy is ubiquitous among angiosperms and often results in better adaptation to stress conditions. Although copper (Cu) is an essential trace element, excess amounts can inhibit plant growth and even result in death. Here, we report that autotetraploid Arabidopsis thaliana esd and 4COL exhibit higher tolerance to Cu stress. Under such conditions, tetraploid plants had lower Cu contents and significantly more biomass compared with diploid plants. When exposed to excess Cu for 24 h, levels of superoxide anions, hydrogen peroxide, and malondialdehyde were lower in tetraploids than in diploids. Moreover, activities of the antioxidant enzymes superoxide dismutase and peroxidase were stimulated and glutathione content was maintained at a relative higher level in the tetraploids. The expression of genes related to Cu transport and chelation was altered in autotetraploid Arabidopsis under Cu stress, and several key genes involved in the response to abscisic acid (ABA) were significantly up-regulated. Our results indicate that tetraploid Arabidopsis esd and 4COL acquire improved tolerance to Cu stress through enhanced activation of antioxidative defense mechanisms, altered expression of genes related to Cu transport and chelation, and positive regulation of expression for ABA-responsive genes.

OsSGL, a novel pleiotropic stress-related gene enhances grain length and yield in rice.

Abiotic stress seriously affects the yield of rice (Oryza sativa L.). Grain yield in rice is multiplicatively determined by the number of panicles, number of grains per panicle, and grain weight. Here, we describe the molecular and functional characterization of STRESS_tolerance and GRAIN_LENGTH (OsSGL), a rice gene strongly up-regulated by a wide spectrum of abiotic stresses. OsSGL encodes a putative member of the DUF1645 protein family of unknown function. Overexpression of OsSGL significantly altered certain development processes greatly and positively affecting an array of traits in transgenic rice plants, including increased grain length, grain weight and grain number per panicle, resulting in a significant increase in yield. Microscopical analysis showed that the enhanced OsSGL expression promoted cell division and grain filling. Microarray and quantitative real-time PCR (qRT-PCR) analyses revealed that a large number of genes involved in stress-response, cell cycle and cytokinin signaling processes were induced or suppressed in OsSGL-overexpressing plants. Together, our results suggest that OsSGL may regulate stress-tolerance and cell growth by acting via a cytokinin signaling pathway. This study not only contributes to our understanding of the underlying mechanism regulating rice stress-tolerance and grain length, but also provides a strategy for tailor-made crop yield improvement.

OsSGL, a Novel DUF1645 Domain-Containing Protein, Confers Enhanced Drought Tolerance in Transgenic Rice and Arabidopsis.

Drought is a major environmental factor that limits plant growth and crop productivity. Genetic engineering is an effective approach to improve drought tolerance in various crops, including rice (Oryza sativa). Functional characterization of relevant genes is a prerequisite when identifying candidates for such improvements. We investigated OsSGL (Oryza sativa Stress tolerance and Grain Length), a novel DUF1645 domain-containing protein from rice. OsSGL was up-regulated by multiple stresses and localized to the nucleus. Transgenic plants over-expressing or hetero-expressing OsSGL conferred significantly improved drought tolerance in transgenic rice and Arabidopsis thaliana, respectively. The overexpressing plants accumulated higher levels of proline and soluble sugars but lower malondialdehyde (MDA) contents under osmotic stress. Our RNA-sequencing data demonstrated that several stress-responsive genes were significantly altered in transgenic rice plants. We unexpectedly observed that those overexpressing rice plants also had extensive root systems, perhaps due to the altered transcript levels of auxin- and cytokinin-associated genes. These results suggest that the mechanism by which OsSGL confers enhanced drought tolerance is due to the modulated expression of stress-responsive genes, higher accumulations of osmolytes, and enlarged root systems.

Expression of Selenoprotein Genes Is Affected by Obesity of Pigs Fed a High-Fat Diet.

BACKGROUND: Relations of the 25 mammalian selenoprotein genes with obesity and the associated inflammation remain unclear. OBJECTIVE: This study explored impacts of high-fat diet-induced obesity on inflammation and expressions of selenoprotein and obesity-related genes in 10 tissues of pigs. METHODS: Plasma and 10 tissues were collected from pigs (n = 10) fed a corn-soy-based control diet or that diet containing 3-7% lard from weanling to finishing (180 d). Plasma concentrations (n = 8) of cytokines and thyroid hormones and tissue mRNA abundance (n = 4) of 25 selenoprotein genes and 16 obesity-related genes were compared between the pigs fed the control and high-fat diets. Stepwise regression was applied to analyze correlations among all these measures, including the previously reported body physical and plasma biochemical variables. RESULTS: The high-fat diet elevated (P < 0.05) plasma concentrations of tumor necrosis factor α, interleukin-6, leptin, and leptin receptor by 29-42% and affected (P < 0.05-0.1) tissue mRNA levels of the selenoprotein and obesity-related genes in 3 patterns. Specifically, the high-fat diet up-regulated 12 selenoprotein genes in 6 tissues, down-regulated 13 selenoprotein genes in 7 tissues, and exerted no effect on 5 genes in any tissue. Body weights and plasma triglyceride concentrations of pigs showed the strongest regressions to tissue mRNA abundances of selenoprotein and obesity-related genes. Among the selenoprotein genes, selenoprotein V and I were ranked as the strongest independent variables for the regression of phenotypic and plasma measures. Meanwhile, agouti signaling protein, adiponectin, and resistin genes represented the strongest independent variables of the obesity-related genes for the regression of tissue selenoprotein mRNA. CONCLUSIONS: The high-fat diet induced inflammation in pigs and affected their gene expression of selenoproteins associated with thioredoxin and oxidoreductase systems, local tissue thyroid hormone activity, endoplasmic reticulum protein degradation, and phosphorylation of lipids. This porcine model may be used to study interactive mechanisms between excess fat intake and selenoprotein function.

Porcine serum can be biofortified with selenium to inhibit proliferation of three types of human cancer cells.

Our objectives were to determine if porcine serum could be enriched with selenium (Se) by feeding pigs with high concentrations of dietary Se and if the Se-biofortified serum inhibited proliferation of 3 types of human cancer cells. In Expt. 1, growing pigs (8 wk old, n = 3) were fed 0.02 or 3.0 mg Se/kg (as sodium selenite) for 16 wk and produced serum with 0.5 and 5.4 µmol/L Se, respectively. In Expt. 2, growing pigs (5 wk old, n = 6) were fed 0.3 or 1.0 mg Se/kg (as Se-enriched yeast) for 6 wk and produced serum with 2.6 and 6.2 µmol/L Se, respectively. After the Se-biofortified porcine sera were added at 16% in RPMI 1640 to treat NCI-H446, DU145, and HTC116 cells for 144 h, they decreased (P < 0.05) the viability of the 3 types of human cancer cells by promoting apoptosis, compared with their controls. This effect was replicated only by adding the appropriate amount of methylseleninic acid to the control serum and was mediated by a downregulation of 8 cell cycle arrest genes and an upregulation of 7 apoptotic genes. Along with 6 previously reported selenoprotein genes, selenoprotein T (Selt), selenoprotein M (Selm), selenoprotein H (Selh), selenoprotein K (Selk), and selenoprotein N (Sepn1) were revealed to be strongly associated with the cell death-related signaling induced by the Se-enriched porcine serum. In conclusion, porcine serum could be biofortified with Se to effectively inhibit the proliferation of 3 types of human cancer cells and the action synchronized with a matrix of coordinated functional expression of multiple selenoprotein genes.

Prolonged dietary selenium deficiency or excess does not globally affect selenoprotein gene expression and/or protein production in various tissues of pigs.

We previously determined the effects of dietary selenium (Se) deficiency or excess on mRNA abundance of 12 selenoprotein genes in pig tissues. In this study, we determined the effect of dietary Se on mRNA levels of the remaining porcine selenoprotein genes along with protein production of 4 selenoproteins (Gpx1, Sepp1, Selh, and Sels) and body glucose homeostasis. Weanling male pigs (n = 24) were fed a Se-deficient (<0.02 mg Se/kg), basal diet supplemented with 0, 0.3, or 3.0 mg Se/kg as Se-enriched yeast (Angel Yeast) for 16 wk. Although mRNA abundance of the 13 selenoproteins in 10 tissues responded to dietary Se in 3 patterns, there was no common regulation for any given gene across all tissues or for any given tissue across all genes. Dietary Se affected (P < 0.05) 2, 3, 3, 5, 6, 7, 7, and 8 selenoprotein genes in muscle, hypothalamus, liver, kidney, heart, spleen, thyroid, and pituitary, respectively. Protein abundance of Gpx1, Sepp1, Selh, and Sels in 6 tissues was regulated (P < 0.05) by dietary Se concentrations in 3 ways. Compared with those fed 0.3 mg Se/kg, pigs fed 3.0 mg Se/kg became hyperinsulinemic (P < 0.05) and had lower (P < 0.05) tissue levels of serine/threonine protein kinase. In conclusion, dietary Se exerted no global regulation of gene transcripts or protein levels of individual selenoproteins across porcine tissues. Pigs may be a good model for studying mechanisms related to the potential prodiabetic risk of high-Se intake in humans.

A high-selenium diet induces insulin resistance in gestating rats and their offspring.

Although supranutrition of selenium (Se) is considered a promising anti-cancer strategy, recent human studies have shown an intriguing association between high body Se status and diabetic risk. This study was done to determine if a prolonged high intake of dietary Se actually induced gestational diabetes in rat dams and insulin resistance in their offspring. Forty-five 67-day-old female Wistar rats (n=15/diet) were fed a Se-deficient (0.01 mg/kg) corn-soy basal diet (BD) or BD+Se (as Se-yeast) at 0.3 or 3.0mg/kg from 5 weeks before breeding to day 14 postpartum. Offspring (n=8/diet) of the 0.3 and 3.0mg Se/kg dams were fed with the same respective diet until age 112 days. Compared with the 0.3mg Se/kg diet, the 3.0mg/kg diet induced hyperinsulinemia (P<0.01), insulin resistance (P<0.01), and glucose intolerance (P<0.01) in the dams at late gestation and/or day 14 postpartum and in the offspring at age 112 days. These impairments concurred with decreased (P<0.05) mRNA and/or protein levels of six insulin signal proteins in liver and muscle of dams and/or pups. Dietary Se produced dose-dependent increases in Gpx1 mRNA or GPX1 activity in pancreas, liver, and erythrocytes of dams. The 3.0mg Se/kg diet decreased Selh (P<0.01), Sepp1 (P=0.06), and Sepw1 (P<0.01), but increased Sels (P<0.05) mRNA levels in the liver of the offspring, compared with the 0.3mg Se/kg diet. In conclusion, supranutrition of Se as a Se-enriched yeast in rats induced gestational diabetes and insulin resistance. Expression of six selenoprotein genes, in particular Gpx1, was linked to this metabolic disorder.

Functional complementation of dwf4 mutants of Arabidopsis by overexpression of CYP724A1.

An essential step in the biosynthesis of bioactive brassinosteroids (BRs) in plants is the hydroxylation at C-22, a reaction catalyzed by P450 enzymes of the CYP90B and CYP724B subfamilies. Genes for both types of enzymes are present in many species, and in rice (Oryza sativa) and tomato (Solanum lycopersicum) both CYP90B and CYP724B enzymes contribute to C-22 hydroxylation. In Arabidopsis (Arabidopsis thaliana), C-22 hydroxylation of BRs is catalyzed by CYP90B1 (encoded by DWF4) and null dwf4 mutants show severe symptoms of BR-deficiency. CYP724A1 (At5g14400), an Arabidopsis gene of unknown function and limited expression, encodes a P450 sharing less than 55% sequence identity to CYP724B proteins. We used transgenic plants of the null mutants dwf4-102 and a novel allele, bashful (bsf), ectopically expressing the CYP724A1 gene to investigate the potential activity of CYP724A1 as a C-22 hydroxylase of BRs. Defects associated with BR deficiency were reversed and a normal growth habit restored in transgenic dwf4-102 and bsf plants overexpressing CYP724A1. The vegetative phase was prolonged and the transgenic plants were on average larger than wild type plants with respect to several morphometric parameters. Fertility was restored in the transgenic plants but individual siliques yielded fewer and heavier seeds than those of wild type plants. The implications of these findings with regard to the functions of CYP724A1 and the activity of its encoded enzyme are discussed.

The selenium deficiency disease exudative diathesis in chicks is associated with downregulation of seven common selenoprotein genes in liver and muscle.

Fast-growing broiler chicks are susceptible to Se deficiency diseases including exudative diathesis (ED). Our objective was to determine if ED could be induced by feeding a current, practical diet and if the incidence was related to selenogenome expression in liver and muscle of chicks. Four groups of day-old broiler chicks (n = 60/group) were fed a corn-soy basal diet (BD; 14 µg Se/kg; produced in the Se-deficient area of Sichuan, China and not supplemented with Se or vitamin E), the BD and all-rac-α-tocopheryl acetate at 50 mg/kg and Se (as sodium selenite) at 0.3 mg/kg, or both of these nutrients for 6 wk. A high incidence of ED and mortality of chicks were induced by the BD. The incidences and mortality were completely prevented by supplemental dietary Se but were only partially decreased by supplemental α-tocopherol acetate. Dietary Se deficiency decreased (P < 0.05) mRNA levels of 7 common selenoprotein genes (Gpx1, Gpx4, Sepw1, Sepn1, Sepp1, Selo, and Selk) in muscle and liver. Whereas supplementing α-tocopherol acetate enhanced (P < 0.05) only the muscle Sepx1 mRNA level, it actually decreased (P < 0.05) hepatic Gpx1, Seli, Txnrd1, and Txnrd2 mRNA levels. In conclusion, dietary Se protected chicks from the Se deficiency disease ED, probably by upregulating selenoprotein genes coding for oxidation- and/or lesion-protective proteins. The protection by vitamin E might be mediated via selenoproteins not assayed in this study and/or Se-independent mechanisms. The inverse relationship between hepatic expression of 4 redox-related selenoprotein genes and vitamin E status revealed a novel interaction between Se and vitamin E in vivo.

A novel rice calmodulin-like gene, OsMSR2, enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis.

Many abiotic stimuli, such as drought and salt stresses, elicit changes in intracellular calcium levels that serve to convey information and activate adaptive responses. Ca²âº signals are perceived by different Ca²âº sensors, and calmodulin (CaM) is one of the best-characterized Ca²âº sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants, but their functions at the physiological and molecular levels are largely unknown. In this report, we present data on OsMSR2 (Oryza sativa L. Multi-Stress-Responsive gene 2), a novel calmodulin-like protein gene isolated from rice Pei'ai 64S (Oryza sativa L.). Expression of OsMSR2 was strongly up-regulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice, as revealed by both microarray and quantitative real-time RT-PCR analyses. Analysis of the recombinant OsMSR2 protein demonstrated its potential ability to bind Ca²âº in vitro. Expression of OsMSR2 conferred enhanced tolerance to high salt and drought in Arabidopsis (Arabidopsis thaliana) accompanied by altered expression of stress/ABA-responsive genes. Transgenic plants also exhibited hypersensitivity to ABA during the seed germination and post-germination stages. The results suggest that expression of OsMSR2 modulated salt and drought tolerance in Arabidopsis through ABA-mediated pathways.

Enhanced water-holding capacity of meat was associated with increased Sepw1 gene expression in pigs fed selenium-enriched yeast.

To study the effect of selenium-enriched yeast (SeY) level on selenoprotein genes expression and the relation between gene expression and antioxidant status and meat quality, 30 selenium (Se)-depleted pigs (7-week old, 10.30±0.68 kg) were randomly divided into 3 groups and fed a basal diet plus 0, 0.3 and 3.0 mg Se/kg as SeY for 8 weeks. Results showed that dietary SeY supplementation improved the antioxidant status in muscle. The increased levels of SeY decreased (P<0.05) the drip loss and the concentration of thiobarbituric acid reactive substances in the muscle and meat. However, increased dietary SeY intake quadratically increased (P<0.01) the mRNA level of Sepw1 gene among the 12 selenoprotein genes examined in muscle. Statistical analysis showed drip loss was negatively correlated with the mRNA level of Sepw1 gene. These suggested that the enhanced water-holding capacity of meat was associated with the increased expression of Sepw1 gene.

Selenoprotein gene expression in thyroid and pituitary of young pigs is not affected by dietary selenium deficiency or excess.

Expression and function of selenoproteins in endocrine tissues remain unclear, largely due to limited sample availability. Pigs have a greater metabolic similarity and tissue size than rodents as a model of humans for that purpose. We conducted 2 experiments: 1) we cloned 5 novel porcine selenoprotein genes; and 2) we compared the effects of dietary selenium (Se) on mRNA levels of 12 selenoproteins, activities of 4 antioxidant enzymes, and Se concentrations in testis, thyroid, and pituitary with those in liver of pigs. In Experiment 1, porcine Gpx2, Sephs2, Sep15, Sepn1, and Sepp1 were cloned and demonstrated 84-94% of coding sequence homology to human genes. In Experiment 2, weanling male pigs (n = 30) were fed a Se-deficient (0.02 mg Se/kg) diet added with 0, 0.3, or 3.0 mg Se/kg as Se-enriched yeast for 8 wk. Although dietary Se resulted in dose-dependent increases (P < 0.05) in Se concentrations and GPX activities in all 4 tissues, it did not affect the mRNA levels of any selenoprotein gene in thyroid or pituitary. Testis mRNA levels of Txnrd1 and Sep15 were decreased (P < 0.05) by increasing dietary Se from 0.3 to 3.0 mg/kg. Comparatively, expressions of Gpx2, Gpx4, Dio3, and Sep15 were high in pituitary and Dio1, Sepp1, Sephs2, and Gpx1 were high in liver. In conclusion, the mRNA abundances of the 12 selenoprotein genes in thyroid and pituitary of young pigs were resistant to dietary Se deficiency or excess.

Real-time PCR quantification of the predominant bacterial divisions in the distal gut of Meishan and Landrace pigs.

Firmicutes and Bacteroidetes are closely related to body fat in humans and mice, which are the two dominant bacterial divisions of gut microbiota in mammals. Here real-time PCR analysis indicated that Meishan pigs had a 34% reduction in percentage Bacteroidetes (P = 0.008) and a significantly lower proportion of Bacteroides (P = 0.013) than Landrace pigs. The percentage of Bacteroidetes or Bacteroides had a negative correlation with body fat (R2 was 0.63 for Bacteroidetes and 0.57 for Bacteroides, P < 0.05). There was a trend that the percentage of Firmicutes in Meishan pigs was higher in numerical value than in Landrace pigs, although this difference was not statistically significant (P = 0.290) between the two breeds. These suggested that body fat correlated with the percentage of Bacteroidetes division of the gut microbiota in the common pig breeds, and the differences of gut microbial ecology in obese versus lean animals may be analogous.