Thiourea mediated regulation in the expression profile of aquaporins and its impact on water homeostasis under salinity stress in Brassica juncea roots

Bioregulatory molecules such as thiourea (TU) play an important role in imparting stress tolerance to crops. However, the molecular mechanism involved in the TU-mediated tolerance has not been elucidated. Towards this endeavour, the expression profile of various PIPs (plasma membrane intrinsic proteins) was studied under salt stress (NaCl; 700 mM) with/without thiourea (TU; 6.5 mM) at different time periods in roots of Brassica juncea. Various aquaporin isoforms demonstrated an upregulation upon salinity stress imposition, whereas they were downregulated upon TU supplementation. TU treatment also led to a decrease in the accumulation of reactive oxygen species and delimited the need for an enhanced accumulation of osmolytes. The vacuolar pH was also maintained in NaCl + TU treatment as demonstrated by in vivo ³¹P NMR of roots. In conclusion, TU supplementation to salt stressed seedlings was found to maintain the water homeostasis of roots through coordinated regulation of different PIP isoforms.     

Cold acclimation induces freezing tolerance via antioxidative enzymes, proline metabolism and gene expression changes in two chrysanthemum species

Cold acclimation is necessary for chrysanthemum to achieve its genetically determined maximum freezing tolerance, but the underlying physiological and molecular mechanisms are unclear. The aim of this study was to discover whether changes in antioxidative enzymes, proline metabolism and frost-related gene expression induced by cold acclimation are related to freezing tolerance. Our results showed that the semi-lethal temperature (LT₅₀) decreased from ?7.3 to ?23.5 °C in Chrysanthemum dichrum and ?2.1 to ?7.1 °C in Chrysanthemum makinoi, respectively, after cold acclimation for 21 days. The activities of SOD, CAT and APX showed a rapid and transient increase in the two chrysanthemum species after 1 day of cold acclimation, followed by a gradual increase during the subsequent days and then stabilization. qRT-PCR analysis showed that the expression levels of some isozyme genes (Mn SOD, CAT and APX) were upregulated, which was consistent with the SOD, CAT and APX activities, while others remained relatively constant (Fe SOD and Cu/Zn SOD). P5CS and PDH expression were increased under cold acclimation and the level of P5CS presented similar trends as proline content, indicating proline accumulation was via P5CS and PDH cooperation. Cold acclimation also promoted DREB, COR413 and CSD gene expression. The activities of three enzymes and gene expression were higher in C. dichrum than in C. makinoi after cold acclimation. Our data suggested that cold-inducible freezing-tolerance could be attributed to higher activity of antioxidant enzymes, and increased proline content and frost-related gene expression during different periods.     

Effects of methionine hydroxy analogue supplementation on the expression of antioxidant-related genes of acute heat stress-exposed broilers

We evaluated the effects of heat stress (HS) and methionine supplementation on biological markers of stress and expression of the genes for superoxide dismutase (SOD), thioredoxin (TRx), thioredoxin reductase 1 (TRxR1) and methionine sulfoxide reductase A (MsrA) in broilers aged 1 to 21 or 22 to 42 days. The broilers were divided into two treatments, one with the recommended level of methionine supplementation (MS, supplementation of dl-2-hydroxy-4-methylthiobutanoic acid (dl-HMTBA)) and one without methionine supplementation (MD). The animals were maintained at a temperature of thermal comfort or one of HS (38°C for 24 h). Mortality was only observed in 42-day-old broilers exposed to HS and fed the MD diet, and the rate was 5%. Starter period: we observed an interaction effect between diet and temperature on the gene expression of TRxR1 and MsrA, and expression of these genes was higher in the HS animals that received the MS diet than that in birds with the MD diet. Grower period: the expression of SOD, TRxR1 and MsrA genes, activities of aspartate aminotransferase (AST) and creatine kinase (CK) and content of creatinine were influenced by both study variables. In the HS animals, the expression of these genes, AST activity and creatinine content increased and CK activity decreased. In the animals on the MD diet, the expression of these genes and AST and creatinine values were higher and the CK activity was lower than those for the birds on the MS diet. Our results indicated that under HS conditions, the supplementation with dl-HMTBA could mitigate major damage caused by stress through the action on some genes related to TRx complex activity.     

A Transient Transformation System for the Functional Characterization of Genes Involved in Stress Response

In this study, we developed a simple and efficient transient transformation system, which can be used in homologous expression or reverse genetic study of the plants. A system for characterizing gene function in response to stress tolerance was also developed based on this transformation method. The overexpression and RNAi-silencing of a bZIP gene from Tamarix hispida, ThbZIP1, were performed in T. hispida using this transformation method. Real-time PCR showed that the expression of ThbZIP1 was highly up- and down-regulated in the plants with overexpression and RNAi-silenced expression of ThbZIP1, respectively, when compared with control plants (transiently transformed with empty pROK2). A physiological study showed that ThbZIP1 can enhance the activities of both peroxidase (POD) and superoxide dismutase (SOD), and decrease electrolyte leakage rate and levels of reactive oxygen species (ROS) and malondialdehyde (MDA) under salt stress conditions. Furthermore, ThbZIP1 is found to mediate stress tolerance by regulating the expression of SOD and POD genes. These results suggested that this transient transformation system is an effective method for determining the function of a gene in response to abiotic stress in plants.     

Molecular cloning and functional characterization of a Cu/Zn superoxide dismutase gene (<Emphasis Type="Italic">CsCSD1</Emphasis>) from <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Superoxide dismutase (SOD) proteins, which are widely present in the plant kingdom, play vital roles in response to abiotic stress. However, the functions of cucumber SOD genes in response to environmental stresses remain poorly understood. In this study, a SOD gene CsCSD1 was identified and functionally characterized from cucumber (Cucumis sativus). The CsCSD1 protein was successfully expressed in E. coli, and its overexpression significantly improved the tolerance of host E. coli cells to salinity stress. Besides, overexpression of CsCSD1 enhanced salinity tolerance during germination and seedling development in transgenic Arabidopsis plants. Further analyses showed that the SOD and CAT (catalase) activities of transgenic plants were significantly higher than those of wild-type (WT) plants under normal growth conditions as well as under NaCl treatment. In addition, the expression of stress-response genes RD22, RD29B and LEA4-5 was significantly elevated in transgenic plants. Our results demonstrate that the CsCSD1 gene functions in defense against salinity stress and may be important for molecular breeding of salt-tolerant plants.     

Thiourea mediates alleviation of UV-B stress-induced damage in the Indian mustard (Brassica juncea L.)

Abstract

UV-B radiation (280–320 nm) as an environmental stress has damaging effect on plants and its overexposure can potentially interfere with growth and development. The effect of thiourea (TU) on UV-B stress tolerance of 10-day-old Brassica juncea seedlings subjected to supplementary UV-B for 5 days was investigated. An increase in the UV-B absorbing compounds anthocyanin, flavonoids and phenolic compounds was observed in UV-B + TU treated seedlings as compared to that of control. The enhanced synthesis of UV-B screening compounds resulted in lesser damage to chlorophyll and also gain in fresh weight and dry weight in UV-B + TU as compared to UV-B alone treatment. The enzymatic activities of guaiacol peroxidase (GPX) and superoxide dismutase (SOD) also increased in UV-B + TU. The expression profiling of phenylalanine ammonia lyase (pal) and chalcone synthase (chs) indicated an upregulation under UV-B + TU treatment, compared to that of control, suggesting that TU treatment ensured an early and efficient induction of flavonoid biosynthetic pathway. The results indicate that TU helps in ameliorating the damaging effects of UV-B stress by efficiently maintaining the antioxidant status and attenuating the penetration of the UV-B.     

Overexpression of soybean <Emphasis Type="Italic">GmERF9</Emphasis> enhances the tolerance to drought and cold in the transgenic tobacco

Ethylene response factors (ERFs) are widespread in plants, which are widely involved in plant response to biotic and abiotic stress. In this research, a soybean gene, GmERF9, was identified and the function was characterized. The results showed that GmERF9 contained a typical AP2/ERF binding domain and a putative nuclear localization signal sequence. The real-time fluorescence quantitative PCR (qPCR) revealed that the expression of GmERF9 could be induced by ethylene (ET), abscisic acid (ABA), drought, salt and cold stresses. GmERF9 protein could specifically bind to the GCC-box and activate the expression of the reporter gene in the yeast cells and tobacco leaves. Overexpression of GmERF9 enhanced the expression of pathogenesis-related (PR) genes, including PR1, PR2, Osmotin (PR5), and SAR8.2. Also, the overexpression of GmERF9 increased the accumulation of proline and soluble carbohydrate, and decreased the accumulation of malondialdehyde under drought and cold stresses in the transgenic tobacco compared to the wild type (WT) tobacco, which indicated that GmERF9 enhanced the tolerance to drought and cold stresses in the transgenic tobacco. In summary, the function of GmERF9 is involved in the response to environmental stresses for plants, which can be used as a candidate gene for genetic engineering of crops.     

Identification of Reference Genes for Quantitative Real-Time PCR in Date Palm (Phoenix dactylifera L.) Subjected to Drought and Salinity

Date palm is an important crop plant in the arid and semi-arid regions supporting human population in the Middle East and North Africa. These areas have been largely affected by drought and salinity due to insufficient rainfall and improper irrigation practices. Date palm is a relatively salt- and drought-tolerant plant and more recently efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Quantitative real-time PCR (qPCR) is a promising technique for the analysis of stress-induced differential gene expression, which involves the use of stable reference genes for normalizing gene expression. In an attempt to find the best reference genes for date palm’s drought and salinity research, we evaluated the stability of 12 most commonly used reference genes using the geNorm, NormFinder, BestKeeper statistical algorithms and the comparative ΔC_T method. The comprehensive results revealed that HEAT SHOCK PROTEIN (HSP), UBIQUITIN (UBQ) and YTH domain-containing family protein (YT521) were stable in drought-stressed leaves whereas GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH), ACTIN and TUBULIN were stable in drought-stressed roots. On the other hand, SMALL SUBUNIT RIBOSOMAL RNA (25S), YT521 and 18S ribosomal RNA (18S); and UBQ, ACTIN and ELONGATION FACTOR 1-ALPHA (eEF1a) were stable in leaves and roots, respectively, under salt stress. The stability of these reference genes was verified by using the abiotic stress-responsive CYTOSOLIC Cu/Zn SUPEROXIDE DISMUTASE (Cyt-Cu/Zn SOD), an ABA RECEPTOR, and a PROLINE TRANSPORTER 2 (PRO) genes. A combination of top 2 or 3 stable reference genes were found to be suitable for normalization of the target gene expression and will facilitate gene expression analysis studies aimed at identifying functional genes associated with drought and salinity tolerance in date palm.