Metabolism of abscisic acid in two contrasting rice genotypes submitted to recurrent water deficit.

Drought is the main constrain for crops worldwide, however, the effects of recurrent water deficit remain still hidden. We analysed two rice genotypes, 'BRS-Querência' (lowlands) and 'AN-Cambará' (uplands), after 7 days of recurrent drought followed by 24 h of rehydration, hypothesising that genotypes grown in regions with different water availabilities respond differently to water deficits, and that a previous exposure to stress could alter abscisic acid (ABA) metabolism. The results showed that both genotypes reduced stomatal conductance and increased ABA concentration. After rehydration, the ABA levels decreased, mainly in the plants of BRS-Querência subjected to recurrent stress. However, the levels of ABA were higher in plants in recurrent water deficit compared to non-recurrent stress plants in both genotypes. Remarkably in the lowland genotype, the ABA glucosyl-ester (ABA-GE) concentration increased after recovery in the plants under recurrent stress. Regarding of gene expression, the genes associated in ABA biosynthesis with the highest expression levels were NCED2, NCED3, NCED4 and AAO2. However, 'AN-Cambará' showed less transcriptional activation. Taking into account the genes involved in ABA catabolism, ABAH1 appears to play an important role related to the recurrent stress in upland plants. These results indicate that one of the factors that can promote greater tolerance for the upland genotype is the tradeoff between ABA and ABA-GE when plants are subjected to water deficits. In addition, they indicate that abscisic acid metabolism is altered due to the genotype (upland or lowland) and pre-exposure to stress can also modify adaptive responses in rice varieties (recurrent stress).

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.

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.

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.

Drought stress memory in rice guard cells: Proteome changes and genomic stability of DNA.

Drought is one of the major threats for crop plants among them rice, worldwide. The effects of drought vary depending on the plant growth phase and the occurrence of a previous stress, which can leave a memory of the stress. Stomata guard cells perform many essential functions and are highly responsive to hormonal and environmental stimuli. Therefore, information on how guard cells respond to drought might be useful for selecting drought tolerant plants. In this work, physiological analysis, comparative proteomics, gene expression and 5 - methylcytosine (%) analysis were used to elucidate the effects of drought in single stress event at vegetative or reproductive stage or recurrent at both stages in guard cells from rice plants. Photosynthesis and stomatal conductance decreased when drought was applied at reproductive stage in single and recurrent event. Twelve drought-responsive proteins were identified, belonging to photosynthesis pathway, response to oxidative stress, stress signalling and others. The expression of their encoding genes showed a positive relation with the protein abundance. Drought stress increased the total DNA methylation when applied at vegetative stage in single (35%) and recurrent event (18%) and decreased it in plants stressed at reproductive stage (9.8%), with respect to the levels measured in well-watered ones (13.84%). In conclusion, a first drought event seems to induce adaptation to water-deficit conditions through decreasing energy dissipation, increasing ATP energy provision, reducing oxidative damage in GC. Furthermore, the stress memory is associated with epigenetic markers.

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.

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.