Long-term somatic memory of salinity unveiled from physiological, biochemical and epigenetic responses in two contrasting rice genotypes.

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.

Validation of reference genes for RT-qPCR studies in Stevia rebaudiana in response to elicitor agents.

Stevia rebaudiana is an important source of natural steviol glycosides and is of increasing interest in various fields of study. Therefore, understanding the molecular processes regulating its metabolism is of great importance. In this study, the stability of seven reference genes (18S ribosomal RNA, Actin, Aquaporin, Calmodulin, Eukaryote elongation factor 1-α, Malate dehydrogenase, and Ubiquitin) under the effect of three stress-related elicitors (methyl jasmonate, salicylic acid, and spermidine) was evaluated in stevia plants. We used RefFinder software, which makes use of the four main currently available algorithms for reference gene selection: geNorm, NormFinder, BestKeeper, and the Comparative ∆Ct method. The results indicated that Ubiquitin and Actin can be used as reference genes under all tested experimental conditions. The genes, 18S ribosomal RNA, traditionally used as a reference gene, along with Calmodulin, showed the lowest stability. The expression of Deoxyxylulose-5-phosphate synthase and Kaurenoic acid hydroxylase genes was used to confirm the validated reference genes, showing that inadequacy of the reference gene may lead to erroneous results. This is the first study on the stability of reference genes in Stevia rebaudiana plants, and is of great relevance for further analysis of the gene expression of the steviol glycoside biosynthetic pathway.

Molecular responses to recurrent drought in two contrasting rice genotypes.

MAIN CONCLUSION: The set of variables analyzed as integrated by multivariate analysis of principal components consistently showed a memory effect induced by the drought pre-treatment in AN Cambará plants. The effects of drought can vary ddepending on many factors. Among these the occurrence of a previous water stress may leave a residual effect (memory), influencing the future performance of a plant in response to a new drought event. This study tested the hypothesis that plants experiencing recurrent drought would show more active mechanisms of water deficit tolerance, mainly plants of the genotype that is cultivated often experiencing water shortages periods. Additionally, all the plants subjected to water deficit were rehydrated by 24 h and the expression of transcription factors related to drought responses was re-evaluated. To this end, the water status of two rice genotypes, BRS Querência (flooded) and AN Cambará (dryland), was evaluated to identify molecular alterations likely underpinning drought-memory. In growth stage V5, some plants were exposed to water stress (10% VWC soil moisture-pre-treatment). Thereafter, the pots were rehydrated at the same level as the control pots and maintained under this condition until drought was reapplied (10% VWC) at the reproductive stage (R1-R2). Then, the plants were rehydrated and maintained at pot capacity for 24 h. Overall, the set of variables analyzed integrally by multivariate analysis of principal components consistently showed a memory effect induced by the drought pre-treatment in AN Cambará plants (the dryland genotype). This conclusion, based on data of the biochemical and molecular analyses, was supported by the greater capacity of maintenance of the water status by stomatal regulation of the pre-treated and rehydrated plants after the second drought stimulus.

Gene expression and activity of antioxidant enzymes in rice plants, cv. BRS AG, under saline stress.

The rice cultivar (Oryza sativa 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 OsSOD3-CU/Zn, OsSOD2-Cu/Zn, OsSOD-Cu/Zn, OsSOD4-Cu/Zn, OsSODCc1-Cu/Zn, OsSOD-Fe, OsAPX1, OsCATB and OsGR2 were the most responsive. The increase in the transcription of all genes among evaluated isoforms, except for OsAPX6, 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.

Comparative transcriptomics of rice plants under cold, iron, and salt stresses.

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.

Biosynthesis of Betalains Elicited by Methyl Jasmonate in Two Species of Alternanthera Genus: Antagonistic Regulations Result in Increase of Pigments.

Natural pigments are components very important in the dye industry. The betalains are pigments found in plants from Caryophyllales order and are relevant in the food manufacturing. The main source of betalains is beetroot, which has unfavorable aftertaste. Therefore, the demand for alternative species producing betalains has increased. Elicitor molecules such as methyl jasmonate (MeJA) induce metabolic reprogramming acting in the biosynthesis of specialized metabolites and can enhance pigment concentrations. Here, we used this strategy to identify if treatment with MeJA at 100 µM can promote the accumulation of betalains and other bioactive compounds in Alternanthera philoxeroides and Alternanthera sessilis. We performed the gene expression, concentration of betalains, phenols, flavonoids, amino acids (phenylalanine and tyrosine), and antioxidant activity. The results showed that MeJA treatment increased betalains and other bioactive compounds in the two Alternanthera species but A. sessilis had a better performance. One key factor in this pathway is related to the phenylalanine and tyrosine concentration. However, the species have distinct metabolic regulation: in A. philoxeroides, high concentrations of betalain pigments increase the tyrosine concentration and gene expression (include ADH) under MeJA and in A. sessilis, high concentrations of betalain pigments reduce the gene expression and tyrosine concentration after 2 days under MeJA. This study brings new questions about betalain biosynthesis and sheds light on the evolution of this pathway in Caryophyllales.

Stress memory of physiological, biochemical and metabolomic responses in two different rice genotypes under drought stress: The scale matters.

Drought is a pivotal cause for crop yield reductions. When subjected to recurrent external stimuli, plants can develop memory of stress responses that, eventually, enables improved plant tolerance to environmental changes. In addition, despite causal relationships, these responses may vary according to hierarchical levels of observation. Thus, this study aims to check the responses of recurrent and non-recurrent stresses in two rice genotypes observing their drought memory responses at different levels of organization, that is, on a physiological, biochemical and metabolomic scale and for end in global PCA. For this, seventy variables were measured on the scales described in order to obtain a large number of observations. The memory responses were evident in almost all scales observed. The lowland genotype, especially plants not subjected to recurrent water shortage, showed higher damage to the photosynthetic apparatus under drought conditions, although it has exhibited more evident memory response effect after rehydration. On the other hand, the upland genotype appears to be more tolerant to drought insofar lower biochemical damage was observed. Specific behaviors of each genotype at biochemical and metabolomics levels and similar behavior at physiological level were observed. This study demonstrates the importance of observation at different hierarchical levels.

Cold tolerance response mechanisms revealed through comparative analysis of gene and protein expression in multiple rice genotypes.

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.

Evaluation of stability and validation of reference genes for RT-qPCR expression studies in rice plants under water deficit.

Many studies use strategies that allow for the identification of a large number of genes expressed in response to different stress conditions to which the plant is subjected throughout its cycle. In order to obtain accurate and reliable results in gene expression studies, it is necessary to use reference genes, which must have uniform expression in the majority of cells in the organism studied. RNA isolation of leaves and expression analysis in real-time quantitative polymerase chain reaction (RT-qPCR) were carried out. In this study, nine candidate reference genes were tested, actin 11 (ACT11), ubiquitin conjugated to E2 enzyme (UBC-E2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta tubulin (ß-tubulin), eukaryotic initiation factor 4α (eIF-4α), ubiquitin 10 (UBQ10), ubiquitin 5 (UBQ5), aquaporin TIP41 (TIP41-Like) and cyclophilin, in two genotypes of rice, AN Cambará and BRS Querência, with different levels of soil moisture (20%, 10% and recovery) in the vegetative (V5) and reproductive stages (period preceding flowering). Currently, there are different softwares that perform stability analyses and define the most suitable reference genes for a particular study. In this study, we used five different methods: geNorm, BestKeeper, ΔCt method, NormFinder and RefFinder. The results indicate that UBC-E2 and UBQ5 can be used as reference genes in all samples and softwares evaluated. The genes ß-tubulin and eIF-4α, traditionally used as reference genes, along with GAPDH, presented lower stability values. The gene expression of basic leucine zipper (bZIP23 and bZIP72) was used to validate the selected reference genes, demonstrating that the use of an inappropriate reference can induce erroneous results.