A GDSL-motif Esterase/Lipase Affects Wax and Cutin Deposition and Controls Hull-Caryopsis Attachment in Barley.

The cuticle covering aerial organs of land plants is well known to protect against desiccation. Cuticles also play diverse and specialized functions, including organ separation, depending on plant and tissue. Barley shows a distinctive cuticular wax bloom enriched in ß-diketones on leaf sheaths, stem nodes and internodes and inflorescences. Barley also develops a sticky surface on the outer pericarp layer of its grain fruit leading to strongly adhered hulls, 'covered grain', important for embryo protection and seed dispersal. While the transcription factor-encoding gene HvNUDUM (HvNUD) appears essential for adherent hulls, little is understood about how the pericarp cuticle changes during adhesion or whether changes in pericarp cuticles contribute to another phenotype where hulls partially shed, called 'skinning'. To that end, we screened barley lines for hull adhesion defects, focussing on the Eceriferum (= waxless, cer) mutants. Here, we show that the cer-xd allele causes defective wax blooms and compromised hull adhesion, and results from a mutation removing the last 10 amino acids of the GDS(L) [Gly, Asp, Ser, (Leu)]-motif esterase/lipase HvGDSL1. We used severe and moderate HvGDSL1 alleles to show that complete HvGDSL1 function is essential for leaf blade cuticular integrity, wax bloom deposition over inflorescences and leaf sheaths and pericarp cuticular ridge formation. Expression data suggest that HvGDSL1 may regulate hull adhesion independently of HvNUD. We found high conservation of HvGDSL1 among barley germplasm, so variation in HvGDSL1 unlikely leads to grain skinning in cultivated barley. Taken together, we reveal a single locus which controls adaptive cuticular properties across different organs in barley.

Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley (Hordeum vulgare) and analysis of the cuticular barrier functions.

The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3. Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding ß-ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54. The amounts of intracuticular waxes were strongly decreased in cer-ye.267. The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.

Conserved signalling components coordinate epidermal patterning and cuticle deposition in barley.

Faced with terrestrial threats, land plants seal their aerial surfaces with a lipid-rich cuticle. To breathe, plants interrupt their cuticles with adjustable epidermal pores, called stomata, that regulate gas exchange, and develop other specialised epidermal cells such as defensive hairs. Mechanisms coordinating epidermal features remain poorly understood. Addressing this, we studied two loci whose allelic variation causes both cuticular wax-deficiency and misarranged stomata in barley, identifying the underlying genes, Cer-g/ HvYDA1, encoding a YODA-like (YDA) MAPKKK, and Cer-s/ HvBRX-Solo, encoding a single BREVIS-RADIX (BRX) domain protein. Both genes control cuticular integrity, the spacing and identity of epidermal cells, and barley's distinctive epicuticular wax blooms, as well as stomatal patterning in elevated CO2 conditions. Genetic analyses revealed epistatic and modifying relationships between HvYDA1 and HvBRX-Solo, intimating that their products participate in interacting pathway(s) linking epidermal patterning with cuticular properties in barley. This may represent a mechanism for coordinating multiple adaptive features of the land plant epidermis in a cultivated cereal.

Mapping-by-sequencing identifies HvPHYTOCHROME C as a candidate gene for the early maturity 5 locus modulating the circadian clock and photoperiodic flowering in barley.

Phytochromes play an important role in light signaling and photoperiodic control of flowering time in plants. Here we propose that the red/far-red light photoreceptor HvPHYTOCHROME C (HvPHYC), carrying a mutation in a conserved region of the GAF domain, is a candidate underlying the early maturity 5 locus in barley (Hordeum vulgare L.). We fine mapped the gene using a mapping-by-sequencing approach applied on the whole-exome capture data from bulked early flowering segregants derived from a backcross of the Bowman(eam5) introgression line. We demonstrate that eam5 disrupts circadian expression of clock genes. Moreover, it interacts with the major photoperiod response gene Ppd-H1 to accelerate flowering under noninductive short days. Our results suggest that HvPHYC participates in transmission of light signals to the circadian clock and thus modulates light-dependent processes such as photoperiodic regulation of flowering.

HvLUX1 is a candidate gene underlying the early maturity 10 locus in barley: phylogeny, diversity, and interactions with the circadian clock and photoperiodic pathways.

Photoperiodic flowering is a major factor determining crop performance and is controlled by interactions between environmental signals and the circadian clock. We proposed Hvlux1, an ortholog of the Arabidopsis circadian gene LUX ARRHYTHMO, as a candidate underlying the early maturity 10 (eam10) locus in barley (Hordeum vulgare L.). The link between eam10 and Hvlux1 was discovered using high-throughput sequencing of enriched libraries and segregation analysis. We conducted functional, phylogenetic, and diversity studies of eam10 and HvLUX1 to understand the genetic control of photoperiod response in barley and to characterize the evolution of LUX-like genes within barley and across monocots and eudicots. We demonstrate that eam10 causes circadian defects and interacts with the photoperiod response gene Ppd-H1 to accelerate flowering under long and short days. The results of phylogenetic and diversity analyses indicate that HvLUX1 was under purifying selection, duplicated at the base of the grass clade, and diverged independently of LUX-like genes in other plant lineages. Taken together, these findings contribute to improved understanding of the barley circadian clock, its interaction with the photoperiod pathway, and evolution of circadian systems in barley and across monocots and eudicots.

Expression conservation within the circadian clock of a monocot: natural variation at barley Ppd-H1 affects circadian expression of flowering time genes, but not clock orthologs.

BACKGROUND: The circadian clock is an endogenous mechanism that coordinates biological processes with daily changes in the environment. In plants, circadian rhythms contribute to both agricultural productivity and evolutionary fitness. In barley, the photoperiod response regulator and flowering-time gene Ppd-H1 is orthologous to the Arabidopsis core-clock gene PRR7. However, relatively little is known about the role of Ppd-H1 and other components of the circadian clock in temperate crop species. In this study, we identified barley clock orthologs and tested the effects of natural genetic variation at Ppd-H1 on diurnal and circadian expression of clock and output genes from the photoperiod-response pathway. RESULTS: Barley clock orthologs HvCCA1, HvGI, HvPRR1, HvPRR37 (Ppd-H1), HvPRR73, HvPRR59 and HvPRR95 showed a high level of sequence similarity and conservation of diurnal and circadian expression patterns, when compared to Arabidopsis. The natural mutation at Ppd-H1 did not affect diurnal or circadian cycling of barley clock genes. However, the Ppd-H1 mutant was found to be arrhythmic under free-running conditions for the photoperiod-response genes HvCO1, HvCO2, and the MADS-box transcription factor and vernalization responsive gene Vrn-H1. CONCLUSION: We suggest that the described eudicot clock is largely conserved in the monocot barley. However, genetic differentiation within gene families and differences in the function of Ppd-H1 suggest evolutionary modification in the angiosperm clock. Our data indicates that natural variation at Ppd-H1 does not affect the expression level of clock genes, but controls photoperiodic output genes. Circadian control of Vrn-H1 in barley suggests that this vernalization responsive gene is also controlled by the photoperiod-response pathway. Structural and functional characterization of the barley circadian clock will set the basis for future studies of the adaptive significance of the circadian clock in Triticeae species.

Functional characterisation of HvCO1, the barley (Hordeum vulgare) flowering time ortholog of CONSTANS.

Variation in photoperiod response is a major factor determining plant development and the agronomic performance of crops. The genetic control of photoperiodic flowering has been elucidated in the model plant Arabidopsis, and many of the identified genes are structurally conserved in the grasses. In this study, HvCO1, the closest barley ortholog of the key photoperiod response gene CONSTANS in Arabidopsis, was over-expressed in the spring barley Golden Promise. Over-expression of HvCO1 accelerated time to flowering in long- and short-day conditions and caused up-regulation of HvFT1 mRNA under long-day conditions. However, the transgenic plants retained a response to photoperiod, suggesting the presence of photoperiod response factors acting downstream of HvCO1 transcription. Analysis of a population segregating for HvCO1 over-expression and natural genetic variation at Ppd-H1 demonstrated that Ppd-H1 acts downstream of HvCO1 transcription on HvFT1 expression and flowering. Furthermore, variation at Ppd-H1 did not affect diurnal expression of HvCO1 or HvCO2. Over-expression of HvCO1 increased transcription of the spring allele of Vrn-H1 in long- and short-day conditions, while genetic variation at Ppd-H1 did not affect Vrn-H1 expression. Over-expression of HvCO1 and natural genetic variation at Ppd-H1 accelerated inflorescence development and stem elongation. Thus, HvCO1 probably induces flowering by activating HvFT1 whilst Ppd-H1 regulates HvFT1 independently of HvCO1 mRNA, and all three genes also appear to have a strong effect in promoting inflorescence development.

Parallel pigment and transcriptomic analysis of four barley albina and xantha mutants reveals the complex network of the chloroplast-dependent metabolism.

We investigated the pigment composition and the transcriptome of albina (alb-e ( 16 ) and alb-f ( 17 )) and xantha (xan-s ( 46 ) and xan-b ( 12 )) barley mutants to provide an overall transcriptional picture of genes whose expression is interconnected with chloroplast activities and to search for candidate genes associated with the mutations. Beside those encoding plastid-localized proteins, more than 3,000 genes involved in non-chloroplast localized metabolism were up-/down-regulated in the mutants revealing the network of chloroplast-dependent metabolic pathways. The alb-e ( 16 ) mutant was characterized by overaccumulation of protoporphyrin IX upon ALA (5-amino levulinic acid) feeding and down-regulation of the gene encoding one subunit of Mg-chelatase, suggesting a block of the chlorophyll biosynthetic pathway before Mg-protoporphyrin IX biosynthesis, while alb-f ( 17 ) overaccumulated Mg-protoporphyrin IX and repressed PorA expression, without alterations in Mg-chelatase mRNA level. The alb-f ( 17 )mutant also showed overexpression of several genes involved in phytochrome and in phytochrome-dependent pathways. The results indicate that the down-regulation of Lhcb genes in alb-e ( 16 ) cannot be mediated by the accumulation of Mg-protoporphyrin IX. After ALA treatment, xan-s ( 46 ) showed overaccumulation of Mg-protoporphyrin IX, while the relative porphyrin composition of xan-b ( 12 ) was similar to wild type. The transcripts encoding the components of several mitochondrial metabolic pathways were up-regulated in albina/xantha leaves to compensate for the absence of active chloroplasts. The mRNAs encoding gun3, gun4, and gun5 barley homologous genes showed significant expression variations and were used to search for co-expressed genes across all samples. These analyses provide additional evidences on a chloroplast-dependent covariation of large sets of nuclear genes.

Comparative expression of Cbf genes in the Triticeae under different acclimation induction temperatures.

In plants, the C-repeat binding factors (Cbfs) are believed to regulate low-temperature (LT) tolerance. However, most functional studies of Cbfs have focused on characterizing expression after an LT shock and have not quantified differences associated with variable temperature induction or the rate of response to LT treatment. In the Triticeae, rye (Secale cereale L.) is one of the most LT-tolerant species, and is an excellent model to study and compare Cbf LT induction and expression profiles. Here, we report the isolation of rye Cbf genes (ScCbfs) and compare their expression levels in spring- and winter-habit rye cultivars and their orthologs in two winter-habit wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) cultivars. Eleven ScCbfs were isolated spanning all four major phylogenetic groups. Nine of the ScCbfs mapped to 5RL and one to chromosome 2R. Cbf expression levels were variable, with stronger expression in winter- versus spring-habit rye cultivars but no clear relationship with cultivar differences in LT, down-stream cold-regulated gene expression and Cbf expression were detected. Some Cbfs were expressed only at warmer acclimation temperatures in all three species and their expression was repressed at the end of an 8-h dark period at warmer temperatures, which may reflect a temperature-dependent, light-regulated diurnal response. Our work indicates that Cbf expression is regulated by complex genotype by time by induction-temperature interactions, emphasizing that sample timing, induction-temperature and light-related factors must receive greater consideration in future studies involving functional characterization of LT-induced genes in cereals.

Photosynthetic antenna size in higher plants is controlled by the plastoquinone redox state at the post-transcriptional rather than transcriptional level.

We analyze the effect of the plastoquinone redox state on the regulation of the light-harvesting antenna size at transcriptional and post-transcriptional levels. This was approached by studying transcription and accumulation of light-harvesting complexes in wild type versus the barley mutant viridis zb63, which is depleted in photosystem I and where plastoquinone is constitutively reduced. We show that the mRNA level of genes encoding antenna proteins is almost unaffected in the mutant; this stability of messenger level is not a peculiarity of antenna-encoding genes, but it extends to all photosynthesis-related genes. In contrast, analysis of protein accumulation by two-dimensional PAGE shows that the mutant undergoes strong reduction of its antenna size, with individual gene products having different levels of accumulation. We conclude that the plastoquinone redox state plays an important role in the long term regulation of chloroplast protein expression. However, its modulation is active at the post-transcriptional rather than transcriptional level.

Transcriptome analysis of cold acclimation in barley albina and xantha mutants.

Previously, we have shown that barley (Hordeum vulgare) plants carrying a mutation preventing chloroplast development are completely frost susceptible as well as impaired in the expression of several cold-regulated genes. Here we investigated the transcriptome of barley albina and xantha mutants and the corresponding wild type to assess the effect of the chloroplast on expression of cold-regulated genes. First, by comparing control wild type against cold-hardened wild-type plants 2,735 probe sets with statistically significant changes (P = 0.05; > or = 2-fold change) were identified. Expression of these wild-type cold-regulated genes was then analyzed in control and cold-hardened mutants. Only about 11% of the genes cold regulated in wild type were regulated to a similar extent in all genotypes (chloroplast-independent cold-regulated genes); this class includes many genes known to be under C-repeat binding factor control. C-repeat binding factor genes were also equally induced in mutants and wild-type plants. About 67% of wild-type cold-regulated genes were not regulated by cold in any mutant (chloroplast-dependent cold-regulated genes). We found that the lack of cold regulation in the mutants is due to the presence of signaling pathway(s) normally cold activated in wild type but constitutively active in the mutants, as well as to the disruption of low-temperature signaling pathway(s) due to the absence of active chloroplasts. We also found that photooxidative stress signaling pathway is constitutively active in the mutants. These results demonstrate the major role of the chloroplast in the control of the molecular adaptation to cold.