Small RNA, Transcriptome and Degradome Analysis of the Transgenerational Heat Stress Response Network in Durum Wheat.

Heat stress is a major limiting factor of grain yield and quality in crops. Abiotic stresses have a transgenerational impact and the mechanistic basis is associated with epigenetic regulation. The current study presents the first systematic analysis of the transgenerational effects of post-anthesis heat stress in tetraploid wheat. Leaf physiological traits, harvest components and grain quality traits were characterized under the impact of parental and progeny heat stress. The parental heat stress treatment had a positive influence on the offspring for traits including chlorophyll content, grain weight, grain number and grain total starch content. Integrated sequencing analysis of the small RNAome, mRNA transcriptome and degradome provided the first description of the molecular networks mediating heat stress adaptation under transgenerational influence. The expression profile of 1771 microRNAs (733 being novel) and 66,559 genes was provided, with differentially expressed microRNAs and genes characterized subject to the progeny treatment, parental treatment and tissue-type factors. Gene Ontology and KEGG pathway analysis of stress responsive microRNAs-mRNA modules provided further information on their functional roles in biological processes such as hormone homeostasis, signal transduction and protein stabilization. Our results provide new insights on the molecular basis of transgenerational heat stress adaptation, which can be used for improving thermo-tolerance in breeding.

Integrated Analysis of Small RNA, Transcriptome, and Degradome Sequencing Reveals the Water-Deficit and Heat Stress Response Network in Durum Wheat.

Water-deficit and heat stress negatively impact crop production. Mechanisms underlying the response of durum wheat to such stresses are not well understood. With the new durum wheat genome assembly, we conducted the first multi-omics analysis with next-generation sequencing, providing a comprehensive description of the durum wheat small RNAome (sRNAome), mRNA transcriptome, and degradome. Single and combined water-deficit and heat stress were applied to stress-tolerant and -sensitive Australian genotypes to study their response at multiple time-points during reproduction. Analysis of 120 sRNA libraries identified 523 microRNAs (miRNAs), of which 55 were novel. Differentially expressed miRNAs (DEMs) were identified that had significantly altered expression subject to stress type, genotype, and time-point. Transcriptome sequencing identified 49,436 genes, with differentially expressed genes (DEGs) linked to processes associated with hormone homeostasis, photosynthesis, and signaling. With the first durum wheat degradome report, over 100,000 transcript target sites were characterized, and new miRNA-mRNA regulatory pairs were discovered. Integrated omics analysis identified key miRNA-mRNA modules (particularly, novel pairs of miRNAs and transcription factors) with antagonistic regulatory patterns subject to different stresses. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis revealed significant roles in plant growth and stress adaptation. Our research provides novel and fundamental knowledge, at the whole-genome level, for transcriptional and post-transcriptional stress regulation in durum wheat.

Multi-Omics Analysis of Small RNA, Transcriptome, and Degradome in T. turgidum-Regulatory Networks of Grain Development and Abiotic Stress Response.

Crop reproduction is highly sensitive to water deficit and heat stress. The molecular networks of stress adaptation and grain development in tetraploid wheat (Triticum turgidum durum) are not well understood. Small RNAs (sRNAs) are important epigenetic regulators connecting the transcriptional and post-transcriptional regulatory networks. This study presents the first multi-omics analysis of the sRNAome, transcriptome, and degradome in T. turgidum developing grains, under single and combined water deficit and heat stress. We identified 690 microRNAs (miRNAs), with 84 being novel, from 118 sRNA libraries. Complete profiles of differentially expressed miRNAs (DEMs) specific to genotypes, stress types, and different reproductive time-points are provided. The first degradome sequencing report for developing durum grains discovered a significant number of new target genes regulated by miRNAs post-transcriptionally. Transcriptome sequencing profiled 53,146 T. turgidum genes, swith differentially expressed genes (DEGs) enriched in functional categories such as nutrient metabolism, cellular differentiation, transport, reproductive development, and hormone transduction pathways. miRNA-mRNA networks that affect grain characteristics such as starch synthesis and protein metabolism were constructed on the basis of integrated analysis of the three omics. This study provides a substantial amount of novel information on the post-transcriptional networks in T. turgidum grains, which will facilitate innovations for breeding programs aiming to improve crop resilience and grain quality.

Water-deficit stress-responsive microRNAs and their targets in four durum wheat genotypes.

MicroRNAs (miRNAs) guide regulation at the post-transcriptional level by inducing messenger RNA (mRNA) degradation or translational inhibition of their target protein-coding genes. Durum wheat miRNAs may contribute to the genotypic water-deficit stress response in different durum varieties. Further investigation of the interactive miRNA-target regulatory modules and experimental validation of their response to water stress will contribute to our understanding of the small RNA-mediated molecular networks underlying stress adaptation in durum wheat. In this study, a comprehensive genome-wide in silico analysis using the updated Triticum transcriptome assembly identified 2055 putative targets for 113 conserved durum miRNAs and 131 targets for four novel durum miRNAs that putatively contribute to genotypic stress tolerance. Predicted mRNA targets encode various transcription factors, binding proteins and functional enzymes, which play vital roles in multiple biological pathways such as hormone signalling and metabolic processes. Quantitative PCR profiling further characterised 43 targets and 5 miRNAs with stress-responsive and/or genotype-dependent differential expression in two stress-tolerant and two stress-sensitive durum genotypes subjected to pre-anthesis water-deficit stress. Furthermore, a 5' RLM-RACE approach validated nine mRNA targets cleaved by water-deficit stress-responsive miRNAs, which, to our knowledge, has not been previously reported in durum wheat. The present study provided experimental evidence of durum miRNAs and target genes in response to water-deficit stress in contrasting durum varieties, providing new insights into the regulatory roles of the miRNA-guided RNAi mechanism underlying stress adaptation in durum wheat.

Secretome analysis of virulent Pyrenophora teres f. teres isolates.

Pyrenophora teres f. teres (Ptt) causes net form net blotch disease of barley, partially by producing necrosis-inducing proteins. The protein profiles of the culture filtrates of 28 virulent isolates were compared by a combination of 2DE and 1D-PAGE with 105 spots and 51 bands chosen for analysis by liquid chromatography electrospray ionization tandem mass spectrometry. A total of 259 individual proteins were identified with 63 of these proteins being common to the selected virulent isolates. Ptt secretes a broad spectrum of proteins including cell wall degrading enzymes; virulence factors and effectors; proteins associated with fungal pathogenesis and development; and proteins related to oxidation-reduction processes. Potential virulence factors and effectors identified included proteins with glucosidase activity, ricin B and concanavalin A-like lectins, glucanases, spherulin, cutinase, pectin lyase, leucine-rich repeat protein, and ceratoplatanin. Small proteins with unknown function but cysteine-rich, common to effectors, were also identified. Differences in the secretion profile of the Ptt isolates have also provided important insight into the different mechanisms contributing to virulence and the development of net form net blotch symptoms.

Characterisation of ethylene pathway components in non-climacteric capsicum.

BACKGROUND: Climacteric fruit exhibit high ethylene and respiration levels during ripening but these levels are limited in non-climacteric fruit. Even though capsicum is in the same family as the well-characterised climacteric tomato (Solanaceae), it is non-climacteric and does not ripen normally in response to ethylene or if harvested when mature green. However, ripening progresses normally in capsicum fruit when they are harvested during or after what is called the 'Breaker stage'. Whether ethylene, and components of the ethylene pathway such as 1-aminocyclopropane 1-carboxylate (ACC) oxidase (ACO), ACC synthase (ACS) and the ethylene receptor (ETR), contribute to non-climacteric ripening in capsicum has not been studied in detail. To elucidate the behaviour of ethylene pathway components in capsicum during ripening, further analysis is therefore needed. The effects of ethylene or inhibitors of ethylene perception, such as 1-methylcyclopropene, on capsicum fruit ripening and the ethylene pathway components may also shed some light on the role of ethylene in non-climacteric ripening. RESULTS: The expression of several isoforms of ACO, ACS and ETR were limited during capsicum ripening except one ACO isoform (CaACO4). ACS activity and ACC content were also low in capsicum despite the increase in ACO activity during the onset of ripening. Ethylene did not stimulate capsicum ripening but 1-methylcyclopropene treatment delayed the ripening of Breaker-harvested fruit. Some of the ACO, ACS and ETR isoforms were also differentially expressed upon treatment with ethylene or 1-methylcyclopropene. CONCLUSIONS: ACS activity may be the rate limiting step in the ethylene pathway of capsicum which restricts ACC content. The differential expression of several ethylene pathway components during ripening and upon ethylene or 1-methylclopropene treatment suggests that the ethylene pathway may be regulated differently in non-climacteric capsicum compared to the climacteric tomato. Ethylene independent pathways may also exist in non-climacteric ripening as evidenced by the up-regulation of CaACO4 during ripening onset despite being negatively regulated by ethylene exposure. However, some level of ethylene perception may still be needed to induce ripening especially during the Breaker stage. A model of capsicum ripening is also presented to illustrate the probable role of ethylene in this non-climacteric fruit.

Natural variation for Fe-efficiency is associated with upregulation of Strategy I mechanisms and enhanced citrate and ethylene synthesis in Pisum sativum L.

Iron (Fe)-deficiency is a common abiotic stress in Pisum sativum L. grown in many parts of the world. The aim of the study was to investigate variation in tolerance to Fe deficiency in two pea genotypes, Santi (Fe-efficient) and Parafield (Fe-inefficient). Fe deficiency caused greater declines in chlorophyll score, leaf Fe concentration and root-shoot development in Parafield compared to Santi, suggesting greater Fe-efficiency in Santi. Fe chelate reductase activity and ethylene production were increased in the roots of Santi and to a lesser extent in Parafield under Fe deficiency, while proton extrusion was only occurred in Santi. Moreover, expression of the Fe chelate reductase gene, FRO1, and Fe transporter, RIT1 were upregulated in Fe-deficient roots of Santi. Expression of HA1 (proton extrusion) was also significantly higher in Santi when compared to Parafield grown in Fe-deficient conditions. Furthermore, the application of the ethylene biosynthesis inhibitor, 1-aminoisobutyric acid reduced the Fe chelate reductase activity, supporting a direct role for ethylene in its induction. A significant increase in root citrate was only observed in Santi under Fe deficiency indicating a role for citrate in the Fe-efficiency mechanism. Taken together, our physiological and molecular data indicate that genotypic variation in tolerance to Fe deficiency in Santi and Parafield plants is a result of variation in a number of Strategy I mechanisms and also suggest a direct role for ethylene in Fe reductase activity. The pea cultivar, Santi provides a new source of Fe-efficiency that can be exploited to breed more Fe-efficient peas.

Priming crops for the future: rewiring stress memory.

The agricultural sector must produce resilient and climate-smart crops to meet the increasing needs of global food production. Recent advancements in elucidating the mechanistic basis of plant stress memory have provided new opportunities for crop improvement. Stress memory-coordinated changes at the organismal, cellular, and various omics levels prepare plants to be more responsive to reoccurring stress within or across generation(s). The exposure to a primary stress, or stress priming, can also elicit a beneficial impact when encountering a secondary abiotic or biotic stress through the convergence of synergistic signalling pathways, referred to as cross-stress tolerance. 'Rewired plants' with stress memory provide a new means to stimulate adaptable stress responses, safeguard crop reproduction, and engineer climate-smart crops for the future.

Nitrogen Starvation-Responsive MicroRNAs Are Affected by Transgenerational Stress in Durum Wheat Seedlings.

Stress events have transgenerational effects on plant growth and development. In Mediterranean regions, water-deficit and heat (WH) stress is a frequent issue that negatively affects crop yield and quality. Nitrogen (N) is an essential plant macronutrient and often a yield-limiting factor for crops. Here, the response of durum wheat seedlings to N starvation under the transgenerational effects of WH stress was investigated in two genotypes. Both genotypes showed a significant reduction in seedling height, leaf number, shoot and root weight (fresh and dry), primary root length, and chlorophyll content under N starvation stress. However, in the WH stress-tolerant genotype, the percentage reduction of most traits was lower in progeny from the stressed parents than progeny from the control parents. Small RNA sequencing identified 1534 microRNAs in different treatment groups. Differentially expressed microRNAs (DEMs) were characterized subject to N starvation, parental stress and genotype factors, with their target genes identified in silico. GO and KEGG enrichment analyses revealed the biological functions, associated with DEM-target modules in stress adaptation processes, that could contribute to the phenotypic differences observed between the two genotypes. The study provides the first evidence of the transgenerational effects of WH stress on the N starvation response in durum wheat.

Small RNAs and their targets are associated with the transgenerational effects of water-deficit stress in durum wheat.

Water-deficit stress negatively affects wheat yield and quality. Abiotic stress on parental plants during reproduction may have transgenerational effects on progeny. Here we investigated the transgenerational influence of pre-anthesis water-deficit stress by detailed analysis of the yield components, grain quality traits, and physiological traits in durum wheat. Next-generation sequencing analysis profiled the small RNA-omics, mRNA transcriptomics, and mRNA degradomics in first generation progeny. Parental water-deficit stress had positive impacts on the progeny for traits including harvest index and protein content in the less stress-tolerant variety. Small RNA-seq identified 1739 conserved and 774 novel microRNAs (miRNAs). Transcriptome-seq characterised the expression of 66,559 genes while degradome-seq profiled the miRNA-guided mRNA cleavage dynamics. Differentially expressed miRNAs and genes were identified, with significant regulatory patterns subject to trans- and inter-generational stress. Integrated analysis using three omics platforms revealed significant biological interactions between stress-responsive miRNA and targets, with transgenerational stress tolerance potentially contributed via pathways such as hormone signalling and nutrient metabolism. Our study provides the first confirmation of the transgenerational effects of water-deficit stress in durum wheat. New insights gained at the molecular level indicate that key miRNA-mRNA modules are candidates for transgenerational stress improvement.

Transgenerational Effects of Water-Deficit and Heat Stress on Germination and Seedling Vigour-New Insights from Durum Wheat microRNAs.

Water deficiency and heat stress can severely limit crop production and quality. Stress imposed on the parents during reproduction could have transgenerational effects on their progeny. Seeds with different origins can vary significantly in their germination and early growth. Here, we investigated how water-deficit and heat stress on parental durum wheat plants affected seedling establishment of the subsequent generation. One stress-tolerant and one stress-sensitive Australian durum genotype were used. Seeds were collected from parents with or without exposure to stress during reproduction. Generally, stress on the previous generation negatively affected seed germination and seedling vigour, but to a lesser extent in the tolerant variety. Small RNA sequencing utilising the new durum genome assembly revealed significant differences in microRNA (miRNA) expression in the two genotypes. A bioinformatics approach was used to identify multiple miRNA targets which have critical molecular functions in stress adaptation and plant development and could therefore contribute to the phenotypic differences observed. Our data provide the first confirmation of the transgenerational effects of reproductive-stage stress on germination and seedling establishment in durum wheat. New insights gained on the epigenetic level indicate that durum miRNAs could be key factors in optimising seed vigour for breeding superior germplasm and/or varieties.

Genotypic performance of Australian durum under single and combined water-deficit and heat stress during reproduction.

In Mediterranean environments, water deficiency and heat during reproduction severely limit cereal crop production. Our research investigated the effects of single and combined pre-anthesis water-deficit stress and post-anthesis heat stress in ten Australian durum genotypes, providing a systematic evaluation of stress response at the molecular, physiological, grain quality and yield level. We studied leaf physiological traits at different reproductive stages, evaluated the grain yield and quality, and the associations among them. We profiled the expression dynamics of two durum microRNAs and their protein-coding targets (auxin response factors and heat shock proteins) involved in stress adaptation. Chlorophyll content, stomatal conductance and leaf relative water content were mostly reduced under stress, however, subject to the time-point and genotype. The influence of stress on grain traits (e.g., protein content) also varied considerably among the genotypes. Significant positive correlations between the physiological traits and the yield components could be used to develop screening strategies for stress improvement in breeding. Different expression patterns of stress-responsive microRNAs and their targets in the most stress-tolerant and most stress-sensitive genotype provided some insight into the complex defense molecular networks in durum. Overall, genotypic performance observed indicates that different stress-coping strategies are deployed by varieties under various stresses.

Induction of a grapevine germin-like protein (VvGLP3) gene is closely linked to the site of Erysiphe necator infection: a possible role in defense?

Germin-like proteins (GLP) have various proposed roles in plant development and defense. Seven novel GLP cDNA clones were isolated from grapevine (Vitis vinifera cv. Chardonnay). Reverse transcriptase-polymerase chain reaction expression analysis revealed that the VvGLP genes exhibit diverse and highly specific patterns of expression in response to a variety of abiotic and biotic treatments, including challenge by Erysiphe necator, Plasmopara viticola, and Botrytis cinerea, suggesting a diversity of roles for each of the GLP family members. Significantly, one of the grapevine GLP genes, VvGLP3, is induced specifically by E. necator infection and expression is closely linked to the site of infection. Subcellular localization of VvGLP3 determined by transient expression of a VvGLP3:GFP fusion construct in onion cells indicated that the recombinant protein was targeted to the cell wall. Recombinant VvGLP3 was successfully expressed in Arabidopsis thaliana and the partially purified recombinant protein was demonstrated to have superoxide dismutase activity. This data has provided an insight into the diverse nature of the GLP family in grapevine and suggests that VvGLP3 may be involved in the defense response against E. necator.

Proteinaceous Metabolites from Pyrenophora teres Contribute to Symptom Development of Barley Net Blotch.

ABSTRACT Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), induces a combination of necrosis and extensive chlorosis in susceptible barley cultivars. Cell-free filtrates from both net and spot forms of P. teres; P. teres f. sp. teres, and P. teres f. sp. maculata were found to contain phytotoxic low molecular weight compounds (LMWCs) and proteinaceous metabolites which appear to be responsible for different components of the symptoms induced by the two forms of the pathogen in a susceptible cultivar of barley (cv. Sloop). Proteins induced only brown necrotic spots or lesions similar to those induced by the pathogens 72 h after inoculation. In contrast, LMWCs induced general chlorosis seen 240 h after inoculation but not the localized necrosis. Neither hydrolyzed or heat- or protease-treated proteinaceous metabolites induced the symptoms. This is the first report of the involvement of proteins produced by P. teres in symptom development during net blotch disease of barley.

Genotypic water-deficit stress responses in durum wheat: association between physiological traits, microRNA regulatory modules and yield components.

In Mediterranean environments, water-deficit stress that occurs before anthesis significantly limits durum wheat (Triticum turgidum L. ssp. durum) production. Stress tolerant and stress sensitive durum varieties exhibit genotypic differences in their response to pre-anthesis water-deficit stress as reflected by yield performance, but our knowledge of the mechanisms underlying tolerance is limited. We have previously identified stress responsive durum microRNAs (miRNAs) that could contribute to water-deficit stress tolerance by mediating post-transcriptional silencing of genes that lead to stress adaptation (e.g. miR160 and its targets ARF8 (auxin response factor 8) and ARF18). However, the temporal regulation pattern of miR160-ARFs after induction of pre-anthesis water-deficit stress in sensitive and tolerant varieties remains unknown. Here, the physiological responses of four durum genotypes are described by chlorophyll content, leaf relative water content, and stomatal conductance at seven time-points during water-deficit stress from booting to anthesis. qPCR examination of miR160, ARF8 and ARF18 at these time-points revealed a complex stress responsive regulatory pattern, in the flag leaf and the head, subject to genotype. Harvest components and morphological traits measured at maturity confirmed the stress tolerance level of these four varieties for agronomic performance, and their potential association with the physiological responses. In general, the distinct regulatory pattern of miR160-ARFs among stress tolerant and sensitive durum varieties suggests that miRNA-mediated molecular pathways may contribute to the genotypic differences in the physiological traits, ultimately affecting yield components (e.g. the maintenance of harvest index and grain number).

The role of a cytosolic superoxide dismutase in barley-pathogen interactions.

Reactive oxygen species (ROS), including superoxide ( O2·-/ HO2·) and hydrogen peroxide (H2 O2 ), are differentially produced during resistance responses to biotrophic pathogens and during susceptible responses to necrotrophic and hemi-biotrophic pathogens. Superoxide dismutase (SOD) is responsible for the catalysis of the dismutation of O2·-/ HO2· to H2 O2 , regulating the redox status of plant cells. Increased SOD activity has been correlated previously with resistance in barley to the hemi-biotrophic pathogen Pyrenophora teres f. teres (Ptt, the causal agent of the net form of net blotch disease), but the role of individual isoforms of SOD has not been studied. A cytosolic CuZnSOD, HvCSD1, was isolated from barley and characterized as being expressed in tissue from different developmental stages. HvCSD1 was up-regulated during the interaction with Ptt and to a greater extent during the resistance response. Net blotch disease symptoms and fungal growth were not as pronounced in transgenic HvCSD1 knockdown lines in a susceptible background (cv. Golden Promise), when compared with wild-type plants, suggesting that cytosolic O2·-/ HO2· contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi-biotrophic rice blast pathogen Magnaporthe oryzae or the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei, but HvCSD1 also played a role in the regulation of lesion development by methyl viologen. Together, these results suggest that HvCSD1 could be important in the maintenance of the cytosolic redox status and in the differential regulation of responses to pathogens with different lifestyles.

SMARTER De-Stressed Cereal Breeding.

In cereal breeding programs, improved yield potential and stability are ultimate goals when developing new varieties. To facilitate achieving these goals, reproductive success under stressful growing conditions is of the highest priority. In recent times, small RNA (sRNA)-mediated pathways have been associated with the regulation of genes involved in stress adaptation and reproduction in both model plants and several cereals. Reproductive and physiological traits such as flowering time, reproductive branching, and root architecture can be manipulated by sRNA regulatory modules. We review sRNA-mediated pathways that could be exploited to expand crop diversity with adaptive traits and, in particular, the development of high-yielding stress-tolerant cereals: SMARTER cereal breeding through 'Small RNA-Mediated Adaptation of Reproductive Targets in Epigenetic Regulation'.

Enrichment of Antioxidant Capacity and Vitamin E in Pita Made from Barley.

This study aimed to enhance total antioxidant and vitamin E content of pita bread, by replacing 50% of the standard baker's flour with flours milled from covered (WI2585 and Harrington) or hulless (Finniss) barley genotypes, previously shown to have high antioxidant and vitamin E levels at harvest. Pita breads were made from either 100% baker's flour (control) or 50% malt flour, whole-grain flour, or flour from barley grains pearled at 10%, 15%, and 20% grain weight. Antioxidant capacity and vitamin E content of flours and pitas were determined by their ability to scavenge 2,2-diphenyl-1-picrylhydrazyl radicals and high performance liquid chromatography, respectively. The physical and sensory properties of the pitas were also assessed. All pitas made from either whole grain or pearled barley flour had a higher antioxidant capacity and most also had higher vitamin E content than standard pita. The antioxidant and vitamin E levels were reduced in pearled compared to whole grains, however the extent of that reduction varied among genotypes. The greatest antioxidant and vitamin E levels were found in pita made from malt flour or Finniss whole grain flour. Furthermore, sensory analysis suggested these pitas were acceptable to consumers and retained similar physical and sensory properties to those in the control pita.

Antioxidant capacity and vitamin E in barley: Effect of genotype and storage.

Antioxidants, including vitamin E, may have a positive effect on human health and prolong storage of food items. Vitamin E content and antioxidant capacity were measured in 25 barley genotypes before and after 4 months storage at 10 °C using high performance liquid chromatography (HPLC) and ability to scavenge DPPH radicals, respectively. As expected, α-tocotrienol (α-T3) and α-tocopherol (α-T) were the predominant tocol isomers. Vitamin E content and antioxidant capacity varied significantly among genotypes. Vitamin E ranged from 8.5 to 31.5 µg/g dry weight (DW) while ascorbic acid equivalent antioxidant capacity (AEAC) varied from 57.2 to 158.1 mg AEAC/100 g fresh weight (FW). Generally, lower vitamin E content or antioxidant capacity was observed in hulless or coloured genotypes. These results suggest that some genotypes are potential candidates for breeding of barley cultivars with high vitamin E content or antioxidant capacity at harvest, even after storage.

Genome-Wide Identification of MicroRNAs in Leaves and the Developing Head of Four Durum Genotypes during Water Deficit Stress.

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in plant development and abiotic stress responses. The miRNA transcriptome (miRNAome) under water deficit stress has been investigated in many plant species, but is poorly characterised in durum wheat (Triticum turgidum L. ssp. durum). Water stress during early reproductive stages can result in significant yield loss in durum wheat and this study describes genotypic differences in the miRNAome between water deficit tolerant and sensitive durum genotypes. Small RNA libraries (96 in total) were constructed from flag leaf and developing head tissues of four durum genotypes, with or without water stress to identify differentially abundant miRNAs. Illumina sequencing detected 110 conserved miRNAs and 159 novel candidate miRNA hairpins with 66 conserved miRNAs and five novel miRNA hairpins differentially abundant under water deficit stress. Ten miRNAs (seven conserved, three novel) were validated through qPCR. Several conserved and novel miRNAs showed unambiguous inverted regulatory profiles between the durum genotypes. Several miRNAs also showed differential abundance between two tissue types regardless of treatment. Predicted mRNA targets (130) of four novel durum miRNAs were characterised using Gene Ontology (GO) which revealed functions common to stress responses and plant development. Negative correlation was observed between several target genes and the corresponding miRNA under water stress. For the first time, we present a comprehensive study of the durum miRNAome under water deficit stress. The identification of differentially abundant miRNAs provides molecular evidence that miRNAs are potential determinants of water stress tolerance in durum wheat. GO analysis of predicted targets contributes to the understanding of genotypic physiological responses leading to stress tolerance capacity. Further functional analysis of specific stress responsive miRNAs and their interaction with targets is ongoing and will assist in developing future durum wheat varieties with enhanced water deficit stress tolerance.