Stacked mutations in wheat homologues of rice SEMI-DWARF1 confer a novel semi-dwarf phenotype.

BACKGROUND: Semi-dwarfing alleles are used widely in cereals to confer improved lodging resistance and assimilate partitioning. The most widely deployed semi-dwarfing alleles in rice and barley encode the gibberellin (GA)-biosynthetic enzyme GA 20-OXIDASE2 (GA20OX2). The hexaploid wheat genome carries three homoeologous copies of GA20OX2, and because of functional redundancy, loss-of-function alleles of a single homoeologue would not be selected in wheat breeding programmes. Instead, approximately 70% of wheat cultivars carry gain-of-function mutations in REDUCED HEIGHT 1 (RHT1) genes that encode negative growth regulators and are degraded in response to GA. Semi-dwarf Rht-B1b or Rht-D1b alleles encode proteins that are insensitive to GA-mediated degradation. However, because RHT1 is expressed ubiquitously these alleles have pleiotropic effects that confer undesirable traits in some environments. RESULTS: We have applied reverse genetics to combine loss-of-function alleles in all three homoeologues of wheat GA20OX2 and its paralogue GA20OX1 and evaluated their performance in three years of field trials. ga20ox1 mutants exhibited a mild height reduction (approximately 3%) suggesting GA20OX1 plays a minor role in stem elongation in wheat. ga20ox2 mutants have reduced GA1 content and are 12-32% shorter than their wild-type segregants, comparable to the effect of the Rht-D1b 'Green Revolution' allele. The ga20ox2 mutants showed no significant negative effects on yield components in the spring wheat variety 'Cadenza'. CONCLUSIONS: Our study demonstrates that chemical mutagenesis can expand genetic variation in polyploid crops to uncover novel alleles despite the difficulty in identifying appropriate mutations for some target genes and the negative effects of background mutations. Field experiments demonstrate that mutations in GA20OX2 reduce height in wheat, but it will be necessary to evaluate the effect of these alleles in different genetic backgrounds and environments to determine their value in wheat breeding as alternative semi-dwarfing alleles.

Identification and validation of a QTL for spikelet number on chromosome arm 6BL of common wheat (Triticum aestivum L.).

To provide food security for a growing world population, it will be necessary to increase yields of staple crops such as wheat (Triticum aestivum L.). Yield is a complex, polygenic trait influenced by grain weight and number, which are negatively correlated with one another. Spikelet number is an important determinant of grain number, but allelic variants impacting its expression are often associated with heading date, constraining their use in wheat germplasm that must be adapted for specific environments. Identification and characterization of genetic variants affecting spikelet number will increase selection efficiency through their deployment in breeding programs. In this study, a quantitative trait locus (QTL) on chromosome arm 6BL for spikelet number was identified and validated using an association mapping panel, a recombinant inbred line population, and seven derived heterogeneous inbred families. The superior allele, QSn.csu-6Bb, was associated with an increase of 0.248 to 0.808 spikelets per spike across multiple environments that varied for mean spikelet number. Despite epistatic interactions between QSn.csu-6B and three other loci (WAPO-A1, VRN-D3, and PPD-B1), genotypes with a greater number of superior alleles at these loci consistently exhibit higher spikelet number. The frequency of superior alleles at these loci varies among winter wheat varieties adapted to different latitudes of the US Great Plains, revealing opportunities for breeders to select for increased spikelet number using simple molecular markers. This work lays the foundation for the positional cloning of the genetic variant underlying the QSn.csu-6B QTL to strengthen our understanding of spikelet number determination in wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01288-7.

Perimortem fractures in Lucy suggest mortality from fall out of tall tree.

The Pliocene fossil 'Lucy' (Australopithecus afarensis) was discovered in the Afar region of Ethiopia in 1974 and is among the oldest and most complete fossil hominin skeletons discovered. Here we propose, on the basis of close study of her skeleton, that her cause of death was a vertical deceleration event or impact following a fall from considerable height that produced compressive and hinge (greenstick) fractures in multiple skeletal elements. Impacts that are so severe as to cause concomitant fractures usually also damage internal organs; together, these injuries are hypothesized to have caused her death. Lucy has been at the centre of a vigorous debate about the role, if any, of arboreal locomotion in early human evolution. It is therefore ironic that her death can be attributed to injuries resulting from a fall, probably out of a tall tree, thus offering unusual evidence for the presence of arborealism in this species.

Extensive structural variation in the Bowman-Birk inhibitor family in common wheat (Triticum aestivum L.).

BACKGROUND: Bowman-Birk inhibitors (BBI) are a family of serine-type protease inhibitors that modulate endogenous plant proteolytic activities during different phases of development. They also inhibit exogenous proteases as a component of plant defense mechanisms, and their overexpression can confer resistance to phytophagous herbivores and multiple fungal and bacterial pathogens. Dicot BBIs are multifunctional, with a "double-headed" structure containing two separate inhibitory loops that can bind and inhibit trypsin and chymotrypsin proteases simultaneously. By contrast, monocot BBIs have a non-functional chymotrypsin inhibitory loop, although they have undergone internal duplication events giving rise to proteins with multiple BBI domains. RESULTS: We used a Hidden Markov Model (HMM) profile-based search to identify 57 BBI genes in the common wheat (Triticum aestivum L.) genome. The BBI genes are unevenly distributed, with large gene clusters in the telomeric regions of homoeologous group 1 and 3 chromosomes that likely arose through a series of tandem gene duplication events. The genomes of wheat progenitors also contain contiguous clusters of BBI genes, suggesting this family underwent expansion before the domestication of common wheat. However, the BBI gene family varied in size among different cultivars, showing this family remains dynamic. Because of these expansions, the BBI gene family is larger in wheat than other monocots such as maize, rice and Brachypodium. We found BBI proteins in common wheat with intragenic homologous duplications of cysteine-rich functional domains, including one protein with four functional BBI domains. This diversification may expand the spectrum of target substrates. Expression profiling suggests that some wheat BBI proteins may be involved in regulating endogenous proteases during grain development, while others were induced in response to biotic and abiotic stresses, suggesting a role in plant defense. CONCLUSIONS: Genome-wide characterization reveals that the BBI gene family in wheat is subject to a high rate of homologous tandem duplication and deletion events, giving rise to a diverse set of encoded proteins. This information will facilitate the functional characterization of individual wheat BBI genes to determine their role in wheat development and stress responses, and their potential application in breeding.

Reduced free asparagine in wheat grain resulting from a natural deletion of TaASN-B2: investigating and exploiting diversity in the asparagine synthetase gene family to improve wheat quality.

BACKGROUND: Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties. RESULTS: Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency. CONCLUSIONS: Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain.

Differential Stem Proteomics and Metabolomics Profiles for Four Wheat Cultivars in Response to the Insect Pest Wheat Stem Sawfly.

Common wheat (Triticum aestivum L.) is a global staple crop, and insect pests can impact grain yield. The wheat stem sawfly (Cephus cinctus, WSS) is a major wheat pest, and while partial resistance has been deployed by breeding for a solid-stem trait, this trait is affected by environment. Here, a proteomics and metabolomics study was performed on four wheat cultivars to characterize a molecular response to WSS infestation. The cultivars Hatcher (hollow-stem partially tolerant), Conan (semisolid-stem-resistant), and Denali and Reeder (hollow-stem-susceptible) were infested with WSS, and changes in stem proteins and metabolites were characterized using liquid chromatography-mass spectrometry. The proteome was characterized as 1830 proteins that included five major biological processes, including metabolic processes and response to stimuli, and the metabolome (1823 metabolites) spanned eight chemical superclasses, including alkaloids, benzenoids, and lipids. All four varieties had a molecular response to WSS following infestation. Hatcher had the most distinct changes, whereby 62 proteins and 29 metabolites varied in metabolic pathways involving enzymatic detoxification, proteinase inhibition, and antiherbivory compound production via benzoxazinoids, neolignans, and phenolics. Taken together, these data demonstrate variation in the wheat stem molecular response to WSS infestation and support breeding for molecular resistance in hollow-stem cultivars.

Effect of phyB and phyC loss-of-function mutations on the wheat transcriptome under short and long day photoperiods.

BACKGROUND: Photoperiod signals provide important cues by which plants regulate their growth and development in response to predictable seasonal changes. Phytochromes, a family of red and far-red light receptors, play critical roles in regulating flowering time in response to changing photoperiods. A previous study showed that loss-of-function mutations in either PHYB or PHYC result in large delays in heading time and in the differential regulation of a large number of genes in wheat plants grown in an inductive long day (LD) photoperiod. RESULTS: We found that under non-inductive short-day (SD) photoperiods, phyB-null and phyC-null mutants were taller, had a reduced number of tillers, longer and wider leaves, and headed later than wild-type (WT) plants. The delay in heading between WT and phy mutants was greater in LD than in SD, confirming the importance of PHYB and PHYC in accelerating heading date in LDs. Both mutants flowered earlier in SD than LD, the inverse response to that of WT plants. In both SD and LD photoperiods, PHYB regulated more genes than PHYC. We identified subsets of differentially expressed and alternatively spliced genes that were specifically regulated by PHYB and PHYC in either SD or LD photoperiods, and a smaller set of genes that were regulated in both photoperiods. We found that photoperiod had a contrasting effect on transcript levels of the flowering promoting genes VRN-A1 and PPD-B1 in phyB and phyC mutants compared to the WT. CONCLUSIONS: Our study confirms the major role of both PHYB and PHYC in flowering promotion in LD conditions. Transcriptome characterization revealed an unexpected reversion of the wheat LD plants into SD plants in the phyB-null and phyC-null mutants and identified flowering genes showing significant interactions between phytochromes and photoperiod that may be involved in this phenomenon. Our RNA-seq data provides insight into light signaling pathways in inductive and non-inductive photoperiods and a set of candidate genes to dissect the underlying developmental regulatory networks in wheat.

Oxidative Catabolism of (+)-Pinoresinol Is Initiated by an Unusual Flavocytochrome Encoded by Translationally Coupled Genes within a Cluster of (+)-Pinoresinol-Coinduced Genes in Pseudomonas sp. Strain SG-MS2.

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a Km of 1.17 µM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes.IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the ß-ß' linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

Genomic changes associated with adaptation to arid environments in cactophilic Drosophila species.

BACKGROUND: Insights into the genetic capacities of species to adapt to future climate change can be gained by using comparative genomic and transcriptomic data to reconstruct the genetic changes associated with such adaptations in the past. Here we investigate the genetic changes associated with adaptation to arid environments, specifically climatic extremes and new cactus hosts, through such an analysis of five repleta group Drosophila species. RESULTS: We find disproportionately high rates of gene gains in internal branches in the species' phylogeny where cactus use and subsequently cactus specialisation and high heat and desiccation tolerance evolved. The terminal branch leading to the most heat and desiccation resistant species, Drosophila aldrichi, also shows disproportionately high rates of both gene gains and positive selection. Several Gene Ontology terms related to metabolism were enriched in gene gain events in lineages where cactus use was evolving, while some regulatory and developmental genes were strongly selected in the Drosophila aldrichi branch. Transcriptomic analysis of flies subjected to sublethal heat shocks showed many more downregulation responses to the stress in a heat sensitive versus heat resistant species, confirming the existence of widespread regulatory as well as structural changes in the species' differing adaptations. Gene Ontology terms related to metabolism were enriched in the differentially expressed genes in the resistant species while terms related to stress response were over-represented in the sensitive one. CONCLUSION: Adaptations to new cactus hosts and hot desiccating environments were associated with periods of accelerated evolutionary change in diverse biochemistries. The hundreds of genes involved suggest adaptations of this sort would be difficult to achieve in the timeframes projected for anthropogenic climate change.

Mal de Río Cuarto virus infection causes hormone imbalance and sugar accumulation in wheat leaves.

BACKGROUND: Mal de Río Cuarto virus (MRCV) infects several monocotyledonous species including maize and wheat. Infected plants show shortened internodes, partial sterility, increased tillering and reduced root length. To better understand the molecular basis of the plant-virus interactions leading to these symptoms, we combined RNA sequencing with metabolite and hormone measurements. RESULTS: More than 3000 differentially accumulated transcripts (DATs) were detected in MRCV-infected wheat plants at 21 days post inoculation compared to mock-inoculated plants. Infected plants exhibited decreased levels of TaSWEET13 transcripts, which are involved in sucrose phloem loading. Soluble sugars, starch, trehalose 6-phosphate (Tre6P), and organic and amino acids were all higher in MRCV-infected plants. In addition, several transcripts related to plant hormone metabolism, transport and signalling were increased upon MRCV infection. Transcripts coding for GA20ox, D14, MAX2 and SMAX1-like proteins involved in gibberellin biosynthesis and strigolactone signalling, were reduced. Transcripts involved in jasmonic acid, ethylene and brassinosteroid biosynthesis, perception and signalling and in auxin transport were also altered. Hormone measurements showed that jasmonic acid, brassinosteroids, abscisic acid and indole-3-acetic acid were significantly higher in infected leaves. CONCLUSIONS: Our results indicate that MRCV causes a profound hormonal imbalance that, together with alterations in sugar partitioning, could account for the symptoms observed in MRCV-infected plants.

Identification of a candidate gene for a QTL for spikelet number per spike on wheat chromosome arm 7AL by high-resolution genetic mapping.

KEY MESSAGE: A high-resolution genetic map combined with haplotype analyses identified a wheat ortholog of rice gene APO1 as the best candidate gene for a 7AL locus affecting spikelet number per spike. A better understanding of the genes controlling differences in wheat grain yield components can accelerate the improvements required to satisfy future food demands. In this study, we identified a promising candidate gene underlying a quantitative trait locus (QTL) on wheat chromosome arm 7AL regulating spikelet number per spike (SNS). We used large heterogeneous inbred families ( > 10,000 plants) from two crosses to map the 7AL QTL to an 87-kb region (674,019,191-674,106,327 bp, RefSeq v1.0) containing two complete and two partial genes. In this region, we found three major haplotypes that were designated as H1, H2 and H3. The H2 haplotype contributed the high-SNS allele in both H1 × H2 and H2 × H3 segregating populations. The ancestral H3 haplotype is frequent in wild emmer (48%) but rare (~ 1%) in cultivated wheats. By contrast, the H1 and H2 haplotypes became predominant in modern cultivated durum and common wheat, respectively. Among the four candidate genes, only TraesCS7A02G481600 showed a non-synonymous polymorphism that differentiated H2 from the other two haplotypes. This gene, designated here as WHEAT ORTHOLOG OF APO1 (WAPO1), is an ortholog of the rice gene ABERRANT PANICLE ORGANIZATION 1 (APO1), which affects spikelet number. Taken together, the high-resolution genetic map, the association between polymorphisms in the different mapping populations with differences in SNS, and the known role of orthologous genes in other grass species suggest that WAPO-A1 is the most likely candidate gene for the 7AL SNS QTL among the four genes identified in the candidate gene region.

Detoxification Genes Differ Between Cactus-, Fruit-, and Flower-Feeding Drosophila.

We use annotated genomes of 14 Drosophila species covering diverse host use phenotypes to test whether 4 gene families that often have detoxification functions are associated with host shifts among species. Bark, slime flux, flower, and generalist necrotic fruit-feeding species all have similar numbers of carboxyl/cholinesterase, glutathione S-transferase, cytochrome P450, and UDP-glucuronosyltransferase genes. However, species feeding on toxic Morinda citrifolia fruit and the fresh fruit-feeding Drosophila suzukii have about 30 and 60 more, respectively. ABC transporters show a different pattern, with the flower-feeding D. elegans and the generalist necrotic fruit and cactus feeder D. hydei having about 20 and >100 more than the other species, respectively. Surprisingly, despite the complex secondary chemistry we find that 3 cactophilic specialists in the mojavensis species cluster have variably fewer genes than any of the other species across all 4 families. We also find 82 positive selection events across the 4 families, with the terminal D. suzukii and M. citrifolia-feeding D. sechellia branches again having the highest number of such events in proportion to their respective branch lengths. Many of the genes involved in these host-use-specific gene number differences or positive selection events lie in specific clades of the gene families that have been recurrently associated with detoxification. Several genes are also found to be involved in multiple duplication and/or positive selection events across the species studied regardless of their host use phenotypes; the most frequently involved are the ABC transporter CG1718, which is not in a specific clade associated with detoxification, and the α-esterase gene cluster, which is.

Phenotypic and transcriptomic characterization of a wheat tall mutant carrying an induced mutation in the C-terminal PFYRE motif of RHT-B1b.

BACKGROUND: As central regulators of the gibberellic acid (GA) signaling pathway in plants, DELLA proteins function as growth repressors and affect diverse biological processes. The wheat RHT-B1b and RHT-D1b semi-dwarfing alleles, which encode GA-insensitive DELLA proteins, have been widely adopted in modern wheat varieties to improve lodging tolerance and harvest index. However, the molecular mechanisms by which DELLA modulates these responses in wheat remain largely unknown. RESULTS: We identified a tall tetraploid wheat mutant line carrying an induced missense mutation (E529K) in the PFYRE motif of RHT-B1b that partially suppressed the semi-dwarf phenotype. The height-increasing effect of RHT-B1bE529K relative to RHT-B1b (19 cm or 21% increase) was significantly smaller than the effect of RHT-B1a (33 cm or 34% increase) relative to RHT-B1b in the same field experiment. The RHT-B1bE529K mutation was also associated with length increases in coleoptiles, seedling shoots, and stem internodes relative to the RHT-B1b allele. We detected no significant differences in germination rate, seedling root length, tiller number, flag leaf size, spike length, or yield components. Using RNA-seq, we compared gene expression profiles of plants encoding RHT-B1b and RHT-B1bE529K in coleoptile, first leaf, and elongating peduncles. We detected limited overlap among tissues of the genes differentially regulated by the two genotypes, and more genes upregulated (77%) than downregulated (23%) in RHT-B1bE529K relative to RHT-B1b. These results suggest that the wheat DELLA protein affects the transcriptome in a tissue-specific manner and that the mutation mainly eliminates or reduces repression functions of the RHT-B1 protein. Our study identified distinct sets of potential DELLA direct or indirect target genes involved in cell wall and carbohydrate metabolisms, cell cycle/division, and hormone pathways. CONCLUSIONS: We identified the hypomorphic RHT-B1bE529K allele that confers an intermediate plant height and coleoptile elongation. This allele can be useful in rain-fed wheat breeding programs where the strong reduction in height and biomass associated with RHT-B1b has detrimental effects. Transcriptomic characterization of different tissues from the plants encoding RHT-B1bE529K and RHT-B1b provided valuable information for identifying DELLA downstream GA response genes in wheat.

Mapping causal mutations by exome sequencing in a wheat TILLING population: a tall mutant case study.

Forward genetic screens of induced mutant plant populations are powerful tools to identify genes underlying phenotypes of interest. Using traditional techniques, mapping causative mutations from forward screens is a lengthy, multi-step process, requiring the identification of a broad genetic region followed by candidate gene sequencing to characterize the causal variant. Mapping by whole genome sequencing accelerates the identification of causal mutations by simultaneously defining a mapping region and providing information on the induced genetic variants. In wheat, although the availability of a high-quality draft genome assembly facilitates mapping and mutation calling, whole genome resequencing remains prohibitively expensive due to its large genome. In the current study, we used exome sequencing as a complexity reduction strategy to detect mutations associated with a target phenotype. In a segregating wheat EMS population, we identified a clear peak region on chromosome arm 4BS associated with increased plant height. Although none of the significant SNPs seemed causative for the mutant phenotype, they were sufficient to identify a linked ~ 1.9 Mb deletion encompassing nine genes. These genes included Rht-B1, which is known to have a strong effect on plant height and is a strong candidate for the observed phenotype. We performed simulation experiments to determine the impacts of sequencing depth and bulk size and discuss the importance of considering each factor when designing mapping-by-sequencing experiments in wheat. This approach can accelerate the identification of candidate causal point mutations or linked deletions underlying important phenotypes.

Night-Break Experiments Shed Light on the Photoperiod1-Mediated Flowering.

Plants utilize variation in day length (photoperiod) to anticipate seasonal changes. They respond by modulating their growth and development to maximize seed production, which in cereal crops is directly related to yield. In wheat (Triticum aestivum), the acceleration of flowering under long days (LD) is dependent on the light induction of PHOTOPERIOD1 (PPD1) by phytochromes. Under LD, PPD1 activates FLOWERING LOCUS T1 (FT1), a mobile signaling protein that travels from the leaves to the shoot apical meristem to promote flowering. Here, we show that the interruption of long nights by short pulses of light ("night-break" [NB]) accelerates wheat flowering, suggesting that the duration of the night is critical for wheat photoperiodic response. PPD1 transcription was rapidly upregulated by NBs, and the magnitude of this induction increased with the length of darkness preceding the NB Cycloheximide abolished the NB up-regulation of PPD1, suggesting that this process is dependent on active protein synthesis during darkness. While one NB was sufficient to induce PPD1, more than 15 NBs were required to induce high levels of FT1 expression and a strong acceleration of flowering. Multiple NBs did not affect the expression of core circadian clock genes. The acceleration of flowering by NB disappeared in ppd1-null mutants, demonstrating that this response is mediated by PPD1 The acceleration of flowering was strongest when NBs were applied in the middle of the night, suggesting that in addition to PPD1, other circadian-controlled factors are required for the up-regulation of FT1 expression and the acceleration of flowering.

Identification and characterization of Rht25, a locus on chromosome arm 6AS affecting wheat plant height, heading time, and spike development.

KEY MESSAGE: This study identified Rht25, a new plant height locus on wheat chromosome arm 6AS, and characterized its pleiotropic effects on important agronomic traits. Understanding genes regulating wheat plant height is important to optimize harvest index and maximize grain yield. In modern wheat varieties grown under high-input conditions, the gibberellin-insensitive semi-dwarfing alleles Rht-B1b and Rht-D1b have been used extensively to confer lodging tolerance and improve harvest index. However, negative pleiotropic effects of these alleles (e.g., poor seedling emergence and reduced biomass) can cause yield losses in hot and dry environments. As part of current efforts to diversify the dwarfing alleles used in wheat breeding, we identified a quantitative trait locus (QHt.ucw-6AS) affecting plant height in the proximal region of chromosome arm 6AS (< 0.4 cM from the centromere). Using a large segregating population (~ 2800 gametes) and extensive progeny tests (70-93 plants per recombinant family), we mapped QHt.ucw-6AS as a Mendelian locus to a 0.2 cM interval (144.0-148.3 Mb, IWGSC Ref Seq v1.0) and show that it is different from Rht18. QHt.ucw-6AS is officially designated as Rht25, with Rht25a representing the height-increasing allele and Rht25b the dwarfing allele. The average dwarfing effect of Rht25b was found to be approximately half of the effect observed for Rht-B1b and Rht-D1b, and the effect is greater in the presence of the height-increasing Rht-B1a and Rht-D1a alleles than in the presence of the dwarfing alleles. Rht25b is gibberellin-sensitive and shows significant pleiotropic effects on coleoptile length, heading date, spike length, spikelet number, spikelet density, and grain weight. Rht25 represents a new alternative dwarfing locus that should be evaluated for its potential to improve wheat yield in different environments.

RNA-seq studies using wheat PHYTOCHROME B and PHYTOCHROME C mutants reveal shared and specific functions in the regulation of flowering and shade-avoidance pathways.

BACKGROUND: In cereal crops such as wheat, an optimal timing of developmental transitions is required to maximize grain yield. Many of these developmental changes are precisely regulated by changes in the duration, intensity or quality of light. Phytochromes are dimeric photoreceptors that absorb light maximally in the red and far-red wavelengths and induce large-scale transcriptional changes in response to variation in light quality. In wheat, PHYC is required for early flowering under long days. However, it is currently unknown whether this function requires the presence of PHYB. In this study, we characterized the role of PHYB in wheat development and used RNA-seq to analyze and compare the transcriptomes of phyB-null and phyC-null TILLING mutants. RESULTS: Under long-day photoperiods, phyB-null plants exhibit a severe delay in flowering comparable to the delay observed in phyC-null plants. These results demonstrate that both genes are required for the induction of wheat flowering under long days. Using replicated RNA-seq studies we identified 82 genes that are significantly up or down regulated in both the phyB-null and phyC-null mutant relative to their respective wild-type controls. Among these genes are several well-characterized positive regulators of flowering, including PPD1, FT1 and VRN1. Eight-fold more genes were differentially regulated only in the phyB-null mutant (2202) than only in the phyC-null mutant (261). The PHYB-regulated genes were enriched in components of the auxin, gibberellin and brassinosteroid biosynthesis and signaling pathways, and in transcription factors with putative roles in regulating vegetative development and shade-avoidance responses. Several genes involved in abiotic stress tolerance pathways were also found to be regulated by PHYB. CONCLUSIONS: PHYB and PHYC are both required for the photoperiodic induction of wheat flowering, whereas PHYB alone regulates a large number of genes involved in hormone biosynthesis and signaling, shade-avoidance response, and abiotic stress tolerance. Our analysis provides a comprehensive overview of the PHYB- and PHYC-mediated transcriptional changes during light signaling, and an initial step towards the dissection of this regulatory gene network in wheat. This further dissection will be required to explore the individual phytochrome-mediated developmental responses and to evaluate their potential to improve wheat adaptation to changing environments.

WheatExp: an RNA-seq expression database for polyploid wheat.

BACKGROUND: For functional genomics studies, it is important to understand the dynamic expression profiles of transcribed genes in different tissues, stages of development and in response to environmental stimuli. The proliferation in the use of next-generation sequencing technologies by the plant research community has led to the accumulation of large volumes of expression data. However, analysis of these datasets is complicated by the frequent occurrence of polyploidy among economically-important crop species. In addition, processing and analyzing such large volumes of sequence data is a technical and time-consuming task, limiting their application in functional genomics studies, particularly for smaller laboratories which lack access to high-powered computing infrastructure. Wheat is a good example of a young polyploid species with three similar genomes (97 % identical among homoeologous genes), rapidly accumulating RNA-seq datasets and a large research community. DESCRIPTION: We present WheatExp, an expression database and visualization tool to analyze and compare homoeologue-specific transcript profiles across a broad range of tissues from different developmental stages in polyploid wheat. Beginning with publicly-available RNA-seq datasets, we developed a pipeline to distinguish between homoeologous transcripts from annotated genes in tetraploid and hexaploid wheat. Data from multiple studies is processed and compiled into a database which can be queried either by BLAST or by searching for a known gene of interest by name or functional domain. Expression data of multiple genes can be displayed side-by-side across all expression datasets providing immediate access to a comprehensive panel of expression data for specific subsets of wheat genes. CONCLUSIONS: The development of a publicly accessible expression database hosted on the GrainGenes website - http://wheat.pw.usda.gov/WheatExp/ - coupled with a simple and readily-comparable visualization tool will empower the wheat research community to use RNA-seq data and to perform functional analyses of target genes. The presented expression data is homoeologue-specific allowing for the analysis of relative contributions from each genome to the overall expression of a gene, a critical consideration for breeding applications. Our approach can be expanded to other polyploid species by adjusting sequence mapping parameters according to the specific divergence of their genomes.