Proteomic analysis of the meristematic root zone in contrasting genotypes reveals new insights in drought tolerance in rice.

Drought is responsible for major losses in rice production. Root tips contain meristematic and elongation zones that play major roles in determination of root traits and adaptive strategies to drought. In this study we analysed two contrasting genotypes of rice: IR64, a lowland, drought-susceptible, and shallow-rooting genotype; and Azucena, an upland, drought-tolerant, and deep-rooting genotype. Samples were collected of root tips of plants grown under control and water deficit stress conditions. Quantitative proteomics analysis resulted in the identification of 7294 proteins from the root tips of IR64 and 6307 proteins from Azucena. Data are available via ProteomeXchange with identifier PXD033343. Using a Partial Least Square Discriminant Analysis on 4170 differentially abundant proteins, 1138 statistically significant proteins across genotypes and conditions were detected. Twenty two enriched biological processes showing contrasting patterns between two genotypes in response to stress were detected through gene ontology enrichment analysis. This included identification of novel proteins involved in root elongation with specific expression patterns in Azucena, including four Expansins and seven Class III Peroxidases. We also detected an antioxidant network and a metallo-sulfur cluster assembly machinery in Azucena, with roles in reactive oxygen species and iron homeostasis, and positive effects on root cell cycle, growth and elongation.

Genome-Wide Expression Analysis of Root Tips in Contrasting Rice Genotypes Revealed Novel Candidate Genes for Water Stress Adaptation.

Root system architecture (RSA) is an important agronomic trait with vital roles in plant productivity under water stress conditions. A deep and branched root system may help plants to avoid water stress by enabling them to acquire more water and nutrient resources. Nevertheless, our knowledge of the genetics and molecular control mechanisms of RSA is still relatively limited. In this study, we analyzed the transcriptome response of root tips to water stress in two well-known genotypes of rice: IR64, a high-yielding lowland genotype, which represents a drought-susceptible and shallow-rooting genotype; and Azucena, a traditional, upland, drought-tolerant and deep-rooting genotype. We collected samples from three zones (Z) of root tip: two consecutive 5 mm sections (Z1 and Z2) and the following next 10 mm section (Z3), which mainly includes meristematic and maturation regions. Our results showed that Z1 of Azucena was enriched for genes involved in cell cycle and division and root growth and development whereas in IR64 root, responses to oxidative stress were strongly enriched. While the expansion of the lateral root system was used as a strategy by both genotypes when facing water shortage, it was more pronounced in Azucena. Our results also suggested that by enhancing meristematic cell wall thickening for insulation purposes as a means of confronting stress, the sensitive IR64 genotype may have reduced its capacity for root elongation to extract water from deeper layers of the soil. Furthermore, several members of gene families such as NAC, AP2/ERF, AUX/IAA, EXPANSIN, WRKY, and MYB emerged as main players in RSA and drought adaptation. We also found that HSP and HSF gene families participated in oxidative stress inhibition in IR64 root tip. Meta-quantitative trait loci (QTL) analysis revealed that 288 differentially expressed genes were colocalized with RSA QTLs previously reported under drought and normal conditions. This finding warrants further research into their possible roles in drought adaptation. Overall, our analyses presented several major molecular differences between Azucena and IR64, which may partly explain their differential root growth responses to water stress. It appears that Azucena avoided water stress through enhancing growth and root exploration to access water, whereas IR64 might mainly rely on cell insulation to maintain water and antioxidant system to withstand stress. We identified a large number of novel RSA and drought associated candidate genes, which should encourage further exploration of their potential to enhance drought adaptation in rice.

Seasonal Metabolic Investigation in Pomegranate (Punica granatum L.) Highlights the Role of Amino Acids in Genotype- and Organ-Specific Adaptive Responses to Freezing Stress.

Every winter, temperate woody plants have to cope with freezing stress. Winter hardiness is of crucial importance for pomegranate survival and productivity. A comparative morphological and metabolic study was conducted on the stems and buds of 15 field-grown mature pomegranate genotypes in seven time-points during two developmental cycles. Seasonal changes of frost hardiness, as determined by electrolyte leakage method, and metabolite analysis by HPLC and GC revealed the variability in frost hardiness and metabolic contents result from genetic background and organ, as well as seasonal condition. Morphological adaptations, as well as metabolic remodeling, are the distinct features of the hardy genotypes. Larger buds with a greater number of compressed scales and the higher number of protective leaves, together with the higher number and content of changed metabolites, especially amino acids, seem to provide a higher frost resistance for those trees. We recorded two-times the change in metabolites and several-times accumulation of amino acids in the stem compared with buds. A better potential of stem for metabolome adjustment during the hardening period and a higher level of tolerance to stress is therefore suggested. High levels of arginine, proline, glutamine, and asparagine, and particularly the accumulation of alanine, tryptophan, and histidine are responsible for excellent tolerance of the stem of tolerant genotypes. With regard to the protective roles of amino acids, a relation between stress tolerance and the level of amino acids is proposed. This points both to the importance of amino acids in the winter survival of pomegranate trees, and to the evaluation of frost tolerance in other plants, by these specific markers.

Melittin inhibits the expression of key genes involved in tumor microenvironment formation by suppressing HIF-1α signaling in breast cancer cells.

HIF-1α has critical roles in the formation of tumor microenvironment by regulating genes involved in angiogenesis and anaerobic respiration. TME fuels tumors' growth and metastasis and presents therapy with several challenges. Therefore, we aimed to investigate if Melittin disrupts HIF-1α signaling pathway in breast adenocarcinoma cell line MDA-MB-231. Breast adenocarcinoma cell line MDA-MB-231 was cultured in the presence of different doses of Melittin, and MTT assay was carried out to measure Melittin's cytotoxic effects. Cells were exposed to 5% O2 to mimic hypoxic conditions and Melittin. Western blot was used to measure HIF-1α protein levels. Gene expression analysis was performed using real-time PCR to measure relative mRNA abundance of genes involved in tumor microenvironment formation. Our results revealed that Melittin effectively inhibits HIF-1α at transcriptional and translational/post-translational level. HIF-1α protein and mRNA level were significantly decreased in Melittin-treated groups. It is found that inhibition of HIF-1α by Melittin is through downregulation of NFκB gene expression. Furthermore, gene expression analysis showed a downregulation in VEGFA and LDHA expression due to inhibition of HIF-1α protein by Melittin. In addition, cell toxicity assay showed that Melittin inhibits the growth of MDA-MB-231 cell line through activation of extrinsic and intrinsic apoptotic pathways by upregulating TNFA and BAX expression. Melittin suppresses the expression of genes responsible for formation of TME physiological hallmarks by suppressing HIF-1α signaling pathway. Our results suggest that Melittin can modulate tumor microenvironment by inhibition of VEGFA and LDHA.

Differential adaptation strategies to different levels of soil water deficit in two upland and lowland genotypes of rice: a physiological and metabolic approach.

BACKGROUND: Upland genotypes of rice are less sensitive to soil water deficit (SWD), making them suitable candidates for revealing the strategies underlying plant tolerance. The physiological factors, the biochemical traits needed to withstand oxidative stress, and the metabolite fluctuations of an upland genotype (Azucena) and an intolerant lowland genotype (IR64) genotype were measured under two levels of SWD (withholding water for 7- or 14 days) to identify SWD-responsive strategies associated with tolerance. RESULTS: After withholding water for 7 days, no significant changes in physiological and biochemical traits of Azucena were observed, whereas in IR64, significant decreases in physiological factors were recorded along with increases in oxidative-stress indicators. However, the root length of Azucena increased significantly, showing a clear stress avoidance strategy. Under a prolonged treatment (14 days), IR64 entered an oxidative-damage stage, whereas Azucena exhibited a highly efficient antioxidant system. Our metabolite analysis also revealed two different enriched pathways. After a 7-day SWD, the sugar levels were decreased in the leaves of Azucena but increased in IR64. The reduction in the sugar levels (up to 1.79-log2FC) in the Azucena leaves may be indicative of their transport to the roots, supplying the carbon source needed for root elongation. Under a 14-day treatment, proline and aspartate family members accumulated to the highest levels in Azucena, whereas an increase in the levels of aromatic amino acids with key roles in the biosynthesis of secondary metabolites was detected in IR64. CONCLUSION: The adaptation strategies identified in two types of rice genotypes in confronting SWD may assist researchers in finding the proper indicators for screening more tolerant genotypes. © 2019 Society of Chemical Industry.