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.

Optimizing genome editing efficiency in wheat: Effects of heat treatments and different promoters for single guide RNA expression.

Genome editing is a promising method for simultaneously mutagenizing homoeologs in the three subgenomes of wheat (Triticum aestivum L.). However, the mutation rate via genome editing must be improved in order to analyze gene function and to quickly modify agronomic traits in wheat. Here, we examined the Cas9-induced mutation rates in wheat plants using two promoters for single guide RNA (sgRNA) expression and applying heat treatment during Agrobacterium tumefaciens-mediated transformation. Using the TaU6 promoter instead of the OsU6 promoter from rice (Oryza sativa L.) to drive sgRNA expression greatly improved the Cas9-induced mutation rate. Moreover, a heat treatment of 30°C for 1 day during tissue culture increased the Cas9-induced mutation rate and the variety of mutations obtained compared to tissue culture at the normal temperature (25°C). The same heat treatment did not affect the regeneration rates of transgenic plants but tended to increase the number of transgene integration sites in each transgenic plant. These results lay the foundation for improving the Cas9-induced mutation rate in wheat to enhance research on gene function and crop improvement.

Modulation of wheat grain dormancy by introducing the recombinant abscisic acid-stimulated abscisic acid biosynthesis gene.

Pre-harvest sprouting of cereals greatly reduces yield and quality of the grains. Abscisic acid (ABA) is an essential phytohormone for the induction and maintenance of seed dormancy. In this study, the ABA responsive promoter-driven ABA biosynthesis gene system was introduced to common wheat (Triticum aestivum L.) to enhance ABA production in the embryos and pre-harvest sprouting tolerance of the grains. This system consists of a wheat ABA responsive element containing Early-Methionine-labelled (EM) promoter and a sorghum 9-cis-epoxycarotenoid dioxygenase (SbNCED) gene which encodes an ABA biosynthesis rate-limiting enzyme. Twenty-three independent single-insertion lines were obtained, from which five homozygous lines showing various SbNCED expression levels were selected. Correlations were observed between SbNCED expression, ABA accumulation in the embryos and enhanced dormancy levels of the grains. The engineered wheat grains exhibited a few day-delay in germination, which should be effective in reducing pre-harvest sprouting damage. However, the increase in ABA levels in the recombinant grains was moderate, which explains why germination was not completely suppressed. Further analysis indicated a concomitant increase in the expression of the ABA catabolic enzyme gene TaABA8'OH1 and in the levels of isoleucine-conjugated jasmonic acid, implying the presence of possible negative feedback regulation in the innate system, which should be overcome for future technology development. These findings advance an understanding of the regulatory mechanisms of hormone metabolism in seeds and facilitate the development of pre-harvest sprouting tolerance in cereal grains.

Regulation of germination by targeted mutagenesis of grain dormancy genes in barley.

High humidity during harvest season often causes pre-harvest sprouting in barley (Hordeum vulgare). Prolonged grain dormancy prevents pre-harvest sprouting; however, extended dormancy can interfere with malt production and uniform germination upon sowing. In this study, we used Cas9-induced targeted mutagenesis to create single and double mutants in QTL FOR SEED DORMANCY 1 (Qsd1) and Qsd2 in the same genetic background. We performed germination assays in independent qsd1 and qsd2 single mutants, as well as in two double mutants, which revealed a strong repression of germination in the mutants. These results demonstrated that normal early grain germination requires both Qsd1 and Qsd2 function. However, germination of qsd1 was promoted by treatment with 3% hydrogen peroxide, supporting the notion that the mutants exhibit delayed germination. Likewise, exposure to cold temperatures largely alleviated the block of germination in the single and double mutants. Notably, qsd1 mutants partially suppress the long dormancy phenotype of qsd2, while qsd2 mutant grains failed to germinate in the light, but not in the dark. Consistent with the delay in germination, abscisic acid accumulated in all mutants relative to the wild type, but abscisic acid levels cannot maintain long-term dormancy and only delay germination. Elucidation of mutant allele interactions, such as those shown in this study, are important for fine-tuning traits that will lead to the design of grain dormancy through combinations of mutant alleles. Thus, these mutants will provide the necessary germplasm to study grain dormancy and germination in barley.

Targeted genome modifications in cereal crops.

The recent advent of customizable endonucleases has led to remarkable advances in genetic engineering, as these molecular scissors allow for the targeted introduction of mutations or even precisely predefined genetic modifications into virtually any genomic target site of choice. Thanks to its unprecedented precision, efficiency, and functional versatility, this technology, commonly referred to as genome editing, has become an effective force not only in basic research devoted to the elucidation of gene function, but also for knowledge-based improvement of crop traits. Among the different platforms currently available for site-directed genome modifications, RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases have proven to be the most powerful. This review provides an application-oriented overview of the development of customizable endonucleases, current approaches to cereal crop breeding, and future opportunities in this field.

Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar 'Fielder'.

We have established a high-quality, chromosome-level genome assembly for the hexaploid common wheat cultivar 'Fielder', an American, soft, white, pastry-type wheat released in 1974 and known for its amenability to Agrobacterium tumefaciens-mediated transformation and genome editing. Accurate, long-read sequences were obtained using PacBio circular consensus sequencing with the HiFi approach. Sequence reads from 16 SMRT cells assembled using the hifiasm assembler produced assemblies with N50 greater than 20 Mb. We used the Omni-C chromosome conformation capture technique to order contigs into chromosome-level assemblies, resulting in 21 pseudomolecules with a cumulative size of 14.7 and 0.3 Gb of unanchored contigs. Mapping of published short reads from a transgenic wheat plant with an edited seed-dormancy gene, TaQsd1, identified four positions of transgene insertion into wheat chromosomes. Detection of guide RNA sequences in pseudomolecules provided candidates for off-target mutation induction. These results demonstrate the efficiency of chromosome-scale assembly using PacBio HiFi reads and their application in wheat genome-editing studies.

In planta Genome Editing in Commercial Wheat Varieties.

Limitations for the application of genome editing technologies on elite wheat (Triticum aestivum L.) varieties are mainly due to the dependency on in vitro culture and regeneration capabilities. Recently, we developed an in planta particle bombardment (iPB) method which has increased process efficiency since no culture steps are required to create stably genome-edited wheat plants. Here, we report the application of the iPB method to commercially relevant Japanese elite wheat varieties. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaQsd1 were bombarded into the embryos of imbibed seeds with their shoot apical meristem (SAM) exposed. Mutations in the target gene were subsequently analyzed within flag leaf tissue by using cleaved amplified polymorphic sequence (CAPS) analysis. A total of 9/358 (2.51%) of the bombarded plants (cv. "Haruyokoi," spring type) carried mutant alleles in the tissue. Due to the chimeric nature of the T0 plants, only six of them were inherited to the next (T1) generation. Genotypic analysis of the T2 plants revealed a single triple-recessive homozygous mutant of the TaQsd1 gene. Compared to wild type, the homozygous mutant exhibited a 7 days delay in the time required for 50% seed germination. The iPB method was also applied to two elite winter cultivars, "Yumechikara" and "Kitanokaori," which resulted in successful genome editing at slightly lower efficiencies as compared to "Haruyokoi." Taken together, this report demonstrates that the in planta genome editing method through SAM bombardment can be applicable to elite wheat varieties that are otherwise reluctant to callus culture.

Protocol for Genome Editing to Produce Multiple Mutants in Wheat.

Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat (Triticum aestivum) cv. Fielder. Using Agrobacterium-delivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated. The technique for acceleration of generation advancement with embryo culture reduces time for mutant production. The mutants produced by this protocol can be used for the analysis of gene function and crop improvement. For complete details on the use and execution of this protocol, please refer to Abe et al. (2019).

Low-temperature pretreatment of explants and high maltose concentration during callus culture improves particle-bombardment-mediated stable transgene expression in common wheat.

Biolistic transformation systems are widely used to introduce foreign genes into common wheat (Triticum aestivum L.); however, these techniques often generate high transgene copy numbers and complex transgene integration patterns that hinder the stable expression of the transgenes. To improve the efficiency of stable transgene expression, we examined the effect of low-temperature pretreatment of wheat flower spikes and of high maltose concentration (HMC) in the medium during the subsequent callus culture. Tillers of the spring wheat cultivar Bobwhite were stored at 5°C without water for one week before the isolation of their immature scutellar tissues, and the resulting particle-bombarded explants were cultured on 15% maltose for a month. Together, these treatments significantly increased the number of recovered transgenic lines expressing the reporter gene. The low-temperature pretreatment eliminated the negative effects of HMC, and HMC improved the efficiency of stable transgene expression. Southern blot analysis revealed that transgenic lines recovered after HMC treatment integrated a lower copy number of transgenes than those cultured at normal (4%) maltose concentration. These findings suggest that the HMC-mediated reduction of the transgene copy number results from the suppression of plasmid DNA rearrangement before or during transgene integration into the wheat genome.

A rapid method for detection of mutations induced by CRISPR/Cas9-based genome editing in common wheat.

Genome editing using CRISPR/Cas9 is useful for common wheat because common wheat has allohexaploid nature and it can induce mutations simultaneously in three homoeologous genes. Although Agrobacterium-mediated transformation has advantages in genome editing, it still has low efficiency and requires relatively long time in wheat. Therefore, the use of guide RNAs (gRNAs) with efficient mutagenesis in vivo is one of the critical factors for producing genome-edited mutant lines in a short time. In this study, we targeted three genes in common wheat and established a rapid method for detection of mutations induced by the biolistic transient expression system. Biolistic transient expression of the gRNAs and Cas9 was achieved in immature wheat embryos. Mutations were detected a week later using PCR-RFLP and verified by the sequencing of genomic clones. We confirmed several types of mutations that occurred at different rates depending on the target sequences. Furthermore, frequencies of mutations tended to be higher at the targets that were edited at higher rates in the plants transformed by Agrobacterium. These results show that this method of rapid detection of edited mutations could be used for variety of applications, such as screening of target sequences or modified vectors for efficient CRISPR/Cas9 genome editing in wheat.

Genome-Edited Triple-Recessive Mutation Alters Seed Dormancy in Wheat.

Common wheat has three sets of sub-genomes, making mutations difficult to observe, especially for traits controlled by recessive genes. Here, we produced hexaploid wheat lines with loss of function of homeoalleles of Qsd1, which controls seed dormancy in barley, by Agrobacterium-mediated CRISPR/Cas9. Of the eight transformed wheat events produced, three independent events carrying multiple mutations in wheat Qsd1 homeoalleles were obtained. Notably, one line had mutations in every homeoallele. We crossed this plant with wild-type cultivar Fielder to generate a transgene-free triple-recessive mutant, as revealed by Mendelian segregation. The mutant showed a significantly longer seed dormancy period than wild-type, which may result in reduced pre-harvest sprouting of grains on spikes. PCR, southern blotting, and whole-genome shotgun sequencing revealed that this segregant lacked transgenes in its genomic sequence. This technique serves as a model for trait improvement in wheat, particularly for genetically recessive traits, based on locus information from diploid barley.

Root Cortex Provides a Venue for Gas-Space Formation and Is Essential for Plant Adaptation to Waterlogging.

Lysigenous aerenchyma, which develops by death and subsequent lysis of the cortical cells in roots, is essential for internal long-distance oxygen transport from shoot base to root tips of plants in waterlogged soil. Although many studies focus on the amounts of aerenchyma in roots, significance of the size of the root cortex in which aerenchyma forms has received less research attention. In the present study, we evaluated the cross-sectional area of each root tissue in adventitious roots of upland crops, wheat (Triticum aestivum) and maize (Zea mays ssp. mays), and the wetland crop, rice (Oryza sativa) under aerated or stagnant deoxygenated conditions; the latter can mimic the changes in gas composition in waterlogged soils. Our analyses revealed that the areas of whole root and cortex of the three species increased under stagnant conditions. In rice roots, cortex to stele ratio (CSR) and aerenchyma to cortex ratio (ACR), which is associated with the areas of gas spaces, were much higher than those in wheat and maize roots, suggesting that these anatomical features are essential for a high capacity for oxygen transport along roots. To test this hypothesis, rates of radial oxygen loss (ROL), which is the diffusive flux of oxygen from within a root to the external medium, from thick and thin adventitious roots of rice were measured using a cylindrical (root-sleeving) oxygen electrode, for plants with shoots in air and roots in an oxygen-free medium. As expected, the rate of ROL from thick roots, which have larger cortex and aerenchyma areas, was higher than that of thin roots. The rate of ROL was highest at the apical part of rice roots, where aerenchyma was hardly detected, but at which cuboidal cell arrangement in the cortex provides tissue porosity. We conclude that high CSR in combination with large root diameter is a feature which promotes oxygen transport from shoot base to root tips of plants. Moreover, we propose that CSR should be a useful quantitative index for the evaluation and improvement of root traits contributing to tolerance of crops to soil waterlogging.

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.

The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat.

TAD1 (Triticum aestivum defensin 1) is induced during cold acclimation in winter wheat and encodes a plant defensin with antimicrobial activity. In this study, we demonstrated that recombinant TAD1 protein inhibits hyphal growth of the snow mold fungus, Typhula ishikariensis in vitro. Transgenic wheat plants overexpressing TAD1 were created and tested for resistance against T. ishikariensis. Leaf inoculation assays revealed that overexpression of TAD1 confers resistance against the snow mold. In addition, the TAD1-overexpressors showed resistance against Fusarium graminearum, which causes Fusarium head blight, a devastating disease in wheat and barley. These results indicate that TAD1 is a candidate gene to improve resistance against multiple fungal diseases in cereal crops.

Characterization of a wheat pathogenesis-related protein, TaBWPR-1.2, in seminal roots in response to waterlogging stress.

We examined the role of pathogenesis-related protein TaBWPR-1.2 in the context of molecular and physiological responses of wheat (Triticum aestivum) seminal roots under waterlogging stress. Two cDNAs corresponding to the TaBWPR-1.2 gene, TaBWPR-1.2#2 and TaBWPR-1.2#13 were cloned from seminal roots. These cDNAs were predicted to encode proteins of 173 and 172 amino acids, respectively. In a time-course experiment, TaBWPR-1.2 gene expression was highest in whole seminal roots after 1 day of waterlogging treatment and higher than the control for at least 10 days; significantly increased protein abundance was observed after 7 days of waterlogging. Drought, another abiotic stress, did not influence TaBWPR-1.2 gene expression in wheat seminal roots at 5-d-old seedlings. Tissue-specific studies revealed that the highest TaBWPR-1.2 gene expression and protein levels were in the aerenchymatous root zone. TaBWPR-1.2 expression in seminal roots was also increased by the signalling molecules 1-aminocyclopropane-1-carboxylic acid (ACC; an ethylene precursor), H2O2, jasmonic acid (JA), and nitric oxide (NO); however, treatment with abscisic acid (ABA), salicylic acid (SA), and ethanol did not alter its expression. Interestingly, aerenchyma formation in the seminal root cortex was induced only by ACC and H2O2. Taken together, these results indicate that TaBWPR-1.2 is a waterlogging-responsive gene that might be associated with root cortex tissue alteration in wheat plants through ACC and/or H2O2 regulatory mechanisms.

Adventitious roots of wheat seedlings that emerge in oxygen-deficient conditions have increased root diameters with highly developed lysigenous aerenchyma.

Exposing roots of plants to hypoxic conditions is known to greatly improve their anoxic stress tolerance. We previously showed that pre-treatment of wheat seedlings with an ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), enhanced their tolerance of oxygen-deficient conditions. Although ACC-pretreated seminal roots of wheat seedlings grown under oxygen-deficient conditions avoided root tip death, they elongated very little. In the present study, we assessed the effects of ethylene on the responses of adventitious roots of wheat seedlings to oxygen-deficient conditions. Lysigenous aerenchyma formation in the adventitious roots of wheat seedlings pretreated with ACC appeared to reduce tip death under oxygen-deficient conditions, but the adventitious roots, like the seminal roots, hardly elongated. We also found that adventitious roots that emerge in oxygen-deficient conditions continued to elongate even under such conditions. The adventitious roots emerged in oxygen-deficient conditions were found to have thicker root diameters than those emerged in aerated conditions. These results suggest that the adventitious roots with thicker root diameters can better cope with oxygen-deficient conditions. Measurements of the area of the lysigenous aerenchyma confirmed that the increased root diameters have a greater amount of air space generated by lysigenous aerenchyma formation.

A transgenic approach to controlling wheat seed dormancy level by using Triticeae DOG1-like genes.

Seed dormancy is an important agronomic trait: low levels can cause premature germination, while too much can inhibit uniform germination. As an approach to controlling the seed dormancy level in crops, we used Triticeae DOG1-like genes as transgenes. DOG1 is an Arabidopsis gene that underlies natural variation in seed dormancy. We previously showed that although their sequence similarities to DOG1 were low, some cereal DOG1-like genes enhanced seed dormancy in Arabidopsis. Here, we introduced two DOG1-like genes, TaDOG1L4 from wheat and HvDOG1L1 from barley, individually into the wheat cultivar Fielder. Their overexpression under the control of a maize ubiquitin promoter enhanced the seed dormancy level while leaving other traits unchanged. TaDOG1L4 was more effective than HvDOG1L1, which accords with the previously revealed difference in the effectiveness of these two genes in Arabidopsis seed dormancy. Knockdown of endogenous TaDOG1L4 in Fielder using double-strand RNA interference decreased the seed dormancy level by several tens of percent. This result indicates that some degree of seed dormancy inherent in wheat is imparted by DOG1-like genes.

Ethylene and reactive oxygen species are involved in root aerenchyma formation and adaptation of wheat seedlings to oxygen-deficient conditions.

Exposing plants to hypoxic conditions greatly improves their anoxic stress tolerance by enhancing the activities of glycolysis and fermentation in roots. Ethylene may also be involved in these adaptive responses because its synthesis is increased in roots under hypoxic conditions. Here it is reported that pre-treatment of wheat seedlings with an ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), enhanced accumulation of ethylene in the roots of wheat seedlings, and enhanced their tolerance of oxygen-deficient conditions through increasing the expression of genes encoding ethanol fermentation enzymes, alcohol dehydrogenase and pyruvate decarboxylase, in the roots. Lysigenous aerenchyma formation in root was induced by ACC pre-treatment and was further induced by growth under oxygen-deficient conditions. ACC pre-treatment increased the expression of three genes encoding respiratory burst oxidase homologue (a plant homologue of gp91(phox) in NADPH oxidase), which has a role in the generation of reactive oxygen species (ROS), in roots of seedlings. Co-treatment with ACC and an NADPH oxidase inhibitor, diphenyleneiodonium, partly suppressed the ACC-induced responses. These results suggest that ethylene and ROS are involved in adaptation of wheat seedlings to oxygen-deficient conditions through controlling lysigenous aerenchyma formation and the expression of genes encoding ethanol fermentation enzymes.

Quantitative Proteomics of the Root of Transgenic Wheat Expressing TaBWPR-1.2 Genes in Response to Waterlogging.

Once candidate genes are available, the application of genetic transformation plays a major part to study their function in plants for adaptation to respective environmental stresses, including waterlogging (WL). The introduction of stress-inducible genes into wheat remains difficult because of low transformation and plant regeneration efficiencies and expression variability and instability. Earlier, we found two cDNAs encoding WL stress-responsive wheat pathogenesis-related proteins 1.2 (TaBWPR-1.2), TaBWPR-1.2#2 and TaBWPR-1.2#13. Using microprojectile bombardment, both cDNAs were introduced into "Bobwhite". Despite low transformation efficiency, four independent T2 homozygous lines for each gene were isolated, where transgenes were ubiquitously and variously expressed. The highest transgene expression was obtained in Ubi:TaBWPR-1.2#2 L#11a and Ubi:TaBWPR-1.2#13 L#4a. Using quantitative proteomics, the root proteins of L#11a were analyzed to explore possible physiological pathways regulated by TaBWPR-1.2 under normal and waterlogged conditions. In L#11a, the abundance of proteasome subunit alpha type-3 decreased under normal conditions, whereas that of ferredoxin precursor and elongation factor-2 increased under waterlogged conditions in comparison with normal plants. Proteomic results suggest that L#11a is one of the engineered wheat plants where TaBWPR-1.2#2 is most probably involved in proteolysis, protein synthesis and alteration in the energy pathway in root tissues via the above proteins in order to gain metabolic adjustment to WL.