Effects of elevated CO2 concentration and experimental warming on morphological, physiological, and biochemical responses of winter wheat under soil water deficiency.

Global climate change and freshwater scarcity have become two major environmental issues that constrain the sustainable development of the world economy. Climate warming caused by increasing atmospheric CO2 concentration can change global/regional rainfall patterns, leading to uneven global seasonal precipitation distribution and frequent regional extreme drought events, resulting in a drastic reduction of available water resources during the critical crop reproduction period, thus causing many important food-producing regions to face severe water deficiency problems. Understanding the potential processes and mechanisms of crops in response to elevated CO2 concentration and temperature under soil water deficiency may further shed lights on the potential risks of climate change on the primary productivity and grain yield of agriculture. We examined the effects of elevated CO2 concentration (e[CO2]) and temperature (experimental warming) on plant biomass and leaf area, stomatal morphology and distribution, leaf gas exchange and mesophyll anatomy, rubisco activity and gene expression level of winter wheat grown at soil water deficiency with environmental growth chambers. We found that e[CO2] × water × warming sharply reduced plant biomass by 57% and leaf photosynthesis (P n) 50%, although elevated [CO2] could alleviated the stress from water × warming at the amount of gene expression in RbcL3 (128%) and RbcS2 (215%). At ambient [CO2], the combined stress of warming and water deficiency resulted in a significant decrease in biomass (52%), leaf area (50%), P n (71%), and G s (90%) of winter wheat. Furthermore, the total nonstructural carbohydrates were accumulated 10% and 27% and increased R d by 127% and 99% when subjected to water × warming and e[CO2] × water × warming. These results suggest that water × warming may cause irreversible damage in winter wheat and thus the effect of "CO2 fertilization effect" may be overestimated by the current process-based ecological model.

Distribution, expression of hexaploid wheat Fes1s and functional characterization of two TaFes1As in Arabidopsis.

Hexaploid wheat is a major food crop and is sensitive to heat stress. It is necessary to discover genes related to thermotolerance in wheat. Fes1s is a class of nucleotide exchange factor of heat shock protein 70s, proven to be participated in heat response in human, yeast, and Arabidopsis. However, little is known about Fes1s in hexaploid wheat. In this study, we identified nine Fes1s in hexaploid wheat (TaFes1s) and found that they present as three triads. A phylogenetic relationship analysis revealed that these Fes1s grouped into Fes1A, Fes1B and Fes1C subclades, and Fes1As and Fes1Bs were divergent in monocots, but possibly not in dicots. The sequences, gene structures and protein motifs of TaFes1s homoeologues within a triad were highly conserved. Through cis-elements analysis including heat shock elements, and miRNA targets prediction, we found that regulation of three TaFes1s homoeologues may be different, while the expression patterns of three homoeologues were similar. The expression levels of TaFes1As were higher than those of TaFes1Bs and TaFes1Cs, and based on these expressions, TaFes1As were chosen for functional characterization. Intriguingly, neither TaFes1A-5A nor TaFes1A-5D could not rescue the thermotolerance defect of Arabidopsis fes1a mutants at seedling stage, but in the transgenic plants seed germination was accelerated under normal and heat stress condition. The functional characterization indicated that roles of Fes1As would be different in Arabidopsis and hexaploid wheat, and function retention of TaFes1As may occur during wheat evolution. In conclusion, our study comprehensively characterized the distribution and expression of Fes1s in hexaploid wheat and found that two TaFes1As could accelerate seed germination under normal and heat stress condition.

Genome-Wide Analysis of HSP70s in Hexaploid Wheat: Tandem Duplication, Heat Response, and Regulation.

HSP70s play crucial roles in plant growth and development, as well as in stress response. Knowledge of the distribution and heat response of HSP70s is important to understand heat adaptation and facilitate thermotolerance improvement in wheat. In this study, we comprehensively analyzed the distribution of HSP70s in hexaploid wheat (TaHSP70s) and its relatives, and we found an obvious expansion of TaHSP70s in the D genome of hexaploid wheat. Meanwhile, a large portion of tandem duplication events occurred in hexaploid wheat. Among the 84 identified TaHSP70s, more than 64% were present as homeologs. The expression profiles of TaHSP70s in triads tended to be expressed more in non-stressful and heat stress conditions. Intriguingly, many TaHSP70s were especially heat responsive. Tandem duplicated TaHSP70s also participated in heat response and growth development. Further HSE analysis revealed divergent distribution of HSEs in the promoter regions of TaHSP70 homeologs, which suggested a distinct heat regulatory mechanism. Our results indicated that the heat response of TaHSP70s may experience a different regulation, and this regulation, together with the expression of tandem duplicated TaHSP70s, may help hexaploid wheat to adapt to heat conditions.

Alternative Splicing Diversified the Heat Response and Evolutionary Strategy of Conserved Heat Shock Protein 90s in Hexaploid Wheat (Triticum aestivum L.).

Crops are challenged by the increasing high temperature. Heat shock protein 90 (HSP90), a molecular chaperone, plays a critical role in the heat response in plants. However, the evolutionary conservation and divergence of HSP90s homeologs in polyploidy crops are largely unknown. Using the newly released hexaploid wheat reference sequence, we identified 18 TaHSP90s that are evenly distributed as homeologous genes among three wheat subgenomes, and were highly conserved in terms of sequence identity and gene structure among homeologs. Intensive time-course transcriptomes showed uniform expression and transcriptional response profiles among the three TaHSP90 homeologs. Based on the comprehensive isoforms generated by combining full-length single-molecule sequencing and Illumina short read sequencing, 126 isoforms, including 90 newly identified isoforms of TaHSP90s, were identified, and each TaHSP90 generated one to three major isoforms. Intriguingly, the numbers and the splicing modes of the major isoforms generated by three TaHSP90 homeologs were obviously different. Furthermore, the quantified expression profiles of the major isoforms generated by three TaHSP90 homeologs are also distinctly varied, exhibiting differential alternative splicing (AS) responses of homeologs. Our results showed that the AS diversified the heat response of the conserved TaHSP90s and provided a new perspective for understanding about functional conservation and divergence of homologous genes.

Hybrid sequencing reveals insight into heat sensing and signaling of bread wheat.

Wheat (Triticum aestivum L.), a globally important crop, is challenged by increasing temperatures (heat stress, HS). However its polyploid nature, the incompleteness of its genome sequences and annotation, the lack of comprehensive HS-responsive transcriptomes and the unexplored heat sensing and signaling of wheat hinder our full understanding of its adaptations to HS. The recently released genome sequences of wheat, as well as emerging single-molecular sequencing technologies, provide an opportunity to thoroughly investigate the molecular mechanisms of the wheat response to HS. We generated a high-resolution spatio-temporal transcriptome map of wheat flag leaves and filling grain under HS at 0 min, 5 min, 10 min, 30 min, 1 h and 4 h by combining full-length single-molecular sequencing and Illumina short reads sequencing. This hybrid sequencing newly discovered 4947 loci and 70 285 transcripts, generating the comprehensive and dynamic list of HS-responsive full-length transcripts and complementing the recently released wheat reference genome. Large-scale analysis revealed a global landscape of heat adaptations, uncovering unexpected rapid heat sensing and signaling, significant changes of more than half of HS-responsive genes within 30 min, heat shock factor-dependent and -independent heat signaling, and metabolic alterations in early HS-responses. Integrated analysis also demonstrated the differential responses and partitioned functions between organs and subgenomes, and suggested a differential pattern of transcriptional and alternative splicing regulation in the HS response. This study provided comprehensive data for dissecting molecular mechanisms of early HS responses in wheat and highlighted the genomic plasticity and evolutionary divergence of polyploidy wheat.

Metabolic adaptation of wheat grain contributes to a stable filling rate under heat stress.

Wheat (Triticum aestivum) is particularly vulnerable to heat stress during the grain filling stage, and this can adversely affect the final yield. However, the underlying physiological and molecular mechanisms are largely unknown. In this study, the effects of heat stress on grain filling were investigated using wheat varieties with different levels of thermotolerance. Decreased grain weights and filling durations, increased protein contents, and stable filling rates across diverse varieties under different heat regimes suggested a general mechanism for heat adaptation. Proteomic analysis identified 309 heat-responsive proteins (HRPs), and revealed a general decrease in protein synthesis components and metabolic proteins, but a significant increase in stress-response proteins and storage proteins. Metabolomic analysis identified 98 metabolites specifically changed by heat stress, and suggested a global decrease in the content of carbohydrate metabolites, an increased content of amino acids, and stable levels of starch synthesis precursors. The energy-consuming HRPs suggested that less energy was channelled into metabolism and protein synthesis, whereas more energy was allocated to the stress response under elevated heat conditions. Collectively, the data demonstrated a widely distributed mechanism for heat adaptation of metabolism, in which the assimilation and energy required for metabolism and protein synthesis are reallocated to heat protection and deposition of reserves, resulting in increased storage protein accumulation and a stable filling rate.

The heat responsive wheat TaRAD23 rescues developmental and thermotolerant defects of the rad23b mutant in Arabidopsis thaliana.

High temperature severely damage the growth and development of crops with climate change. To effectively screen heat responsive proteins in wheat (Triticum aestivum L.), the isobaric tandem mass tag (TMT)-labeled quantitative proteomic analysis and quantitative real-time PCR (qRT-PCR) were performed. Here, we found that a wheat RADIATION SENSITIVE 23 protein, TaRAD23, was up-regulated at both protein and RNA levels by exposing to heat stress. Sequence homology analysis indicated that the TaRAD23 is a conserved protein, which is closely related to the Arabidopsis thaliana proteins AtRAD23B and AtRAD23A. Genetic knockout of AtRAD23B, but not AtRAD23A, shows multiple developmental defects, as well as sensitivity to heat stress. Meanwhile, we observed that constitutive overexpression of TaRAD23 in rad23b fully rescued developmental and thermotolerant defects of the mutant. Furthermore, qRT-PCR analysis of heat responsive genes in rad23b and its complementary lines suggested that suppression of the heat shock transcription factor AtHSFA2 and heat responsive genes (HSP70, HSP90, HSP17.6 and HSA32) may be the cause of the weaker thermotolerance in rad23b. Taken together, the data suggest that the heat responsive TaRAD23 is a functionally highly conserved protein that plays an important role in development, as well as the regulation in heat stress response network.

Comparative Proteomic Analysis of Flag Leaves Reveals New Insight into Wheat Heat Adaptation.

Hexaploid wheat (Triticum aestivum L.) is an important food crop but it is vulnerable to heat. The heat-responsive proteome of wheat remains to be fully elucidated because of previous technical and genomic limitations, and this has hindered our understanding of the mechanisms of wheat heat adaptation and advances in improving thermotolerance. Here, flag leaves of wheat during grain filling stage were subjected to high daytime temperature stress, and 258 heat-responsive proteins (HRPs) were identified with iTRAQ analysis. Enrichment analysis revealed that chlorophyll synthesis, carbon fixation, protein turnover, and redox regulation were the most remarkable heat-responsive processes. The HRPs involved in chlorophyll synthesis and carbon fixation were significantly decreased, together with severe membrane damage, demonstrating the specific effects of heat on photosynthesis of wheat leaves. In addition, the decrease in chlorophyll content may result from the decrease in HRPs involved in chlorophyll precursor synthesis. Further analysis showed that the accumulated effect of heat stress played a critical role in photosynthesis reduction, suggested that improvement in heat tolerance of photosynthesis, and extending heat tolerant period would be major research targets. The significantly accumulation of GSTs and Trxs in response to heat suggested their important roles in redox regulation, and they could be the promising candidates for improving wheat thermotolerance. In summary, our results provide new insight into wheat heat adaption and provide new perspectives on thermotolerance improvement.