Metabolic and transcriptomic profiling during wheat seed development under progressive drought conditions.

Globally, bread wheat (Triticum aestivum) is one of the most important staple foods; when exposed to drought, wheat yields decline. Although much research has been performed to generate higher yield wheat cultivars, there have been few studies on improving end-product quality under drought stress, even though wheat is processed into flour to produce so many foods, such as bread, noodles, pancakes, cakes, and cookies. Recently, wheat cultivation has been affected by severe drought caused by global climate change. In previous studies, seed shrinkage was observed in wheat exposed to continuous drought stress during seed development. In this study, we investigated how progressive drought stress affected seed development by metabolomic and transcriptomic analyses. Metabolite profiling revealed the drought-sensitive line reduced accumulation of proline and sugar compared with the water-saving, drought-tolerant transgenic line overexpressing the abscisic acid receptor TaPYL4 under drought conditions in spikelets with developing seeds. Meanwhile, the expressions of genes involved in translation, starch biosynthesis, and proline and arginine biosynthesis was downregulated in the drought-sensitive line. These findings suggest that seed shrinkage, exemplifying a deficiency in endosperm, arose from the hindered biosynthesis of crucial components including seed storage proteins, starch, amino acids, and sugars, ultimately leading to their inadequate accumulation within spikelets. Water-saving drought tolerant traits of wheat would aid in supporting seed formation under drought conditions.

Genomic analysis for heat and combined heat-drought resilience in bread wheat under field conditions.

KEY MESSAGE: GWAS on a bread wheat panel with high D genome diversity identified novel alleles and QTLs associated with resilience to combined heat and drought stress under natural field conditions. As heat (H) and drought stresses occur concurrently under field conditions, studying them separately offers limited opportunities for wheat improvement. Here, a wheat diversity panel containing Aegilops tauschii introgressions was evaluated under H and combined heat-drought (HD) stresses to identify quantitative trait loci (QTLs) associated with resilience to the stresses, and to assess the practicability of harnessing Ae. tauschii diversity for breeding for combined stress resilience. Using genome-wide analysis, we identified alleles and QTLs on chromosomes 3D, 5D, and 7A controlling grain yield (GY), kernel number per spike, and thousand-kernel weight, and on 3D (521-549 Mbp) controlling GY alone. A strong marker-trait association (MTA) for GY stability on chromosome 3D (508.3 Mbp) explained 20.3% of the variation. Leaf traits-canopy temperature, vegetation index, and carbon isotope composition-were controlled by five QTLs on 2D (23-96, 511-554, and 606-614 Mbp), 3D (155-171 Mbp), and 5D (407-413 Mbp); some of them were pleiotropic for GY and yield-related traits. Further analysis revealed candidate genes, including GA20ox, regulating GY stability, and CaaX prenyl protease 2, regulating canopy temperature at the flowering stage, under H and HD stresses. As genome-wide association studies under HD in field conditions are scarce, our results provide genomic landmarks for wheat breeding to improve adaptation to H and HD conditions under climate change.

Metabolome Profiling of Heat Priming Effects, Senescence, and Acclimation of Bread Wheat Induced by High Temperatures at Different Growth Stages.

Our previous study described stage-specific responses of 'Norin 61' bread wheat to high temperatures from seedling to tillering (GS1), tillering to flowering (GS2), flowering to full maturity stage (GS3), and seedling to full maturity stage (GS1-3). The grain development phase lengthened in GS1 plants; source tissue decreased in GS2 plants; rapid senescence occurred in GS3 plants; all these effects occurred in GS1-3 plants. The present study quantified 69 flag leaf metabolites during early grain development to reveal the effects of stage-specific high-temperature stress and identify markers that predict grain weight. Heat stresses during GS2 and GS3 showed the largest shifts in metabolite contents compared with the control, followed by GS1-3 and GS1. The GS3 plants accumulated nucleosides related to the nucleotide salvage pathway, beta-alanine, and serotonin. Accumulation of these compounds in GS1 plants was significantly lower than in the control, suggesting that the reduction related to the high-temperature priming effect observed in the phenotype (i.e., inhibition of senescence). The GS2 plants accumulated a large quantity of free amino acids, indicating residual effects of the previous high-temperature treatment and recovery from stress. However, levels in GS1-3 plants tended to be close to those in the control, indicating an acclimation response. Beta-alanine, serotonin, tryptophan, proline, and putrescine are potential molecular markers that predict grain weight due to their correlation with agronomic traits.

Stage-Specific Characterization of Physiological Response to Heat Stress in the Wheat Cultivar Norin 61.

Bread wheat (Triticum aestivum) is less adaptable to high temperatures than other major cereals. Previous studies of the effects of high temperature on wheat focused on the reproductive stage. There are few reports on yield after high temperatures at other growth stages. Understanding growth-stage-specific responses to heat stress will contribute to the development of tolerant lines suited to high temperatures at various stages. We exposed wheat cultivar "Norin 61" to high temperature at three growth stages: seedling-tillering (GS1), tillering-flowering (GS2), and flowering-maturity (GS3). We compared each condition based on agronomical traits, seed maturity, and photosynthesis results. Heat at GS2 reduced plant height and number of grains, and heat at GS3 reduced the grain formation period and grain weight. However, heat at GS1 reduced senescence and prolonged grain formation, increasing grain weight without reducing yield. These data provide fundamental insights into the biochemical and molecular adaptations of bread wheat to high-temperature stresses and have implications for the development of wheat lines that can respond to high temperatures at various times of the year.

Metabolic and physiological responses to progressive drought stress in bread wheat.

Wheat (Tritium aestivum) is vulnerable to future climate change because it is predominantly grown under rain-fed conditions in drought-prone areas. Thus, in-depth understanding of drought effect on wheat metabolism is essential for developing drought-tolerant wheat varieties. Here, we exposed wheat 'Norin 61' plants to progressive drought stress [0 (before drought), 2, 4, 6, 8, and 10 days after withholding water] during the flowering stage to investigate physiological and metabolomic responses. Transcriptional analyses of key abscisic acid-responsive genes indicated that abscisic acid signalling played a major role in the adaptation of wheat to water deficit. Carbon isotope composition had a higher value than the control while canopy temperature (CT) increased under drought stress. The CT depression was tightly correlated with soil water potential (SWP). Additionally, SWP at - 517 kPa was identified as the critical point for increasing CT and inducing reactive oxygen species. Metabolome analysis identified four potential drought-responsive biomarkers, the enhancement of nitrogen recycling through purine and pyrimidine metabolism, drought-induced senescence based on 1-aminocyclopropane-1-carboxylic acid and Asn accumulation, and an anti-senescence response through serotonin accumulation under severe drought stress. Our findings provide in-depth insight into molecular, physiological and metabolite changes involved in drought response which are useful for wheat breeding programs to develop drought-tolerant wheat varieties.

The crystal structures of Thermus thermophilus CMP kinase complexed with a phosphoryl group acceptor and donor.

Nucleoside monophosphate kinases play crucial roles in biosynthesis and regeneration of nucleotides. These are bi-substrate enzymes that catalyze reversible transfers of a phosphoryl group between ATP and nucleoside monophosphate. These enzymes are comprised of the CORE domain, the NMP-binding domain, and the LID domain. Large conformational rearrangement of the three domains occurs during the catalytic cycle. Although many structures of CMP kinase have been determined, only limited structural information has been available on the conformational changes along the reaction pathway. We determined five crystal structures of CMP kinase of Thermus thermophilus HB8 in ligand-free form and the CMP "open", CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms at resolutions of 1.7, 2.2, 1.5, 1.6, and 1.7 Å, respectively. The ligand-free form was in an open conformation, whereas the structures of the CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms were in a closed conformation, in which the shift of the NMP-binding domain and LID domain caused closure of the substrate-binding cleft. Interestingly, the CMP "open" form was in an open conformation even with CMP bound, implying intrinsic conformational fluctuation. The structure of the ADP-CDP complex is the first structure of CMP kinase with a phosphoryl group donor and an acceptor. Upon simultaneous binding of ADP and CDP, the side chains of several residues in the LID domain moved toward the nucleotides without global open-closed conformational changes compared to those in the CMP "closed" and CDP complexes. These global and local conformational changes may be crucial for the substrate recognition and catalysis. The terminal phosphate groups of ADP and CDP had similar geometry to those of two ADP in AMP kinase, suggesting common catalytic mechanisms to other nucleoside monophosphate kinases. Our findings are expected to contribute to detailed understanding of the reaction mechanism of CMP kinase.

Genetic manipulation of abscisic acid receptors enables modulation of water use efficiency.

The phytohormone abscisic acid (ABA) is a key signal molecule for controlling stomatal movement and stress responses such as drought. Genetic manipulation of ABA receptors has recently been reported to improve plant water use efficiency (WUE). We therefore compared details of WUE traits between the ABA receptor quintuple mutant and wheat ABA receptor-overexpressing Arabidopsis lines. Biomass and seed productivities per liter of water were both reduced in the receptor mutant but improved in the transgenic Arabidopsis lines. This result suggests that appropriate modulation of ABA receptors can extend crop potential productivity.

Tuning water-use efficiency and drought tolerance in wheat using abscisic acid receptors.

Water availability is a key determinant of terrestrial plant productivity. Many climate models predict that water stress will increasingly challenge agricultural yields and exacerbate projected food deficits. To ensure food security and increase agricultural efficiency, crop water productivity must be increased. Research over past decades has established that the phytohormone abscisic acid (ABA) is a central regulator of water use and directly regulates stomatal opening and transpiration. In this study, we investigated whether the water productivity of wheat could be improved by increasing its ABA sensitivity. We show that overexpression of a wheat ABA receptor increases wheat ABA sensitivity, which significantly lowers a plant's lifetime water consumption. Physiological analyses demonstrated that this water-saving trait is a consequence of reduced transpiration and a concomitant increase in photosynthetic activity, which together boost grain production per litre of water and protect productivity during water deficit. Our findings provide a general strategy for increasing water productivity that should be applicable to other crops because of the high conservation of the ABA signalling pathway.

Abscinazole-E3M, a practical inhibitor of abscisic acid 8'-hydroxylase for improving drought tolerance.

Abscisic acid (ABA) is an essential phytohormone that regulates plant water use and drought tolerance. However, agricultural applications of ABA have been limited because of its rapid inactivation in plants, which involves hydroxylation of ABA by ABA 8'-hydroxylase (CYP707A). We previously developed a selective inhibitor of CYP707A, (-)-Abz-E2B, by structurally modifying S-uniconazole, which functions as an inhibitor of CYP707A and as a gibberellin biosynthetic enzyme. However, its synthetic yield is too low for practical applications. Therefore, we designed novel CYP707A inhibitors, Abz-T compounds, that have simpler structures in which the 1,2,3-triazolyl ring of (-)-Abz-E2B has been replaced with a triple bond. They were successfully synthesised in shorter steps, resulting in greater yields than that of (-)-Abz-E2B. In the enzymatic assays, one of the Abz-T compounds, (-)-Abz-E3M, acted as a strong and selective inhibitor of CYP707A, similar to (-)-Abz-E2B. Analysis of the biological effects in Arabidopsis revealed that (-)-Abz-E3M enhanced ABA's effects more than (-)-Abz-E2B in seed germination and in the expression of ABA-responsive genes. Treatment with (-)-Abz-E3M induced stomatal closure and improved drought tolerance in Arabidopsis. Furthermore, (-)-Abz-E3M also increased the ABA response in rice and maize. Thus, (-)-Abz-E3M is a more practical and effective inhibitor of CYP707A than (-)-Abz-E2B.

Sustained low abscisic acid levels increase seedling vigor under cold stress in rice (Oryza sativa L.).

Stress-induced abscisic acid (ABA) is mainly catabolized by ABA 8'-hydroxylase (ABA8ox), which also strictly regulates endogenous ABA levels. Although three members of the ABA8ox gene family are conserved in rice, it is not clear which stressors induce expression of these genes. Here, we found that OsABA8ox1 was induced by cold stress within 24 h and that OsABA8ox2 and OsABA8ox3 were not. In contrast, OsABA8ox2 and OsABA8ox3 were ABA-inducible, but OsABA8ox1 was not. OsABA8ox1, OsABA8ox2, and OsABA8ox3 restored germination of a cyp707a1/a2/a3 triple mutant of Arabidopsis to rates comparable to those of the wild type, indicating that OsABA8ox1, OsABA8ox2, and OsABA8ox3 function as ABA-catabolic genes in vivo. Transgenic rice lines overexpressing OsABA8ox1 showed decreased levels of ABA and increased seedling vigor at 15 °C. These results indicate that sustained low levels of ABA lead to increased seedling vigor during cold stress. On the other hand, excessively low endogenous ABA levels caused reduced drought and cold tolerance, although some of the transgenic rice lines expressing OsABA8ox1 at moderate levels did not show these harmful effects. Adequate regulation of endogenous ABA levels is thought to be crucial for maintaining seedling vigor under cold stress and for cold and drought tolerance in rice.

A non-cold-inducible cold shock protein homolog mainly contributes to translational control under optimal growth conditions.

Cold shock proteins (Csps) include both cold-induced and non-cold-induced proteins, contrary to their name. Cold-induced Csps are well studied; they function in cold acclimation by controlling transcription and translation. Some Csps have been reported to contribute to other cellular processes. However, the functions of non-cold-induced Csps under optimal growth conditions remain unknown. To elucidate these functions, we used transcriptome and proteome analyses as comprehensive approaches and have compared the outputs of wild-type and non-cold-induced Csp-deletion mutant cells. As a model organism, we selected Thermus thermophilus HB8 because it has only two csp genes (ttcsp1 and ttcsp2); ttCsp1 is the only non-cold-induced Csp. Surprisingly, the amount of transcripts and proteins upon deletion of the ttcsp1 gene was quite different. DNA microarray analysis revealed that the deletion of ttcsp1 did not affect the amount of transcripts, although the ttcsp1 gene was constantly expressed in the wild-type cell. Nonetheless, proteomic analysis revealed that the expression levels of many proteins were significantly altered when ttcsp1 was deleted. These results suggest that ttCsp1 functions in translation independent of transcription. Furthermore, ttCsp1 is involved in both the stimulation and inhibition of translation of specific proteins. Here, we have determined the crystal structure of ttCsp1 at 1.65 Å. This is the first report to present the structure of a non-cold-inducible cold shock protein. We also report the nucleotide binding affinity of ttCsp1. Finally, we discuss the functions of non-cold-induced Csps and propose how they modulate the levels of specific proteins to suit the prevailing environmental conditions.

An alkyltransferase-like protein from Thermus thermophilus HB8 affects the regulation of gene expression in alkylation response.

Alkylation is a type of stress that is fatal to cells. However, cells have various responses to alkylation. Alkyltransferase-like (ATL) protein is a novel protein involved in the repair of alkylated DNA; however, its repair mechanism at the molecular level is unclear. DNA microarray analysis revealed that the upregulation of 71 genes because of treatment with an alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine was related to the presence of TTHA1564, the ATL protein from Thermus thermophilus HB8. Affinity chromatography showed a direct interaction of purified TTHA1564 with purified RNA polymerase holoenzyme. The amino acid sequence of TTHA1564 is homologous to that of the C-terminal domain of Ada protein, which acts as a transcriptional activator. These results suggest that TTHA1564 might act as a transcriptional regulator. The results of DNA microarray analysis also implied that the alkylating agent induced oxidation stress in addition to alkylation stress.

Structures of hypoxanthine-guanine phosphoribosyltransferase (TTHA0220) from Thermus thermophilus HB8.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase), which is a key enzyme in the purine-salvage pathway, catalyzes the synthesis of IMP or GMP from alpha-D-phosphoribosyl-1-pyrophosphate and hypoxanthine or guanine, respectively. Structures of HGPRTase from Thermus thermophilus HB8 in the unliganded form, in complex with IMP and in complex with GMP have been determined at 2.1, 1.9 and 2.2 A resolution, respectively. The overall fold of the IMP complex was similar to that of the unliganded form, but the main-chain and side-chain atoms of the active site moved to accommodate IMP. The overall folds of the IMP and GMP complexes were almost identical to each other. Structural comparison of the T. thermophilus HB8 enzyme with 6-oxopurine PRTases for which structures have been determined showed that these enzymes can be tentatively divided into groups I and II and that the T. thermophilus HB8 enzyme belongs to group I. The group II enzymes are characterized by an N-terminal extension with additional secondary elements and a long loop connecting the second alpha-helix and beta-strand compared with the group I enzymes.

Very rapid induction of a cold shock protein by temperature downshift in Thermus thermophilus.

A rapid temperature downshift induces the expression of many proteins termed 'cold-induced' proteins. Although some of these proteins are known to participate in metabolism, transcription, translation and protein folding, processes that are affected by cold stress, it has not yet been identified which proteins sense the temperature downshift. Here we analyzed the mRNA expression profiles of genes induced immediately following a temperature downshift in Thermus thermophilus HB8. The cold shock protein gene ttcsp2 displayed the most rapid and drastic increase in mRNA. ttcsp2 mRNA was induced at 30s after temperature downshift, although ttCSP2 protein was first detected at 10 min. A temperature-dependent secondary structure was predicted to form in the 5'-untranslated region, including the Shine-Dalgarno sequence, of ttcsp2 mRNA. Stabilization of this secondary structure at 45 degrees C was assumed to prevent degradation of ttcsp2 mRNA and to slow translation. Thus, ttCSP2 is considered to act as a 'thermosensor' during temperature downshift through changes in its secondary structure.

Two dNTP triphosphohydrolases from Pseudomonas aeruginosa possess diverse substrate specificities.

Nucleotide hydrolases are known to hydrolyze not only noncanonical dNTPs to reduce the risk of mutation, but also canonical dNTPs to maintain the dNTP concentrations in the cell. dGTP triphosphohydrolase from Escherichia coli is known as an enzyme that hydrolyzes dGTP. Recently, we identified a triphosphohydrolase from Thermus thermophilus HB8 that hydrolyzes all canonical dNTPs through a complex activation mechanism. These dNTP triphosphohydrolases are widely distributed in eubacteria, but it is difficult to predict whether they possess hydrolytic activity for dGTP or dNTP. To obtain information concerning the structure-function relationships of this protein family, we characterized two dNTP triphosphohydrolases, PA1124 and PA3043, from Pseudomonas aeruginosa. Molecular phylogenic analysis showed that dNTP triphosphohydrolases can be classified into three groups. Experimentally, PA1124 had a preference for dGTP, similar to the E. coli enzyme, whereas PA3043 displayed a broad substrate specificity. Both enzymes hydrolyzed substrates in the absence of additional dNTP as an activating effector. These kinetic data suggest that PA3043 is a novel type distinct from both the E. coli and T. thermophilus enzymes. On the basis of these results, we propose that the dNTP triphosphohydrolase family should be classified into at least three subfamilies.

Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin.

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR-neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH-pi interactions in the Ls-AChBP-CTD complex than in the Ls-AChBP-IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.