BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq.

BACKGROUND: The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. RESULTS: A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts - BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427-433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20-28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5' and 3' UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. CONCLUSION: A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.

Barley SIX-ROWED SPIKE3 encodes a putative Jumonji C-type H3K9me2/me3 demethylase that represses lateral spikelet fertility.

The barley inflorescence (spike) comprises a multi-noded central stalk (rachis) with tri-partite clusters of uni-floretted spikelets attached alternately along its length. Relative fertility of lateral spikelets within each cluster leads to spikes with two or six rows of grain, or an intermediate morphology. Understanding the mechanisms controlling this key developmental step could provide novel solutions to enhanced grain yield. Classical genetic studies identified five major SIX-ROWED SPIKE (VRS) genes, with four now known to encode transcription factors. Here we identify and characterise the remaining major VRS gene, VRS3, as encoding a putative Jumonji C-type H3K9me2/me3 demethylase, a regulator of chromatin state. Exploring the expression network modulated by VRS3 reveals specific interactions, both with other VRS genes and genes involved in stress, hormone and sugar metabolism. We show that combining a vrs3 mutant allele with natural six-rowed alleles of VRS1 and VRS5 leads to increased lateral grain size and greater grain uniformity.The VRS genes of barley control the fertility of the lateral spikelets on the barley inflorescence. Here, Bull et al. show that VRS3 encodes a putative Jumonji C-type histone demethylase that regulates expression of other VRS genes, and genes involved in stress, hormone and sugar metabolism.

Dawn and Dusk Set States of the Circadian Oscillator in Sprouting Barley (Hordeum vulgare) Seedlings.

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.

Direct links between the vernalization response and other key traits of cereal crops.

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.

Low temperatures induce rapid changes in chromatin state and transcript levels of the cereal VERNALIZATION1 gene.

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.

Adaptation of barley to mild winters: a role for PPDH2.

BACKGROUND: Understanding the adaptation of cereals to environmental conditions is one of the key areas in which plant science can contribute to tackling challenges presented by climate change. Temperature and day length are the main environmental regulators of flowering and drivers of adaptation in temperate cereals. The major genes that control flowering time in barley in response to environmental cues are VRNH1, VRNH2, VRNH3, PPDH1, and PPDH2 (candidate gene HvFT3). These genes from the vernalization and photoperiod pathways show complex interactions to promote flowering that are still not understood fully. In particular, PPDH2 function is assumed to be limited to the ability of a short photoperiod to promote flowering. Evidence from the fields of biodiversity, ecogeography, agronomy, and molecular genetics was combined to obtain a more complete overview of the potential role of PPDH2 in environmental adaptation in barley. RESULTS: The dominant PPDH2 allele is represented widely in spring barley cultivars but is found only occasionally in modern winter cultivars that have strong vernalization requirements. However, old landraces from the Iberian Peninsula, which also have a vernalization requirement, possess this allele at a much higher frequency than modern winter barley cultivars. Under field conditions in which the vernalization requirement of winter cultivars is not satisfied, the dominant PPDH2 allele promotes flowering, even under increasing photoperiods above 12 h. This hypothesis was supported by expression analysis of vernalization-responsive genotypes. When the dominant allele of PPDH2 was expressed, this was associated with enhanced levels of VRNH1 and VRNH3 expression. Expression of these two genes is needed for the induction of flowering. Therefore, both in the field and under controlled conditions, PPDH2 has an effect of promotion of flowering. CONCLUSIONS: The dominant, ancestral, allele of PPDH2 is prevalent in southern European barley germplasm. The presence of the dominant allele is associated with early expression of VRNH1 and early flowering. We propose that PPDH2 promotes flowering of winter cultivars under all non-inductive conditions, i.e. under short days or long days in plants that have not satisfied their vernalization requirement. This mechanism is indicated to be a component of an adaptation syndrome of barley to Mediterranean conditions.

Expression analysis of vernalization and day-length response genes in barley (Hordeum vulgare L.) indicates that VRNH2 is a repressor of PPDH2 (HvFT3) under long days.

The response to vernalization and the expression of genes associated with responses to vernalization (VRNH1, VRNH2, and VRNH3) and photoperiod (PPDH1 and PPDH2) were analysed in four barley (Hordeum vulgare L.) lines: 'Alexis' (spring), 'Plaisant' (winter), SBCC058, and SBCC106 (Spanish inbred lines), grown under conditions of vernalization and short days (VSD) or no vernalization and long days (NVLD). The four genotypes differ in VRNH1. Their growth habits and responses to vernalization correlated with the level of expression of VRNH1 and the length of intron 1. 'Alexis' and 'Plaisant' behaved as expected. SBCC058 and SBCC106 showed an intermediate growth habit and flowered relatively late in the absence of vernalization. VRNH1 expression was induced by cold for all genotypes. Under VSD, VRNH1 expression was detected in the SBCC genotypes later than in 'Alexis' but earlier than in 'Plaisant'. VRNH2 was repressed under short days while VRNH1 expression increased in parallel. VRNH3 was detected only in 'Alexis' under NVLD, whereas it was not expressed in plants with the active allele of VRNH2 (SBCC058 and 'Plaisant'). Under VSD, PPDH2 was expressed in 'Alexis', SBCC058, and SBCC106, but it was only expressed weakly in 'Alexis' under NVLD. Further analysis of PPDH2 expression in two barley doubled haploid populations revealed that, under long days, HvFT3 and VRNH2 expression levels were related inversely. The timing of VRNH2 expression under a long photoperiod suggests that this gene might be involved in repression of PPDH2 and, indirectly, in the regulation of flowering time through an interaction with the day-length pathway.

The promoter of the cereal VERNALIZATION1 gene is sufficient for transcriptional induction by prolonged cold.

The VERNALIZATION1 (VRN1) gene of temperate cereals is transcriptionally activated by prolonged cold during winter (vernalization) to promote flowering. To investigate the mechanisms controlling induction of VRN1 by prolonged cold, different regions of the VRN1 gene were fused to the GREEN FLUORESCENT PROTEIN (GFP) reporter and expression of the resulting gene constructs was assayed in transgenic barley (Hordeum vulgare). A 2 kb segment of the promoter of VRN1 was sufficient for GFP expression in the leaves and shoot apex of transgenic barley plants. Fluorescence increased at the shoot apex prior to inflorescence initiation and was subsequently maintained in the developing inflorescence. The promoter was also sufficient for low-temperature induction of GFP expression. A naturally occurring insertion in the proximal promoter, which is associated with elevated VRN1 expression and early flowering in some spring wheats, did not abolish induction of VRN1 transcription by prolonged cold, however. A translational fusion of the promoter and transcribed regions of VRN1 to GFP, VRN1::GFP, was localised to nuclei of cells at the shoot apex of transgenic barley plants. The distribution of VRN1::GFP at the shoot apex was similar to the expression pattern of the VRN1 promoter-GFP reporter gene. Fluorescence from the VRN1::GFP fusion protein increased in the developing leaves after prolonged cold treatment. These observations suggest that the promoter of VRN1 is targeted by mechanisms that trigger vernalization-induced flowering in economically important temperate cereal crops.