The rice cultivar ( L.) BRS AG, developed by Embrapa Clima Temperado, is the first cultivar designed for purposes other than human consumption. It may be used in ethanol production and animal feed. Different abiotic stresses negatively affect plant growth. Soil salinity is responsible for a serious reduction in productivity. Therefore, the objective of this study was to evaluate the gene expression and the activity of antioxidant enzymes (SOD, CAT, APX and GR) and identify their functions in controlling ROS levels in rice plants, cultivar BRS AG, after a saline stress period. The plants were grown in vitro with two NaCl concentrations (0 and 136 mM), collected at 10, 15 and 20 days of cultivation. The results indicated that the activity of the enzymes evaluated promotes protection against oxidative stress. Although, there was an increase of reactive oxygen species, there was no increase in MDA levels. Regarding genes encoding isoforms of antioxidant enzymes, it was observed that -, -, -, -, -, -, , and were the most responsive. The increase in the transcription of all genes among evaluated isoforms, except for, which remained stable, contributed to the increase or the maintenance of enzyme activity. Thus, it is possible to infer that the cv. BRS AG has defense mechanisms against salt stress.

ABSTRACT.To obtain accurate and reliable results for the expression of genes of interest using quantitative real-time polymerase chain reaction (RT-qPCR) techniques, it is necessary to normalize the data by comparing them to constitutive genes that exhibit uniform expression levels under experimental conditions. In this study, the stability of expression was evaluated for the following ten candidate reference genes in rice leaves (Oryza sativa L.) from the BRS Bojuru and BRS Ligeirinho genotypes that were subjected to salt stress (150 mM): actin 11 (ACT11), beta-tubulin (β-TUB), eukaryote elongation factor 1-α (Eef-1), eukaryotic initiation factor 4-α (eIF-4-α), E2 ubiquitinconjugating enzyme (UBC-E2), ubiquitin 5 (UBQ5), ubiquitin 10 (UBQ10), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TIP41-like, and cyclophilin. The stability of expression for the aforementioned genes was then compared to that of three LTP genes using UBQ10, Eef-1, and eIF-4-α as references. After analyzing the Reference genes for RT-qPCR in rice under salt stress expression levels using analysis of variance tests, the results indicated that UBQ10 was the most stable in all treatments (M = 0.404 and SV = 0.327). Furthermore, the eIF-4-α, TIP41-like, and cyclophilin genes exhibited the highest total coefficient of variation (CV = 269, 169.2, 179.2, respectively), which signifies that they exhibited the least stable expression. The expression levels of each candidate gene (LTP7, LTP10, and LTP13) were in contrast to the reference genes. Therefore, we concluded that UBQ10 is the best reference gene for RT-qPCR reactions under the experimental conditions. The expression analysis of LTP7, LTP10, and LTP13 confirmed the importance of validating reference genes to achieve accurate RT-qPCR results.

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Rice (Oryza sativa L.) is one of the most important species for food production worldwide, besides being an excellent genetic model among the grasses. Cold is one of the major abiotic factors reducing rice yield, primarily affecting germination and reproduction phases. Currently, the RNAseq technique allows the identification of differential expressed genes in response to a given treatment, such as cold stress. In the present work, a transcriptome (RNAseq) analysis was performed in the V3 phase for contrasting genotypes Oro (tolerant) and Tio Taka (sensitive), in response to cold (13°C). A total of 241 and 244 M readings were obtained, resulting in the alignment of 25.703 and 26.963 genes in genotypes Oro and Tio Taka respectively. The analyses revealed 259 and 5579 differential expressed genes in response to cold in the genotypes Oro and Tio Taka respectively. Ontology classes with larger changes were metabolic process ~27%, cellular process ~21%, binding ~30% and catalytic activity ~22%. In the genotype Oro, 141 unique genes were identified, 118 were common between Oro and Tio Taka and 5461 were unique to Tio Taka. Genes involved in metabolic routes of signal transduction, phytohormones, antioxidant system and biotic stress were identified. These results provide an understanding that breeding for a quantitative trait, such as cold tolerance at germination, several gene loci must be simultaneously selected. In general, few genes were identified, but it was not possible to associate only one gene function as responsible for the cultivar tolerance; since different genes from different metabolic routes were identified. The genes described in the present work will be useful for future investigations and for the detailed validation in marker assisted selection projects for cold tolerance in the germination of rice.

Due to its tropical origin and adaptation, rice is significantly impacted by cold stress, and consequently sustains large losses in growth and productivity. Currently, rice is the second most consumed cereal in the world and production losses caused by extreme temperature events in the context of "major climatic changes" can have major impacts on the world economy. We report here an analysis of rice genotypes in response to low-temperature stress, studied through physiological gas-exchange parameters, biochemical changes in photosynthetic pigments and antioxidants, and at the level of gene and protein expression, towards an understanding and identification of multiple low-temperature tolerance mechanisms. The first effects of cold stress were observed on photosynthesis among all genotypes. However, the tropical japonica genotypes Secano do Brazil and Cypress had a greater reduction in gas exchange parameters like photosynthesis and water use efficiency in comparison to the temperate japonica Nipponbare and M202 genotypes. The analysis of biochemical profiles showed that despite the impacts of low temperature on tolerant plants, they quickly adjusted to maintain their cellular homeostasis by an accumulation of antioxidants and osmolytes like phenolic compounds and proline. The cold tolerant and sensitive genotypes showed a clear difference in gene expression at the transcript level for OsGH3-2 , OsSRO1a , OsZFP245 , and OsTPP1 , as well as for expression at the protein level for LRR-RLKs, bHLH, GLYI, and LTP1 proteins. This study exemplifies the cold tolerant features of the temperate japonica Nipponbare and M202 genotypes, as observed through the analysis of physiological and biochemical responses and the associated changes in gene and protein expression patterns. The genes and proteins showing differential expression response are notable candidates towards understanding the biological pathways affected in rice and for engineering cold tolerance, to generate cultivars capable of maintaining growth, development, and reproduction under cold stress. We also propose that the mechanisms of action of the genes analyzed are associated with the tolerance response.

Plants are constantly exposed to environmental fluctuations, that may occur in a single day or over longer periods. In many cases, abiotic stresses are transient and recurrent, impacting how plants respond in subsequent adverse conditions. Adaptation mechanisms may occur at the physiological, biochemical and molecular level, modifying transcriptional response, regulatory proteins, epigenetic marks or metabolites. Here, we aimed to uncover the different strategies that rice uses to respond to recurrent stress. We tested varieties with contrasting behavior towards salinity (tolerance or sensitivity) and imposed salt stress (150 mM NaCl) during 48 h at vegetative and/or reproductive stages. After 48 h of stress in reproductive stage, leaves and roots were harvested separately or otherwise the plants were submitted to a 24 h recovery, prior to sample harvesting. Plants submitted to a recurrent stress responded differently from those suffering a single stress event. In the case of the sensitive genotype, recurrent stress led to lower Na/K ratio in roots and lower hydrogen peroxide accumulation and lipid peroxidation in leaves, but maintenance of global DNA methylation levels. In the tolerant genotype, recurrent stress did neither affect the Na/K ratio nor the stomatal conductance, although the levels of superoxide anion and hydrogen peroxide accumulation were lower, as also observed for global levels of DNA methylation. Our work shows that a short pre‐exposure to salt stress may improve rice tolerance to subsequent stress, trough biochemical, physiological and epigenetic processes, with more significant changes visible in the tolerant genotype.

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Abiotic stresses such as salinity, iron toxicity, and low temperatures are the main limiting factors of rice (Oryza sativa L.) yield. The elucidation of the genes involved in responses to these stresses is extremely important to understand the mechanisms that confer tolerance, as well as for the development of cultivars adapted to these conditions. In this study, the RNA-seq technique was used to compare the transcriptional profile of rice leaves (cv. BRS Querência) in stage V3, exposed to cold, iron, and salt stresses for 24 h. A range of 41 to 51 million reads was aligned, in which a total range of 88.47 to 89.21 % was mapped in the reference genome. For cold stress, 7905 differentially expressed genes (DEGs) were observed, 2092 for salt and 681 for iron stress; 370 of these were common to the three DEG stresses. Functional annotation by software MapMan demonstrated that cold stress usually promoted the greatest changes in the overall metabolism, and an enrichment analysis of overrepresented gene ontology (GO) terms showed that most of them are contained in plastids, ribosome, and chloroplasts. Saline stress induced a more complex interaction network of upregulated overrepresented GO terms with a relatively low number of genes compared with cold stress. Our study demonstrated a high number of differentially expressed genes under cold stress and a greater relationship between salt and iron stress levels. The physiological process most affected at the molecular level by the three stresses seems to be photosynthesis.