Drought stress tolerance strategies revealed by RNA-Seq in two sorghum genotypes with contrasting WUE.

BACKGROUND: Drought stress is the major environmental stress that affects plant growth and productivity. It triggers a wide range of responses detectable at molecular, biochemical and physiological levels. At the molecular level the response to drought stress results in the differential expression of several metabolic pathways. For this reason, exploring the subtle differences in gene expression of drought sensitive and drought tolerant genotypes enables the identification of drought-related genes that could be used for selection of drought tolerance traits. Genome-wide RNA-Seq technology was used to compare the drought response of two sorghum genotypes characterized by contrasting water use efficiency. RESULTS: The physiological measurements carried out confirmed the drought sensitivity of IS20351 and the drought tolerance of IS22330 genotypes, as previously studied. The expression of drought-related genes was more abundant in the drought sensitive genotype IS20351 compared to the tolerant genotype IS22330. Under drought stress Gene Ontology enrichment highlighted a massive increase in transcript abundance in the sensitive genotype IS20351 in "response to stress" and "abiotic stimulus", as well as for "oxidation-reduction reaction". "Antioxidant" and "secondary metabolism", "photosynthesis and carbon fixation process", "lipids" and "carbon metabolism" were the pathways most affected by drought in the sensitive genotype IS20351. In addition, genotype IS20351 showed a lower constitutive expression level of "secondary metabolic process" (GO:0019748) and "glutathione transferase activity" (GO:000004364) under well-watered conditions. CONCLUSIONS: RNA-Seq analysis proved to be a very useful tool to explore differences between sensitive and tolerant sorghum genotypes. Transcriptomics analysis results supported all the physiological measurements and were essential to clarify the tolerance of the two genotypes studied. The connection between differential gene expression and physiological response to drought unequivocally revealed the drought tolerance of genotype IS22330 and the strategy adopted to cope with drought stress.

Integration of Gas Exchange With Metabolomics: High-Throughput Phenotyping Methods for Screening Biostimulant-Elicited Beneficial Responses to Short-Term Water Deficit.

Biostimulants are emerging as a feasible tool for counteracting reduction in climate change-related yield and quality under water scarcity. As they are gaining attention, the necessity for accurately assessing phenotypic variables in their evaluation is emerging as a critical issue. In light of this, high-throughput phenotyping techniques have been more widely adopted. The main bottleneck of these techniques is represented by data management, which needs to be tailored to the complex, often multifactorial, data. This calls for the adoption of non-linear regression models capable of capturing dynamic data and also the interaction and effects between multiple factors. In this framework, a commercial glycinebetaine- (GB-) based biostimulant (Vegetal B60, ED&F Man) was tested and distributed at a rate of 6 kg/ha. Exogenous application of GB, a widely accumulated and documented stress adaptor molecule in plants, has been demonstrated to enhance the plant abiotic stress tolerance, including drought. Trials were conducted on tomato plants during the flowering stage in a greenhouse. The experiment was designed as a factorial combination of irrigation (water-stressed and well-watered) and biostimulant treatment (treated and control) and adopted a mixed phenotyping-omics approach. The efficacy of a continuous whole-canopy multichamber system coupled with generalized additive mixed modeling (GAMM) was evaluated to discriminate between water-stressed plants under the biostimulant treatment. Photosynthetic performance was evaluated by using GAMM, and was then correlated to metabolic profile. The results confirmed a higher photosynthetic efficiency of the treated plants, which is correlated to biostimulant-mediated drought tolerance. Furthermore, metabolomic analyses demonstrated the priming effect of the biostimulant for stress tolerance and detoxification and stabilization of photosynthetic machinery. In support of this, the overaccumulation of carotenoids was particularly relevant, given their photoprotective role in preventing the overexcitation of photosystem II. Metabolic profile and photosynthetic performance findings suggest an increased effective use of water (EUW) through the overaccumulation of lipids and leaf thickening. The positive effect of GB on water stress resistance could be attributed to both the delayed onset of stress and the elicitation of stress priming through the induction of H2O2-mediated antioxidant mechanisms. Overall, the mixed approach supported by a GAMM analysis could prove a valuable contribution to high-throughput biostimulant testing.

Increased water use efficiency in miR396-downregulated tomato plants.

MicroRNAs regulate plant development and responses to biotic and abiotic stresses but their impact on water use efficiency (WUE) is poorly known. Increasing WUE is a major task in crop improvement programs aimed to meet the challenges posed by the reduction in water availability associated with the ongoing climatic change. We have examined the physiological and molecular response to water stress of tomato (Solanum lycopersicum L.) plants downregulated for miR396 by target mimicry. In water stress conditions, miR396-downregulated plants displayed reduced transpiration and a less then proportional decrease in the photosynthetic rate that resulted in higher WUE. The increase in WUE was associated with faster foliar accumulation of abscisic acid (ABA), with the induction of several drought-protective genes and with the activation of the jasmonic acid (JA) and γ-aminobutyric acid (GABA) pathways. We propose a model in which the downregulation of miR396 leads to the activation of a complex molecular response to water stress. This response acts synergistically with a set of leaf morphological modifications to increase stomatal closure and preserve the efficiency of the photosynthetic activity, ultimately resulting in higher WUE.

Bioaugmented Phytoremediation of Metal-Contaminated Soils and Sediments by Hemp and Giant Reed.

We assessed the effects of EDTA and selected plant growth-promoting rhizobacteria (PGPR) on the phytoremediation of soils and sediments historically contaminated by Cr, Ni, and Cu. A total of 42 bacterial strains resistant to these heavy metals (HMs) were isolated and screened for PGP traits and metal bioaccumulation, and two Enterobacter spp. strains were finally selected. Phytoremediation pot experiments of 2 months duration were carried out with hemp (Cannabis sativa L.) and giant reed (Arundo donax L.) grown on soils and sediments respectively, comparing in both cases the effects of bioaugmentation with a single PGPR and EDTA addition on plant and root growth, plant HM uptake, HM leaching, as well as the changes that occurred in soil microbial communities (structure, biomass, and activity). Good removal percentages on a dry mass basis of Cr (0.4%), Ni (0.6%), and Cu (0.9%) were observed in giant reed while negligible values (<100‰) in hemp. In giant reed, HMs accumulated differentially in plant (rhizomes > > roots > leaves > stems) with largest quantities in rhizomes (Cr 0.6, Ni 3.7, and Cu 2.2 g plant-1). EDTA increased Ni and Cu translocation to aerial parts in both crops, despite that in sediments high HM concentrations in leachates were measured. PGPR did not impact fine root diameter distribution of both crops compared with control while EDTA negatively affected root diameter class length (DCL) distribution. Under HM contamination, giant reed roots become shorter (from 5.2 to 2.3 mm cm-3) while hemp roots become shorter and thickened from 0.13 to 0.26 mm. A consistent indirect effect of HM levels on the soil microbiome (diversity and activity) mediated by plant response (root DCL distribution) was observed. Multivariate analysis of bacterial diversity and activity revealed not only significant effects of plant and soil type (rhizosphere vs. bulk) but also a clear and similar differentiation of communities between control, EDTA, and PGPR treatments. We propose root DCL distribution as a key plant trait to understand detrimental effect of HMs on microbial communities. Positive evidence of the soil-microbe-plant interactions occurring when bioaugmentation with PGPR is associated with deep-rooting perennial crops makes this combination preferable over the one with chelating agents. Such knowledge might help to yield better bioaugmented bioremediation results in contaminated sites.

Water- and Nitrogen-Use Efficiencies of Hemp (Cannabis sativa L.) Based on Whole-Canopy Measurements and Modeling.

Interest in hemp (Cannabis sativa L.) as a crop for the biobased economy is growing worldwide because hemp produces a high and valuable biomass while requiring low inputs. To understand the physiological basis of hemp's resource-use efficiency, canopy gas exchange was assessed using a chamber technique on canopies exposed to a range of nitrogen (N) and water levels. Since canopy transpiration and carbon assimilation were very sensitive to variations in microclimate among canopy chambers, observations were adjusted for microclimatic differences using a physiological canopy model, with leaf-level parameters estimated for hemp from our previous study. Canopy photosynthetic water-use efficiency (PWUEc), defined as the ratio of gross canopy photosynthesis to canopy transpiration, ranged from 4.0 mmol CO2 (mol H2O)-1 to 7.5 mmol CO2 (mol H2O)-1. Canopy photosynthetic nitrogen-use efficiency (PNUEc), the ratio of the gross canopy photosynthesis to canopy leaf-N content, ranged from 0.3 mol CO2 d-1 (g N)-1 to 0.7 mol CO2 d-1 (g N)-1. The effect of N-input levels on PWUEc and PNUEc was largely determined by the N effect on canopy size or leaf area index (LAI), whereas the effect of water-input levels differed between short- and long-term stresses. The effect of short-term water stress was reflected by stomatal regulation. The long-term stress increased leaf senescence, decreased LAI but retained total canopy N content; however, the increased average leaf-N could not compensate for the lost LAI, leading to a decreased PNUEc. Although hemp is known as a resource-use efficient crop, its final biomass yield and nitrogen use efficiency may be restricted by water limitation during growth. Our results also suggest that crop models should take stress-induced senescence into account in addition to stomatal effects if crops experience a prolonged water stress during growth.

Real-Time Determination of Photosynthesis, Transpiration, Water-Use Efficiency and Gene Expression of Two Sorghum bicolor (Moench) Genotypes Subjected to Dry-Down.

Plant growth and productivity are strongly affected by limited water availability in drought prone environments. The current climate change scenario, characterized by long periods without precipitations followed by short but intense rainfall, forces plants to implement different strategies to cope with drought stress. Understanding how plants use water during periods of limited water availability is of primary importance to identify and select the best adapted genotypes to a certain environment. Two sorghum genotypes IS22330 and IS20351, previously characterized as drought tolerant and drought sensitive genotypes, were subjected to progressive drought stress through a dry-down experiment. A whole-canopy multi-chamber system was used to determine the in vivo water use efficiency (WUE). This system records whole-canopy net photosynthetic and transpiration rate of 12 chambers five times per hour allowing the calculation of whole-canopy instantaneous WUE daily trends. Daily net photosynthesis and transpiration rates were coupled with gene expression dynamics of five drought related genes. Under drought stress, the tolerant genotype increased expression level for all the genes analyzed, whilst the opposite trend was highlighted by the drought sensitive genotype. Correlation between gene expression dynamics and gas exchange measurements allowed to identify three genes as valuable candidate to assess drought tolerance in sorghum.

Drought tolerance strategies highlighted by two Sorghum bicolor races in a dry-down experiment.

Drought stress is the major environmental stress that affects more and more frequently plant growth and productivity due to the current climate change scenario. Unravelling the physiological mechanism underlying the response of plants to water stress and discover traits related to drought tolerance provide new and powerful tools for the selection in breeding programmes. Four genotypes of Sorghum bicolor (L.) Moench were screened in a dry-down experiment using different approaches to discover physiological and molecular indicators of drought tolerance. Different strategies were identified in response to drought among the four genotypes and the two Sorghum race allowing to state the tolerance of durra race compared to the caudatum one and, within the durra race, the drought tolerance of the genotype IS22330. It retained high biomass production and high tolerance index, it had a low threshold of fraction of transpirable soil water and high capacity to recover leaf apparatus after drought stress. Furthermore in this study, the expression levels of four genes highlighted that they could be used as proxy for drought tolerance. Dehdrine (DHN) could be used for screening drought tolerance both in durra and in caudatum races. NADP-Malic Enzyme, Carbonic Anhydrase (CA) and Plasma membrane Intrinsic Protein (PIP2-5), being up-regulated by drought stress only in durra race, have a more limited, though nonetheless useful application. In the tolerant durra genotype IS22330 in particular, the regulation of stomatal openings was strongly related to NADP-Malic Enzyme expression.

Microarray analysis of differentially expressed mRNAs and miRNAs in young leaves of sorghum under dry-down conditions.

Sorghum is a C4 plant adapted to semi-arid environments, and characterized by high water-use efficiency. To better understand the molecular and physiological basis of drought response the sorghum genotype IS19453, selected as a drought tolerant line during field trials, was evaluated in a "dry-down" experiment under controlled conditions. The incoming stress was monitored by determining the water potential available for 4-leaf-old plants. Control plants were maintained at approximately 2.5 pF, while water stressed plants were sampled at 3.12, 3.65 and 4.14 pF. Transcriptome analysis was monitored using a high density microarray containing all available sorghum TC sequences. Drought affected gene expression at 4.14 pF; 1205 genes resulted up-regulated. Most of the differentially expressed genes were involved in regulation of transcription (bZIPs, MYBs, HOXs), signal transduction (phosphoesterases, kinases, phosphatases), carbon metabolism (NADP-ME), detoxification (CYPs, GST, AKRs), osmoprotection mechanisms (P5CS) and stability of protein membranes (DHN1, LEA, HSPs). Several of them could be located in stay green QTLs. Eight were selected and validated by qRT-PCR. A dedicated miRNA microarray allowed the identification of four families of miRNAs up-regulated in the earlier phase of stress, while one family was down-regulated. The selected drought related genes could be used to screen for potential drought tolerance in other sorghum genotypes.