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Context Wheat (Triticum aestivum L.) adaptation is highly dependent on crop lifecycle duration, particularly the time at which flowering occurs in a specific environment. Frost, low solar radiation, heat and drought can significantly reduce yield if a crop flowers too early or late. Wheat genotypes have different lifecycle durations determined by plant responses to temperature (thermal time accumulation and vernalisation) and photoperiod. These responses are largely controlled by five phenology genes (two PPD1 and three VRN1 genes). Advances in crop phenology modelling suggest that flowering time under field conditions could be accurately predicted with parameters derived from photoperiod and vernalisation responses obtained in controlled environments. Aims This study quantified photoperiod and vernalisation responses of 69 Australian wheat genotypes selected for diversity at the PPD1 and VRN1 loci. Methods Spring and winter genotypes were grown in four controlled environments at a constant temperature of 22°C with photoperiod (17 or 8 h) and vernalisation (0 or 8 weeks) treatments as factors. Key results Thermal time from coleoptile emergence to flowering in spring genotypes was typically decreased more by long photoperiod than by vernalisation; the opposite was true for winter genotypes. Spring genotypes that were sensitive to vernalisation contained a sensitive allele at the Vrn-A1 locus. Conclusions There is large diversity in phenological responses of wheat genotypes to photoperiod and vernalisation, including among those with matching multi-locus genotype. Implications Data from this study will be used to parameterise and test a wheat phenology model in a future study.

Wheat is a major food crop, with around 765 million tonnes produced globally. The largest wheat producers include the European Union, China, India, Russia, United States, Canada, Pakistan, Australia, Ukraine and Argentina. Cultivation of wheat across such diverse global environments with variation in climate, biotic and abiotic stresses, requires cultivars adapted to a range of growing conditions. One intrinsic way that wheat achieves adaptation is through variation in phenology (seasonal timing of the lifecycle) and related traits (e.g., those affecting plant architecture). It is important to understand the genes that underlie this variation, and how they interact with each other, other traits and the growing environment. This review summarises the current understanding of phenology and developmental traits that adapt wheat to different environments. Examples are provided to illustrate how different combinations of alleles can facilitate breeding of wheat varieties with optimal crop performance for different growing regions or farming systems.

Safflower (Carthamus tinctorius) is a member of the Asteraceae family that is grown in temperate climates as an oil seed crop. Most commercially grown safflower varieties can be sown in late winter or early spring and flower rapidly in the absence of overwintering. There are winter-hardy safflower accessions that can be sown in autumn and survive over-wintering. Here, we show that a winter-hardy safflower possesses a vernalization response, whereby flowering is accelerated by exposing germinating seeds to prolonged cold. The impact of vernalization was quantitative, such that increasing the duration of cold treatment accelerated flowering to a greater extent, until the response was saturated after 2 weeks exposure to low-temperatures. To investigate the molecular-basis of the vernalization-response in safflower, transcriptome activity was compared and contrasted between vernalized versus non-vernalized plants, in both ‘winter hardy’ and ‘spring’ cultivars. These genome-wide expression analyses identified a small set of transcripts that are both differentially expressed following vernalization and that also have different expression levels in the spring versus winter safflowers. Four of these transcripts were quantitatively induced by vernalization in a winter hardy safflower but show high basal levels in spring safflower. Phylogenetic analyses confidently assigned that the nucleotide sequences of the four differentially expressed transcripts are related to FLOWERING LOCUS T (FT), FRUITFUL (FUL), and two genes within the MADS-like clade genes. Gene models were built for each of these sequences by assembling an improved safflower reference genome using PacBio-based long-read sequencing, covering 85% of the genome, with N50 at 594,000 bp in 3000 contigs. Possible evolutionary relationships between the vernalization response of safflower and those of other plants are discussed.

Price surges in staple foods trigger civil unrest and conflict1. The food riots of 2007–2008 and Arab spring uprisings (2010–2012) were, in part, a consequence of price increases due to a tightening supply of staple grains, particularly wheat. Prolonged drought in Australia contributed to the global wheat shortage; Australia accounts for 10% of global wheat exports2. Australian wheat yields have plateaued3 owing to reduced rainfall4,5 and increasing temperatures3 attributed to anthropogenic climate change6. If Australia is to increase wheat production in line with projected global population growth and demand, an increase in yield is required7. Crop simulations reveal that an early sowing system combined with slower-developing wheat genotypes could exploit a longer growing season8. We developed near-isogenic lines and tested this hypothesis in experiments across the grain belt of Australia, and extended the results using whole-farm simulations. Our proposed early sowing system can increase national yields by 0.54 (s.d. = 0.38) t ha−1 representing an additional 7.1 Mt annually under reduced rainfall and increasing temperature regimes. This adaptation could facilitate increasing yields across Australia under climate change with global food security benefits. Crop models suggest that early sowing and slower-developing cultivars could maintain Australian wheat yields despite less-favourable climatic conditions. Field trials now confirm the potential of this adaptation for wheat production across Australia.

Australian wheat (Triticum aestivum) producers have been sowing crops earlier to adapt to reduced autumn rainfall, extreme spring weather and increasing farm size. Analysis of sowing date records indicate a shift of around 1.5 days/year over a 10 year period. The most suitable development patterns to maintain or increase yield at earlier sowing times have not been identified. Field experiments were conducted over two years at a range of sites and times of sowing (TOS), comparing a novel cultivar with fast-winter (FW) development to current elite spring and winter cultivars, and near-isogenic lines that differed only in major development genes. In cooler environments, the FW exhibited a more stable flowering time across a broader range of TOS compared to spring or slower developing winter cultivars. The optimal sowing window was shorter in warmer environments for the FW. Early-sown FW wheat yielded 8% more than fast-developing spring wheat sown later but flowering concurrently. FW wheat yielded 17% more than the elite mid-winter cultivar, and 18% more than elite slower developing spring cultivars when averaged across all TOS. The FW development pattern has potential to extend sowing periods while achieving 10–20% higher yields and flowering time stability. Wheat cultivars with altered development patterns must be developed to ensure crops flower during optimal periods from earlier sowing times.

The number of rachis nodes (spikelets) on a wheat spike is a component of grain yield that correlates with flowering time. The genetic basis regulating flowering in cereals is well understood, but there are reports that flowering time can be modified at a high frequency by selective breeding, suggesting that it may be regulated by both epigenetic and genetic mechanisms. We investigated the role of DNA methylation in regulating spikelet number and flowering time by treating a semi-spring wheat with the demethylating agent, Zebularine. Three lines with a heritable increase in spikelet number were identified. The molecular basis for increased spikelet number was not determined in 2 lines, but the phenotype showed non-Mendelian inheritance, suggesting that it could have an epigenetic basis. In the remaining line, the increased spikelet phenotype behaved as a Mendelian recessive trait and late flowering was associated with a deletion encompassing the floral promoter, FT-B1. Deletion of FT-B1 delayed the transition to reproductive growth, extended the duration of spike development, and increased spikelet number under different temperature regimes and photoperiod. Transiently disrupting DNA methylation can generate novel flowering behaviour in wheat, but these changes may not be sufficiently stable for use in breeding programs.

To adapt to the challenges of climate change and the growing world population, it is vital to increase global crop production. Understanding the function of genes within staple crops will accelerate crop improvement by allowing targeted breeding approaches. Despite the importance of wheat, which provides 20 % of the calories consumed by humankind, a lack of genomic information and resources has hindered the functional characterisation of genes in this species. The recent release of a high-quality reference sequence for wheat underpins a suite of genetic and genomic resources that support basic research and breeding. These include accurate gene model annotations, gene expression atlases and gene networks that provide background information about putative gene function. In parallel, sequenced mutation populations, improved transformation protocols and structured natural populations provide rapid methods to study gene function directly. We highlight a case study exemplifying how to integrate these resources to study gene function in wheat and thereby accelerate improvement in this important crop. We hope that this review provides a helpful guide for plant scientists, especially those expanding into wheat research for the first time, to capitalise on the discoveries made in Arabidopsis and other plants. This will accelerate the improvement of wheat, a complex polyploid crop, of vital importance for food and nutrition security.

The impact of different allelic combinations of VERNALIZATION1 (VRN1) and PHOTOPERIOD1 (PPD1) on the development of reproductive structures, spike dry weight (SDW) and its links to the duration of stem elongation (SE) and yield components was assessed using Near Isogenic Lines (NILs) of the cultivar Sunstate for both genes. Emphasis was on observations on the main shoot and tillers, linking to plant and canopy level in Steinfort at al. (2016). Experiments under controlled and irrigated field conditions relevant to the northern wheat growing region of Australia were designed. In vernalised plants, the number of fertile florets in the main shoot spike was positively related to the number of fertile spikelets, the duration of SE, the SDW at anthesis and the sum of the radiation intercepted during SE, while no associations were found under non vernalised and slow vernalising field conditions. Vernalised plants also produced more grains in the average tiller spike, compared to non vernalised plants, particularly under short days. Both main shoot and tiller grain number per spike contributed to the higher grain number per plant observed in vernalised plants in Steinfort et al. (2016), as spike number was similar. In the field, higher yield of lines with spring alleles of VRN1 was underpinned by longer spikes, with fewer spikelets but more fertile florets per spikelet in the main shoot spike, and more grains per tiller. When non vernalised, winter lines had an apex that grew slower, shorter spikes and higher spikelet density compared to the other genotypes. The utilisation of sensitivity to VRN1 or PPD1 to increase the duration of the SE and the number of fertile florets and grains is a more complex process than originally proposed.

Genetic variation in the VERNALIZATION1 (VRN1) and PHOTOPERIOD1 (PPD1) genes, which control the vernalisation and photoperiod response, underpin wheat adaptation to different environments. Near isogenic lines were used to investigate the role of allelic combinations of VRN1 and PPD-D1, including new alleles for VRN1-A1, on the length of developmental phases, dynamics of leaf and tiller appearance and yield components in complementary irrigated field trials relevant to low latitude wheat growing areas and controlled conditions. Allelic differences in VRN1 had a stronger effect on the duration of the vegetative phase, while photoperiod sensitivity at PPD-D1 lengthened the stem elongation phase (SE) by up to 23%. If a phase was lengthened, flowering was delayed. The level of response to daylength during stem elongation (SE) introduced by photoperiod sensitive alleles was dependent on the VRN1 composition and vernalisation status. A longer SE under short days was achieved by PPD1 sensitive genotypes when one VRN1 spring allele was present and plants were vernalised. The duration of SE was weakly related to spike dry weight m−2 at DC65 in the field but did not translate into higher grain number m−2. In the field, lines with two to three VRN1 spring alleles had shortest development phases, including SE, close flowering dates, sampled similar temperature environments at different stages, and achieved high yields. Yield advantage was explained by higher biomass, harvest index, grain number m−2 and thousand kernel weight. Genotypes with three winter VRN1 alleles were comparatively disadvantaged, with a longer vegetative phase placing SE under higher temperatures. Allelic differences in both genes caused large variation in leaf and tiller number generation but also in tiller mortality and individual leaf size, lessening the impact on leaf area. Changes in plant morphology and yield components that did not seem mediated via the influence of development genes on the duration of different stages and their impact on resource capture deserve further investigation.

We developed a photoperiod-corrected thermal model that can predict wheat phenology based solely on the combination of photoperiod (Ppd) and vernalisation (Vrn) alleles to identify the phenological suitability of germplasm across the cropping region in southern Australia. More than 200 wheat genotypes that vary in combinations of Ppd and Vrn alleles were grown at 17 locations spanning 11° Latitude, thus providing a wide range in temperature and daylength gradients. The phenological sensitivities of a genotype to varying basic temperature, photoperiod and vernalisation requirement was adjusted via optimisation to minimise the least square difference between the measured and predicted dates of both terminal spike (TS) and flowering (AN). The model predicted dates of TS and AN to within 5 days of the field values. Information was used to identify the alleles required to achieve a wheat ideotype defined in a previous study. The optimum allelic combinations required to target the optimum flowering period for different locations when sown on different dates were also identified. The use of allelic based phenological models has the potential to reduce the costs to breeding programs and accelerate the release of better adapted germplasm to new and changing environments.

The number of rachis nodes (spikelets) on a wheat spike is a component of grain yield that correlates with flowering time. The genetic basis regulating flowering in cereals is well understood, but there are reports that flowering time can be modified at a high frequency by selective breeding, suggesting that it may be regulated by both epigenetic and genetic mechanisms. We investigated the role of DNA methylation in regulating spikelet number and flowering time by treating a semi-spring wheat with the demethylating agent, Zebularine. Three lines with a heritable increase in spikelet number were identified. The molecular basis for increased spikelet number was not determined in 2 lines, but the phenotype showed non-Mendelian inheritance, suggesting that it could have an epigenetic basis. In the remaining line, the increased spikelet phenotype behaved as a Mendelian recessive trait and late flowering was associated with a deletion encompassing the floral promoter, FT-B1. Deletion of FT-B1 delayed the transition to reproductive growth, extended the duration of spike development, and increased spikelet number under different temperature regimes and photoperiod. Transiently disrupting DNA methylation can generate novel flowering behaviour in wheat, but these changes may not be sufficiently stable for use in breeding programs.

Increasing biomass accumulation is becoming a major objective of most bread wheat (Triticum aestivum L.) breeding programs worldwide. This study addresses the changes caused by breeding in the pattern of wheat growth and its relationship with the presence of Rht dwarfing alleles. A historical series of 16 varieties representative of the most widely grown during the twentieth century in Spain, including landraces (grown before 1940’s), initial (1947–1955) and modern (1972–2001) cultivars, was characterized for Rht8c, Rht-B1b and Rht-D1b dwarfing alleles. Changes in biomass accumulation and partitioning, leaf area index (LAI) and leaf area duration (LAD) were studied in four field experiments conducted in NE Spain. Biomass and LAI at the beginning of jointing were reduced by ca. 20 and 40 % in varieties carrying one or two dwarfing alleles, respectively. The rates of biomass accumulation from the beginning of jointing to anthesis were similar for wheats from the three historical periods (ca. 1.70 g m−2 GDD−1), and amongst varieties carrying a different number of dwarfing alleles. Almost 80 % of absolute yield gains during the last century were due to increases in the spikes weight per unit area from milk-grain to maturity. Initial varieties maintained and modern varieties increased above-ground biomass from milk-grain stage to maturity. The contribution of photosynthesis during this last phase was enhanced in modern varieties due to increases in LAI at milk-grain stage (0.83 % y−1) and of LAD from it to maturity (0.79 % y−1). Changes on the pattern of biomass accumulation and partitioning caused by the introduction of improved varieties were related to the number of dwarfing alleles present. The contribution of photosynthesis to grain filling was increased in modern varieties due to improved LAI values at milk-grain stage and, subsequently, a longer LAD.

EARLY FLOWERING3 (ELF3) is a circadian clock gene that contributes to photoperiod-dependent flowering in plants, with loss-of-function mutants in barley (Hordeum vulgare), legumes, and Arabidopsis thaliana flowering early under noninductive short-day (SD) photoperiods. The barley elf3 mutant displays increased expression of FLOWERING LOCUS T1 (FT1); however, it remains unclear whether this is the only factor responsible for the early flowering phenotype. We show that the early flowering and vegetative growth phenotypes of the barley elf3 mutant are strongly dependent on gibberellin (GA) biosynthesis. Expression of the central GA biosynthesis gene, GA20oxidase2, and production of the bioactive GA, GA1, were significantly increased in elf3 leaves under SDs, relative to the wild type. Inhibition of GA biosynthesis suppressed the early flowering of elf3 under SDs independently of FT1 and was associated with altered expression of floral identity genes at the developing apex. GA is also required for normal flowering of spring barley under inductive photoperiods, with chemical and genetic attenuation of the GA biosynthesis and signaling pathways suppressing inflorescence development under long-day conditions. These findings illustrate that GA is an important floral promoting signal in barley and that ELF3 suppresses flowering under noninductive photoperiods by blocking GA production and FT1 expression.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited., Transcriptional activation of the VERNALIZATION1 gene mediates the acceleration of flowering by prolonged cold (vernalization) in temperate cereals. This study examined the earliest stages of the transcriptional response of VRN1 to low temperatures. Time-course analyses, using a sensitive quantitative PCR assay, showed that in sprouting barley seedlings VRN1 transcripts begin to accumulate within 24 hours of the onset of cold. The kinetics of the initial transcriptional response of VRN1 to cold was similar to the cold-induced genes DEHYDRIN5 (DHN5) and COLD REGULATED 14B (COR14B), but occurred at lower levels compared to cold acclimation genes or the response to longer cold treatments. Temperatures between 15 and –2 °C induced expression of VRN1 within 24 hours, with a maximal response observed between 2 and –2 °C. Transcriptional induction was also observed in undifferentiated callus cells. There were significant increases in histone acetylation levels at the VRN1 locus in response to 24-hour cold treatment. Sodium butyrate, a histone deacetylation inhibitor, triggered an increase in histone acetylation at VRN1 chromatin and elevated VRN1 transcript levels. The transcriptional response of VRN1 to short-term cold treatment was examined in near-isogenic lines that have different VRN1 genotypes, showing that an allele of the barley VRN1 gene with an insertion in the first intron and high basal expression levels has a reduced transcriptional response to short term cold treatment. This study suggests that low-temperature induction of VRN1 is a cellular response to cold triggered by the same mechanisms that mediate low-temperature induction of cold acclimation genes., This work was supported by an Office of the Chief Executive (OCE) Postdoctoral fellowship (S.N Oliver).

The advantages of free threshing in wheat led to the selection of the domesticated Q allele, which is now present in almost all modern wheat varieties. Q and the pre-domestication allele, q, encode an AP2 transcription factor, with the domesticated allele conferring a free-threshing character and a subcompact (i.e. partially compact) inflorescence (spike). We demonstrate that mutations in the miR172 binding site of the Q gene are sufficient to increase transcript levels via a reduction in miRNA-dependent degradation, consistent with the conclusion that a single nucleotide polymorphism in the miRNA binding site of Q relative to q was essential in defining the modern Q allele. We describe novel gain- and loss-of-function alleles of Q and use these to define new roles for this gene in spike development. Q is required for the suppression of 'sham ramification', and increased Q expression can lead to the formation of ectopic florets and spikelets (specialized inflorescence branches that bear florets and grains), resulting in a deviation from the canonical spike and spikelet structures of domesticated wheat.

The advantages of free threshing in wheat led to the selection of the domesticated

Highlight In barley, GIGANTEA and the PSEUDO RESPONSE REGULATOR clock genes respond rapidly to temperature in an EARLY FLOWERING 3-dependent manner., An increase in global temperatures will impact future crop yields. In the cereal crops wheat and barley, high temperatures accelerate reproductive development, reducing the number of grains per plant and final grain yield. Despite this relationship between temperature and cereal yield, it is not clear what genes and molecular pathways mediate the developmental response to increased temperatures. The plant circadian clock can respond to changes in temperature and is important for photoperiod-dependent flowering, and so is a potential mechanism controlling temperature responses in cereal crops. This study examines the relationship between temperature, the circadian clock, and the expression of flowering-time genes in barley (Hordeum vulgare), a crop model for temperate cereals. Transcript levels of barley core circadian clock genes were assayed over a range of temperatures. Transcript levels of core clock genes CCA1, GI, PRR59, PRR73, PRR95, and LUX are increased at higher temperatures. CCA1 and PRR73 respond rapidly to a decrease in temperature whereas GI and PRR59 respond rapidly to an increase in temperature. The response of GI and the PRR genes to changes in temperature is lost in the elf3 mutant indicating that their response to temperature may be dependent on a functional ELF3 gene.

With 1 figure and 5 tables Abstract Two winter alleles of Vrn-A1 were recently found in wheat cultivars adapted to the Great Plains of the USA. Using a diagnostic marker for a single-nucleotide polymorphism (SNP) in exon 4, which distinguishes the alleles, the allele in the Great Plains cultivar ‘Jagger’ was common in Australian and CIMMYT cultivars, but ‘Veery’ cultivars carried the alternate allele from their Russian ancestor, which was the same as the allele in the Great Plains cultivar ‘Wichita’. The ‘Wichita’ allele was in North American winter cultivars, and chromosome substitution lines with a high level of tolerance to freezing, but not in substitution lines with a lower level of tolerance. The SNP between the alleles alters the predicted Vrn-A1 protein sequence, and this potentially explains differences in freezing tolerance. We suggest that these winter alleles could be coded as Vrn-A1v for the ‘Jagger’ allele and Vrn-A1w for the ‘Wichita’ allele. Cultivars with the spring Vrn-A1a or Vrn-A1b alleles carried the same SNP allele as ‘Jagger’, suggesting that the mutation from winter to spring for these alleles occurred in a Vrn-A1v genotype.

MADS-box transcription factors specify plant meristem identity. In doing so, they determine when floral organs are produced at the shoot apex and control the timing of flowering. The transcriptional activity of key MADS-box genes is controlled by temperature in many plants, and this synchronises flowering with changing seasons. Here we review how seasonal temperature variation influences the developmental programme of plants via transcriptional regulation of MADS-box genes. In particular we examine the role of MADS-box genes in regulating the acceleration of flowering by vernalization (prolonged periods of cold), using FLOWERING LOCUS C of Arabidopsis and VERNALIZATION1 of cereals as examples. A potential role for SHORT VEGETATIVE PHASE-like genes in controlling winter bud dormancy is also examined, as are potential roles for MADS-box genes in regulating developmental responses to elevated growth temperatures. We conclude that understanding how temperature regulates the transcription of MADS-box genes provides insight into how seasonal fluctuations in temperature influence plant development. Plant breeders may be able to use natural variation in temperature-responsive MADS-box genes to breed future crop varieties.

Responses to prolonged low-temperature treatment of imbibed seeds (vernalization) were examined in barley (Hordeum vulgare). These occurred in two phases: the perception of prolonged cold, which occurred gradually at low temperatures, and the acceleration of reproductive development, which occurred after vernalization. Expression of the VERNALIZATION1 gene (HvVRN1) increased gradually in germinating seedlings during vernalization, both at the shoot apex and in the developing leaves. This occurred in darkness, independently of VERNALIZATION2 (HvVRN2), consistent with the hypothesis that expression of HvVRN1 is induced by prolonged cold independently of daylength flowering-response pathways. After vernalization, expression of HvVRN1 was maintained in the shoot apex and leaves. This was associated with accelerated inflorescence initiation and with down-regulation of HvVRN2 in the leaves. The largest determinant of HvVRN1 expression levels in vernalized plants was the length of seed vernalization treatment. Daylength did not influence HvVRN1 expression levels in shoot apices and typically did not affect expression in leaves. In the leaves of plants that had experienced a saturating seed vernalization treatment, expression of HvVRN1 was higher in long days, however. HvFT1 was expressed in the leaves of these plants in long days, which might account for the elevated HvVRN1 expression. Long-day up-regulation of HvVRN1 was not required for inflorescence initiation, but might accelerate subsequent stages of inflorescence development. Similar responses to seed vernalization were also observed in wheat (Triticum aestivum). These data support the hypothesis that VRN1 is induced by cold during winter to promote spring flowering in vernalization-responsive cereals.

Genetic analyses have identified three genes that control the vernalization requirement in wheat and barley; VRN1, VRN2 and FT (VRN3). These genes have now been isolated and shown to regulate not only the vernalization response but also the promotion of flowering by long days. VRN1 is induced by vernalization and accelerates the transition to reproductive development at the shoot apex. FT is induced by long days and further accelerates reproductive apex development. VRN2, a floral repressor, integrates vernalization and day-length responses by repressing FT until plants are vernalized. A comparison of flowering time pathways in cereals and Arabidopsis shows that the vernalization response is controlled by different MADS box genes, but integration of vernalization and long-day responses occurs through similar mechanisms.

Flowering time, vernalization requirement, photoperiod sensitivity and low temperature tolerance are key traits in the Triticeae. We characterized a set of isogenic genetic stocks-representing single and pairwise substitutions of spring alleles at the VRN-H1, VRN-H2 and VRN-H3 loci in a winter barley background-at the structural, functional and phenotypic levels. High density mapping with reference to the barley genome sequence confirmed that in all cases target VRN alleles were present in the near isogenic lines (NILs) and allowed estimates of introgression size (at the genetic and physical levels) and gene content. Expression data corroborated the structural and phenotypic results. The latter confirmed that substitution of a spring allele at any of the VRN loci is sufficient to eliminate vernalization requirement. There was no significant change in low temperature tolerance with substitution of a spring allele at VRN-H2, but there were significant losses in cold tolerance with substitutions at VRN-H1 and VRN-H3. Reductions in cold tolerance are ascribed to an accelerated transition from the vegetative to reproductive state. The set of NILs will be a rich resource for understanding the genetics of vernalization, low temperature tolerance and other traits encoded/regulated by genes within the introgressed intervals.

Timothy is a perennial forage grass grown commonly in Boreal regions. This study explored the effect of vernalization and photoperiod (PP) on flowering and growth characteristics and how this related to changes in expression of three flowering related genes in accessions from different geographic origin. Large variation was found in accessions in their vernalization and PP responses. In southern accessions vernalization response or requirement was not observed, the heading date remained unchanged, and plants flowered without vernalization. On the contrary, northern types had obligatory requirement for vernalization and long PP, but the tiller elongation did not require vernalization at 16-h PP. Longer vernalization or PP treatments reduced the genotypical differences in flowering. Moreover, the vernalization saturation progressed stepwise from main tiller to lateral tillers, and this process was more synchronized in southern accessions. The expression of PpVRN1 was associated with vernalization while PpVRN3 accumulated at long PP. A crucial role for PpVRN3 in the transition to flowering was supported as in southern accession the transcript accumulated in non-vernalized plants after transfer to 16-h PP, and the apices transformed to generative stage. Differences in vernalization requirements were associated with variation in expression levels of PpVRN1 and PpVRN3, with higher expression levels in southern type. Most divergent transcript accumulation of PpMADS10 was found under different vernalization conditions. These differences between accessions can be translated into agronomic traits, such as the tiller composition of canopy, which affects the forage yield. The southern types, with minimal vernalization response, have fast re-growth ability and rapidly decreasing nutritive value, whereas northern types grow slowly and have better quality. This information can be utilized in breeding for new cultivars for longer growing seasons at high latitudes.

The plant circadian clock is an internal timekeeper that coordinates biological processes with daily changes in the external environment. The transcript levels of clock genes, which oscillate to control circadian outputs, were examined during early seedling development in barley (Hordeum vulgare), a model for temperate cereal crops. Oscillations of clock gene transcript levels do not occur in barley seedlings grown in darkness or constant light but were observed with day-night cycles. A dark-to-light transition influenced transcript levels of some clock genes but triggered only weak oscillations of gene expression, whereas a light-to-dark transition triggered robust oscillations. Single light pulses of 6, 12 or 18 hours induced robust oscillations. The light-to-dark transition was the primary determinant of the timing of subsequent peaks of clock gene expression. After the light-to-dark transition the timing of peak transcript levels of clock gene also varied depending on the length of the preceding light pulse. Thus, a single photoperiod can trigger initiation of photoperiod-dependent circadian rhythms in barley seedlings. Photoperiod-specific rhythms of clock gene expression were observed in two week old barley plants. Changing the timing of dusk altered clock gene expression patterns within a single day, showing that alteration of circadian oscillator behaviour is amongst the most rapid molecular responses to changing photoperiod in barley. A barley EARLY FLOWERING3 mutant, which exhibits rapid photoperiod–insensitive flowering behaviour, does not establish clock rhythms in response to a single photoperiod. The data presented show that dawn and dusk cues are important signals for setting the state of the circadian oscillator during early development of barley and that the circadian oscillator of barley exhibits photoperiod-dependent oscillation states.

Analysis of the functions of Short Vegetative Phase (SVP)-like MADS-box genes in barley (Hordeum vulgare) indicated a role in determining meristem identity. Three SVP-like genes are expressed in vegetative tissues of barley: Barley MADS1 (BM1), BM10, and Vegetative to Reproductive Transition gene 2. These genes are induced by cold but are repressed during floral development. Ectopic expression of BM1 inhibited spike development and caused floral reversion in barley, with florets at the base of the spike replaced by tillers. Head emergence was delayed in plants that ectopically express BM1, primarily by delayed development after the floral transition, but expression levels of the barley VRN1 gene (HvVRN1) were not affected. Ectopic expression of BM10 inhibited spike development and caused partial floral reversion, where florets at the base of the spike were replaced by inflorescence-like structures, but did not affect heading date. Floral reversion occurred more frequently when BM1 and BM10 ectopic expression lines were grown in short-day conditions. BM1 and BM10 also inhibited floral development and caused floral reversion when expressed in Arabidopsis (Arabidopsis thaliana). We conclude that SVP-like genes function to suppress floral meristem identity in winter cereals.

Two genetic loci control the vernalization response in winter cereals; VRN1, which encodes an AP1-like MADS-box transcription factor, and VRN2, which has been mapped to a chromosome region containing ZCCT zinc finger transcription factor genes. We examined whether daylength regulates expression of HvVRN1 and HvVRN2. In a vernalization-responsive winter barley (Hordeum vulgare), expression of HvVRN1 is regulated by vernalization and by development, but not by daylength. Daylength affected HvVRN1 expression in only one of six vernalization-insensitive spring barleys examined and so cannot be a general feature of regulation of this gene. In contrast, daylength is the major determinant of expression levels of two ZCCT genes found at the barley VRN2 locus, HvZCCTa and HvZCCTb. In winter barley, high levels of HvZCCTa and HvZCCTb expression were detected only when plants were grown in long days. During vernalization in long-day conditions, HvVRN1 is induced and expression of HvZCCTb is repressed. During vernalization under short days, induction of HvVRN1 occurs without changes in HvZCCTa and HvZCCTb expression. Analysis of HvZCCTa and HvZCCTb expression levels in a doubled haploid population segregating for different vernalization and daylength requirements showed that HvVRN1 genotype determines HvZCCTa and HvZCCTb expression levels. We conclude that the vernalization response is mediated through HvVRN1, whereas HvZCCTa and HvZCCTb respond to daylength cues to repress flowering under long days in nonvernalized plants.

Voltage-dependent anion channels (VDACs) are generally considered as the main pathway for metabolite transport across the mitochondrial outer membrane. Recent proteomic studies on isolated symbiosome membranes from legume nodules indicated that VDACs might also be involved in transport of nutrients between plants and rhizobia. In an attempt to substantiate this, we carried out a detailed molecular and cellular characterization of VDACs in Lotus japonicus and soybean (Glycine max). Database searches revealed at least five genes encoding putative VDACs in each of the legumes L. japonicus, Medicago truncatula, and soybean. We obtained and sequenced cDNA clones from L. japonicus encoding five full-length VDAC proteins (LjVDAC1.1–1.3, LjVDAC2.1, and LjVDAC3.1). Complementation of a yeast (Saccharomyces cerevisiae) mutant impaired in VDAC1, a porin of the mitochondrial outer membrane, showed that LjVDAC1.1, LjVDAC1.2, LjVDAC2.1, and LjVDAC3.1, but not LjVDAC1.3, are functional and targeted to the mitochondrial outer membrane in yeast. Studies of the expression pattern of the five L. japonicus VDAC genes revealed largely constitutive expression of each throughout the plant, including nodules. Antibodies to LjVDAC1.1 of L. japonicus and the related POM36 protein of potato (Solanum tuberosum) recognized several proteins between 30 and 36 kD on western blots, including LjVDAC1.1, LjVDAC1.2, LjVDAC1.3, and LjVDAC2.1. Immunolocalization of VDACs in L. japonicus and soybean root nodules demonstrated their presence on not only mitochondria but also on numerous, small vesicles at the cell periphery. No evidence was found for the presence of VDACs on the symbiosome membrane. Nonetheless, the data indicate that VDACs may play more diverse roles in plants than suspected previously.

By comparing expression levels of MADS box transcription factor genes between near-isogenic winter and spring lines of bread wheat, Triticum aestivum , we have identified WAP1 as the probable candidate for the Vrn-1 gene, the major locus controlling the vernalization flowering response in wheat. WAP1 is strongly expressed in spring wheats and moderately expressed in semispring wheats, but is not expressed in winter wheat plants that have not been exposed to vernalization treatment. Vernalization promotes flowering in winter wheats and strongly induces expression of WAP1. WAP1 is located on chromosome 5 in wheat and, by synteny with other cereal genomes, is likely to be collocated with Vrn-1 . These results in hexaploid bread wheat cultivars extend the conclusion made by Yan et al. [Yan, L., Loukoianov, A., Tranquilli, G., Helguera, M., Fahima, T. & Dubcovsky, J. (2003) Proc. Natl. Acad. Sci. USA 100, 6263–6268] in the diploid wheat progenitor Triticum monococcum that WAP1 ( TmAP1 ) corresponds to the Vrn-1 gene. The barley homologue of WAP1, BM5 , shows a similar pattern of expression to WAP1 and TmAP1. BM5 is not expressed in winter barleys that have not been vernalized, but as with WAP1 , expression of BM5 is strongly induced by vernalization treatment. In spring barleys, the level of BM5 expression is determined by interactions between the Vrn-H1 locus and a second locus for spring habit, Vrn-H2 . There is now evidence that AP1 -like genes determine the time of flowering in a range of cereal and grass species.

Flowering time of wheat (Triticum aestivum L.) is a critical determinant of grain yield. Frost, drought and heat stresses from either overly early or overly late flowering can inflict significant yield penalties. The ability to predict time of flowering from different sowing dates for diverse cultivars across environments in Australia is important for maintaining yield as autumn rainfall events become less reliable. However, currently there are no models that can accurately do this when new cultivars are released. Two major Photoperiod1 and three Vernalisation1 development genes, with alleles identified by molecular markers, are known to be important in regulating phasic development and therefore time to anthesis, in response to the environmental factors of temperature and photoperiod. Allelic information from molecular markers has been used to parameterise models that could predict flowering time, but it is uncertain how much variation in flowering time can be explained by different alleles of the five major genes. This experiment used 13 elite commercial cultivars of wheat, selected for their variation in phenology and in turn allelic variation at the major development genes, and 13 near-isogenic lines (NILs) with matching multi-locus genotypes for the major development genes, to quantify how much response in time to flowering could be explained by alleles of the major genes. Genotypes were grown in four controlled environments at constant temperature of 22°C with factorial photoperiod (long or short day) and vernalisation (±) treatments applied. NILs were able to explain a large proportion of the variation of thermal time to flowering in elite cultivars in the long-day environment with no vernalisation (97%), a moderate amount in the short-day environment with no vernalisation (62%), and less in the short-day (51%) and long-day (47%) environments with vernalisation. Photoperiod was found to accelerate development, as observed in a reduction in phyllochron, thermal time to heading, thermal time to flowering, and decreased final leaf numbers. Vernalisation response was not as great, and rates of development in most genotypes were not significantly increased. The results indicate that the alleles of the five major development genes alone cannot explain enough variation in flowering time to be used to parameterise gene-based models that will be accurate in simulating flowering time under field conditions. Further understanding of the genetics of wheat development, particularly photoperiod response, is required before a model with genetically based parameter estimates can be deployed to assist growers to make sowing-time decisions for new cultivars.

Fr-B2 is a complex locus on chromosome 5B that affects frost tolerance, days to heading, grain yield and probably other traits of commercial importance in wheat (Triticum aestivum L.). It interacts epistatically with other major genes, especially VRN1. There are two known alleles of Fr-B2: an intact, wild-type allele, and an allele with a large deletion. Published methods for identifying these alleles are slow and expensive, making the development of a high-throughput, co-dominant SNP (single-nucleotide polymorphism) marker highly desirable, especially for commercial wheat breeding. A diverse panel of cultivars and breeding lines was characterised for SNPs and alleles of Fr-B2. Four SNP markers co-segregated as a haplotype block with Fr-B2 across unrelated cultivars and related backcrosses differing for alleles of Fr-B2. A robust KASP (Kompetitive allele-specific PCR) assay was developed for one of the SNPs, KASP_IWB26333, which should facilitate the inclusion of Fr-B2 on genotyping platforms for breeding and research.

The domestication of cereal crops such as wheat, maize, rice and barley has included the modification of inflorescence architecture to improve grain yield and ease harvesting(1). Yield increases have often been achieved through modifying the number and arrangement of spikelets, which are specialized reproductive branches that form part of the inflorescence. Multiple genes that control spikelet development have been identified in maize, rice and barley(2-5). However, little is known about the genetic underpinnings of this process in wheat. Here, we describe a modified spikelet arrangement in wheat, termed paired spikelets. Combining comprehensive QTL and mutant analyses, we show that Photoperiod-1 (Ppd-1), a pseudo-response regulator gene that controls photoperiod-dependent floral induction, has a major inhibitory effect on paired spikelet formation by regulating the expression of FLOWERING LOCUS T (FT)(6,7). These findings show that modulated expression of the two important flowering genes, Ppd-1 and FT, can be used to form a wheat inflorescence with a more elaborate arrangement and increased number of grain producing spikelets.

Transcription of the vernalization1 gene (VRN1) is induced by prolonged cold (vernalization) to trigger flowering of cereal crops, such as wheat and barley. VRN1 encodes a MADS box transcription factor that promotes flowering by regulating the expression of other genes. Here we use transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to identify direct targets of VRN1. Over 500 genomic regions were identified as potential VRN1-binding targets by ChIP-seq. VRN1 binds the promoter of flowering locus T-like 1, a promoter of flowering in vernalized plants. VRN1 also targets vernalization2 and ODDSOC2, repressors of flowering that are downregulated in vernalized plants. RNA-seq identified additional VRN1 targets that might play roles in triggering flowering. Other targets of VRN1 include genes that play central roles in low-temperature-induced freezing tolerance, spike architecture and hormone metabolism. This provides evidence for direct regulatory links between the vernalization response pathway and other important traits in cereal crops.

There is an error in Fig 2C. Specifically, the panels for Fig 2C showing expression data for HvTOC1, HvGI and HvPRR73 were duplicates of panels from Fig 1C. The duplication of these images occurred during figure preparation and was an error made by the corresponding author (B. Trevaskis). The correction does not alter the conclusions of the study. The authors apologise for this error and thank the diligent reader who brought this to our attention. Fig 2 Clock gene expression after light/dark transitions or with exogenous sucrose. Please see the complete, correct Fig 2 here.

Overexpression of a class 1 Hb (GLB1) protects Arabidopsis thaliana plants from the effects of severe hypoxia. Overexpression of the bifunctional symbiotic Hb (GLB1S) from Parasponia andersonii in A. thaliana also increases survival after hypoxia. Plants overexpressing the Hb 1 protein, mutated to have a low oxygen affinity, are as susceptible to hypoxia as WT plants, suggesting that the protection against hypoxia depends on the ability of the Hb to bind ligands, such as oxygen, with high affinity. A mild hypoxia pretreatment (5%) induces the Hb gene and increases the survival of plants after severe hypoxic treatment (0.1%). These results with Hb 1 show that plant Hbs have a role other than in nitrogen-fixing root nodules. Plants overexpressing the GLB1 protein show early vigorous growth in nonhypoxic conditions and are 50% larger in weight than the controls at 14 days. The constitutive expression of GLB1 also resulted in a reduced number of root hairs and increased number of laterals in the root system.

Summary Functional genomics is transforming the way biological research is done in the 21st century. Functional genomics brings together high-throughput genetics with multiparallel analyses of gene transcripts, proteins, and metabolites to answer the ultimate question posed by all genome-sequencing projects: what is the biological function of each and every gene? Functional genomics is driving a shift in the research paradigm away from vertical analysis of single genes, proteins, or metabolites, towards horizontal analysis of full suites of genes, proteins, and metabolites. By identifying and measuring many, if not all of the molecular players that participate in a given biological process, functional genomics offers the prospect of obtaining a truly holistic picture of life. This review describes the tools that are currently being used for functional genomics work and considers the impact that this new discipline is likely to have in the future.

Summary Nitrogen-fixing symbioses between legumes and rhizobia are important for sustainable agriculture and contribute significantly to the global nitrogen cycle. The genomes of two rhizobial species, Mesorhizobium loti and Sinorhizobium meliloti have now been completely sequenced. Hundreds of thousands of expressed sequence tags representing tens of thousands of different genes from three major legume species; soybean, Medicago truncatula and Lotus japonicus have also been deposited in the public domain. Lotus japonicus recently became the focus of a genome project that aims to sequence one third of the entire genome over the next 5 yr. With this as a backdrop, the stage is set for a renaissance in symbiosis research, which will provide new insight into the complex molecular interplay that underpins symbiotic nitrogen fixation. This review considers how functional genomics might contribute to this renaissance.

Nitrogen-fixing symbioses between legumes and bacteria of the family Rhizobiaceae involve differentiation of both plant and bacterial cells. Differentiation of plant root cells is required to build an organ, the nodule, which can feed and accommodate a large population of bacteria under conditions conducive to nitrogen fixation. An efficient vascular system is built to connect the nodule to the root, which delivers sugars and other nutrients to the nodule and removes the products of nitrogen fixation for use in the rest of the plant. Cells in the outer cortex differentiate to form a barrier to oxygen diffusion into nodules, which helps to produce the micro-aerobic environment necessary for bacterial nitrogenase activity. Cells of the central, infected zone of nodules undergo multiple rounds of endoreduplication, which may be necessary for colonisation by rhizobia and may enable enlargement and greater metabolic activity of these cells. Infected cells of the nodule contain rhizobia within a unique plant membrane called the peribacteroid or symbiosome membrane, which separates the bacteria from the host cell cytoplasm and mediates nutrient and signal exchanges between the partners. Rhizobia also undergo differentiation during nodule development. Not surprisingly, perhaps, differentiation of each partner is dependent upon interactions with the other. High-throughput methods to assay gene transcripts, proteins, and metabolites are now being used to explore further the different aspects of plant and bacterial differentiation. In this review, we highlight recent advances in our understanding of plant cell differentiation during nodulation that have been made, at least in part, using high-throughput methods.

Grasses of the subfamily Pooideae, which includes important cereal crops and pasture grasses, are widespread in temperate zones. Seasonal regulation of developmental transitions coordinates the life cycles of Pooideae with the passing seasons, so that flowering and seed production coincide with favourable conditions in spring. This review examines the molecular pathways that control the seasonal flowering responses of Pooideae and how variation in the activity of genes controlling these pathways can adapt cereals or grasses to different climates and geographical regions. The possible evolutionary origins of the seasonal flowering responses of the Pooideae are discussed and key questions for future research highlighted. These include the need to develop a better understanding of the molecular basis for seasonal flowering in perennial Pooideae and in temperate grasses outside the core Pooideae group.

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (Km for ammonium and methylammonium of 8 μM and 31 μM, respectively) matched those of AtAMT1;1 expressed in yeast (Km for methylammonium of 32 μM and Ki for ammonium of 1–10 μM). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (Km for ammonium of 46 μM). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a Km of 36 μM, a low-affinity phase with a Km for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.