Assessment of Drought and Zinc Stress Tolerance of Novel Miscanthus Hybrids and Arundo donax Clones Using Physiological, Biochemical, and Morphological Traits.

High-yield potential perennial crops, such as Miscanthus spp. and Arundo donax are amongst the most promising sources of sustainable biomass for bioproducts and bioenergy. Although several studies assessed the agronomic performance of these species on diverse marginal lands, research to date on drought and zinc (Zn) resistance is scarce. Thus, the objective of this study was to investigate the drought and Zn stress tolerance of seven novel Miscanthus hybrids and seven Arundo clones originating from different parts of Italy. We subjected both species to severe drought (less than 30%), and Zn stress (400 mg/kg-1 of ZnSO4) separately, after one month of growth. All plants were harvested after 28 days of stress, and the relative drought and Zn stress tolerance were determined by using a set of morpho-physio-biochemical and biomass attributes in relation to stress tolerance indices (STI). Principal component analysis (PCA), hierarchical clustering analysis (HCA) and stress tolerance indices (STI) were performed for each morpho-physio-biochemical and biomass parameters and showed significant relative differences among the seven genotypes of both crops. Heatmaps of these indices showed how the different genotypes clustered into four groups. Considering PCA ranking value, Miscanthus hybrid GRC10 (8.11) and Arundo clone PC1 (11.34) had the highest-ranking value under both stresses indicating these hybrids and clones are the most tolerant to drought and Zn stress. In contrast, hybrid GRC3 (-3.33 lowest ranking value) and clone CT2 (-5.84) were found to be the most sensitive to both drought and Zn stress.

Field Plant Monitoring from Macro to Micro Scale: Feasibility and Validation of Combined Field Monitoring Approaches from Remote to in Vivo to Cope with Drought Stress in Tomato.

Monitoring plant growth and development during cultivation to optimize resource use efficiency is crucial to achieve an increased sustainability of agriculture systems and ensure food security. In this study, we compared field monitoring approaches from the macro to micro scale with the aim of developing novel in vivo tools for field phenotyping and advancing the efficiency of drought stress detection at the field level. To this end, we tested different methodologies in the monitoring of tomato growth under different water regimes: (i) micro-scale (inserted in the plant stem) real-time monitoring with an organic electrochemical transistor (OECT)-based sensor, namely a bioristor, that enables continuous monitoring of the plant; (ii) medium-scale (<1 m from the canopy) monitoring through red-green-blue (RGB) low-cost imaging; (iii) macro-scale multispectral and thermal monitoring using an unmanned aerial vehicle (UAV). High correlations between aerial and proximal remote sensing were found with chlorophyll-related indices, although at specific time points (NDVI and NDRE with GGA and SPAD). The ion concentration and allocation monitored by the index R of the bioristor during the drought defense response were highly correlated with the water use indices (Crop Water Stress Index (CSWI), relative water content (RWC), vapor pressure deficit (VPD)). A high negative correlation was observed with the CWSI and, in turn, with the RWC. Although proximal remote sensing measurements correlated well with water stress indices, vegetation indices provide information about the crop's status at a specific moment. Meanwhile, the bioristor continuously monitors the ion movements and the correlated water use during plant growth and development, making this tool a promising device for field monitoring.

A Pseudomonas Plant Growth Promoting Rhizobacterium and Arbuscular Mycorrhiza differentially modulate the growth, photosynthetic performance, nutrients allocation, and stress response mechanisms triggered by a mild Zinc and Cadmium stress in tomato.

This study aimed to assess the effectiveness of plant growth-promoting rhizobacteria (PGPR; Pseudomonas strain So_08) and arbuscular mycorrhizal fungi (AMF; Rhizoglomus irregulare BEG72 and Funneliformis mosseae BEG234) in mitigating the detrimental effects of cadmium (Cd) and zinc (Zn) stress in tomato plants. Plant biomass, root morphology, leaf relative water content, membrane stability, photosynthetic performance, chlorophyll content, and heavy metals (HMs) accumulation were determined. Furthermore, an ionomic profile was conducted to investigate whether microbial inoculants affected the uptake and allocation of macro- and micronutrients. Metabolomics with pathway analysis of both roots and leaves was performed to unravel the mechanisms underlying the differential responses to HMs stress. The findings revealed that the levels of HMs did not significantly affect plant growth parameters; however, they affected membrane stability, photosynthetic performance, nutrient allocation, and chlorophyll content. Cadmium was mainly accumulated in roots, whilst Zn exhibited accumulation in various plant organs. Our findings demonstrate the beneficial effects of PGPR and AMF in mitigating Cd and Zn stress in tomato plants. The microbial inoculations improved physiological parameters and induced differential accumulation of macro- and micronutrients, modulating nutrient uptake balance. These results provide insights into the mechanisms underlying the plant-microbe interactions and highlight the differential modulation of the biosynthetic pathways of secondary metabolites related to oxidative stress response, membrane lipids stability, and phytohormone crosstalk.

Novel Miscanthus hybrids: Modelling productivity on marginal land in Europe using dynamics of canopy development determined by light interception.

New biomass crop hybrids for bioeconomic expansion require yield projections to determine their potential for strategic land use planning in the face of global challenges. Our biomass growth simulation incorporates radiation interception and conversion efficiency. Models often use leaf area to predict interception which is demanding to determine accurately, so instead we use low-cost rapid light interception measurements using a simple laboratory-made line ceptometer and relate the dynamics of canopy closure to thermal time, and to measurements of biomass. We apply the model to project the European biomass potentials of new market-ready hybrids for 2020-2030. Field measurements are easier to collect, the calibration is seasonally dynamic and reduces influence of weather variation between field sites. The model obtained is conservative, being calibrated by crops of varying establishment and varying maturity on less productive (marginal) land. This results in conservative projections of miscanthus hybrids for 2020-2030 based on 10% land use conversion of the least (productive) grassland and arable for farm diversification, which show a European potential of 80.7-89.7 Mt year-1 biomass, with potential for 1.2-1.3 EJ year-1 energy and 36.3-40.3 Mt year-1 carbon capture, with seeded Miscanthus sacchariflorus × sinensis displaying highest yield potential. Simulated biomass projections must be viewed in light of the field measurements on less productive land with high soil water deficits. We are attempting to model the results from an ambitious and novel project combining new hybrids across Europe with agronomy which has not been perfected on less productive sites. Nevertheless, at the time of energy sourcing issues, seed-propagated miscanthus hybrids for the upscaled provision of bioenergy offer an alternative source of renewable energy. If European countries provide incentives for growers to invest, seeded hybrids can improve product availability and biomass yields over the current commercial miscanthus variety.

Perennial biomass cropping and use: Shaping the policy ecosystem in European countries.

Demand for sustainably produced biomass is expected to increase with the need to provide renewable commodities, improve resource security and reduce greenhouse gas emissions in line with COP26 commitments. Studies have demonstrated additional environmental benefits of using perennial biomass crops (PBCs), when produced appropriately, as a feedstock for the growing bioeconomy, including utilisation for bioenergy (with or without carbon capture and storage). PBCs can potentially contribute to Common Agricultural Policy (CAP) (2023-27) objectives provided they are carefully integrated into farming systems and landscapes. Despite significant research and development (R&D) investment over decades in herbaceous and coppiced woody PBCs, deployment has largely stagnated due to social, economic and policy uncertainties. This paper identifies the challenges in creating policies that are acceptable to all actors. Development will need to be informed by measurement, reporting and verification (MRV) of greenhouse gas emissions reductions and other environmental, economic and social metrics. It discusses interlinked issues that must be considered in the expansion of PBC production: (i) available land; (ii) yield potential; (iii) integration into farming systems; (iv) R&D requirements; (v) utilisation options; and (vi) market systems and the socio-economic environment. It makes policy recommendations that would enable greater PBC deployment: (1) incentivise farmers and land managers through specific policy measures, including carbon pricing, to allocate their less productive and less profitable land for uses which deliver demonstrable greenhouse gas reductions; (2) enable greenhouse gas mitigation markets to develop and offer secure contracts for commercial developers of verifiable low-carbon bioenergy and bioproducts; (3) support innovation in biomass utilisation value chains; and (4) continue long-term, strategic R&D and education for positive environmental, economic and social sustainability impacts.

Nettle, a Long-Known Fiber Plant with New Perspectives.

The stinging nettle Urticadioica L. is a perennial crop with low fertilizer and pesticide requirements, well adapted to a wide range of environmental conditions. It has been successfully grown in most European climatic zones while also promoting local flora and fauna diversity. The cultivation of nettle could help meet the strong increase in demand for raw materials based on plant fibers as a substitute for artificial fibers in sectors as diverse as the textile and automotive industries. In the present review, we present a historical perspective of selection, harvest, and fiber processing features where the state of the art of nettle varietal selection is detailed. A synthesis of the general knowledge about its biology, adaptability, and genetics constituents, highlighting gaps in our current knowledge on interactions with other organisms, is provided. We further addressed cultivation and processing features, putting a special emphasis on harvesting systems and fiber extraction processes to improve fiber yield and quality. Various uses in industrial processes and notably for the restoration of marginal lands and avenues of future research on this high-value multi-use plant for the global fiber market are described.

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.

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.

The variety, terroir, and harvest types affect the yield and the phenolic and sterolic profiles of hemp seed oil.

In this work, considering the rising interest towards the exploitation of hemp seed oil in human nutrition, 45 hemp seeds from mono-variety fields were analyzed for their yield, oil content, in vitro antioxidant activity, followed by a comprehensive assessment of phenolic and sterolic composition. The results demonstrated that seed dimension is inversely correlated to total oil content, thus being a potential reference for quality assessment of seeds and for further improvement of hemp varieties. The UHPLC-QTOF metabolomic analysis revealed a large abundance of phytosterols, lower-molecular-weight phenolic acids, and lignanamides. Differences across varieties could be described, with Diana hemp seed oil having the highest cumulative abundance of phytochemicals, recording 6.04 mg/g. Overall, the in vitro antioxidant activity results indicated that hemp seed oil antioxidants have a low potential for preventing oil rancidity, with phenolic acids being the most active radical scavengers. Besides, in the group of Futura 75 samples cultivated across Italy, the type of harvesting affected the acidity value significantly as a consequence of mechanical harvest and post-harvest handling. Finally, multivariate statistics following untargeted metabolomic analysis showed that variety, geographical origin, and harvest-type were able to affect the phytochemical profiles with different incidences, with some phytochemicals proposed for the first time as potential discriminant markers.

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.

Genetic Variability of Morphological, Flowering, and Biomass Quality Traits in Hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L.) is a bast-fiber crop well-known for the great potential to produce sustainable fibers. Nevertheless, hemp fiber quality is a complex trait, and little is known about the phenotypic variability and heritability of fiber quality traits in hemp. The aim of this study is to gain insights into the variability in fiber quality within the hemp germplasm and to estimate the genetic components, environmental components, and genotype-by-environment (G×E) interactions on fiber quality traits in hemp. To investigate these parameters, a panel of 123 hemp accessions was phenotyped for 28 traits relevant to fiber quality at three locations in Europe, corresponding to climates of northern, central, and southern Europe. In general, hemp cultivated in northern latitudes showed a larger plant vigor while earlier flowering was characteristic of plants cultivated in southern latitudes. Extensive variability between accessions was observed for all traits. Most cell wall components (contents of monosaccharides derived from cellulose and hemicellulose; and lignin content), bast fiber content, and flowering traits revealed large genetic components with low G×E interactions and high broad-sense heritability values, making these traits suitable to maximize the genetic gains of fiber quality. In contrast, contents of pectin-related monosaccharides, most agronomic traits, and several fiber traits (fineness and decortication efficiency) showed low genetic components with large G×E interactions affecting the rankings across locations. These results suggest that pectin, agronomic traits, and fiber traits are unsuitable targets in breeding programs of hemp, as their large G×E interactions might lead to unexpected phenotypes in untested locations. Furthermore, all environmental effects on the 28 traits were statistically significant, suggesting a strong adaptive behavior of fiber quality in hemp to specific environments. The high variability in fiber quality observed in the hemp panel, the broad range in heritability, and adaptability among all traits prescribe positive prospects for the development of new hemp cultivars of excellent fiber quality.

Highly Predictive Genetic Markers Distinguish Drug-Type from Fiber-Type Cannabis sativa L.

Genetic markers can be used in seeds and in plants to distinguish drug-type from fiber-type Cannabis Sativa L. varieties even at early stages, including pre-germination when cannabinoids are not accumulated yet. With this aim, this paper reports sequencing results for tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes from 21 C. sativa L. varieties. Taking into account that THCAS- and CBDAS-derived enzymes compete for the same substrate, the novelty of this work relies in the identification of markers based on both THCAS and CBDAS rather than THCAS alone. Notably, in our panel, we achieved an adequate degree of discrimination (AUC 100%) between drug-type and fiber-type cannabis samples. Our sequencing approach allowed identifying multiple genetic markers (single-nucleotide polymorphisms-SNPs-and a deletion/insertion) that effectively discriminate between the two subgroups of cannabis, namely fiber type vs. drug type. We identified four functional SNPs that are likely to induce decreased THCAS activity in the fiber-type cannabis plants. We also report the finding on a deletion in the CBDAS gene sequence that produces a truncated protein, possibly resulting in loss of function of the enzyme in the drug-type varieties. Chemical analyses for the actual concentration of cannabinoids confirmed the identification of drug-type rather than fiber-type genotypes. Genetic markers permit an early identification process for forensic applications while simplifying the procedures related to detection of therapeutic or industrial hemp.

Optimizing Hemp Fiber Production for High Performance Composite Applications.

Hemp is a sustainable and environmental friendly crop that can provide valuable raw materials to a large number of industrial applications. Traditionally harvested at full flowering for textile destinations, nowadays hemp is mainly harvested at seed maturity for dual-purpose applications and has a great potential as multipurpose crop. However, the European hemp fiber market is stagnating if compared to the growing market of hemp seeds and phytocannabinoids. To support a sustainable growth of the hemp fiber market, agronomic techniques as well as genotypes and post-harvest processing should be optimized to preserve fiber quality during grain ripening, enabling industrial processing and maintaining, or even increasing, actual fiber applications and improving high-added value applications. In this paper, the effect of genotypes, harvest times, retting methods and processing on the yield and quality of long hemp for wet spun yarns was investigated. Conventional green-stem varieties were compared with yellow-stem ones on two harvesting times: at full flower and seed maturity. Scutching was performed on un-retted stems and dew-retted stems, the un-retted scutched fiber bundles were then bio-degummed before hackling. Both scutching and hackling was performed on flax machines. Quality of hackled hemp, with particular reference to its suitability for high performance composites production, was assessed. The results of fiber extraction indicate that yellow-stem varieties are characterized by higher scutching efficiency than green-stem varieties. Composites strength at breaking point, measured on specimens produced with the Impregnated Fiber Bundle Test, was lower with hemp obtained from stems harvested at seed maturity than at full flowering. On average, back-calculated fiber properties, from hackled hemp-epoxy composites, proved the suitability of long hemp fiber bundles for high performance composites applications, having properties comparable to those of high quality long flax. Highlights: - The trait yellow stem in hemp is an indicator of processability. - Yellow stem varieties have finer hackled fiber bundles. - Controlled dew retting increased yield of hackled fiber compared to bio-degumming. - Retting influenced fiber and composite mechanical properties. - Hemp can achieve properties comparable to high quality long flax for high performance composites.

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 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.

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.