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.

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.

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.

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.

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.

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.

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.

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.