Maria von Korff

Starch as a major integrator in the regulation of plant growth

Rising demand for food and bioenergy makes it imperative to breed for increased crop yield. Vegetative plant growth could be driven by resource acquisition or developmental programs. Metabolite profiling in 94 Arabidopsis accessions revealed that biomass correlates negatively with many metabolites, especially starch. Starch accumulates in the light and is degraded at night to provide a sustained supply of carbon for growth. Multivariate analysis revealed that starch is an integrator of the overall metabolic response. We hypothesized that this reflects variation in a regulatory network that balances growth with the carbon supply. Transcript profiling in 21 accessions revealed coordinated changes of transcripts of more than 70 carbon-regulated genes and identified 2 genes (myo-inositol-1-phosphate synthase, a Kelch-domain protein) whose transcripts correlate with biomass. The impact of allelic variation at these 2 loci was shown by association mapping, identifying them as candidate lead genes with the potential to increase biomass production.

Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage

Drought tolerance is a key trait for increasing and stabilizing barley productivity in dry areas worldwide. Identification of the genes responsible for drought tolerance in barley (Hordeum vulgare L.) will facilitate understanding of the molecular mechanisms of drought tolerance, and also facilitate the genetic improvement of barley through marker-assisted selection or gene transformation. To monitor the changes in gene expression at the transcriptional level in barley leaves during the reproductive stage under drought conditions, the 22K Affymetrix Barley 1 microarray was used to screen two drought-tolerant barley genotypes, Martin and Hordeum spontaneum 41-1 (HS41-1), and one drought-sensitive genotype Moroc9-75. Seventeen genes were expressed exclusively in the two drought-tolerant genotypes under drought stress, and their encoded proteins may play significant roles in enhancing drought tolerance through controlling stomatal closure via carbon metabolism (NADP malic enzyme, NADP-ME, and pyruvate dehydrogenase, PDH), synthesizing the osmoprotectant glycine-betaine (C-4 sterol methyl oxidase, CSMO), generating protectants against reactive-oxygen-species scavenging (aldehyde dehydrogenase,ALDH, ascorbate-dependent oxidoreductase, ADOR), and stabilizing membranes and proteins (heat-shock protein 17.8, HSP17.8, and dehydrin 3, DHN3). Moreover, 17 genes were abundantly expressed in Martin and HS41-1 compared with Moroc9-75 under both drought and control conditions. These genes were possibly constitutively expressed in drought-tolerant genotypes. Among them, seven known annotated genes might enhance drought tolerance through signalling [such as calcium-dependent protein kinase (CDPK) and membrane steroid binding protein (MSBP)], anti-senescence (G2 pea dark accumulated protein, GDA2), and detoxification (glutathione S-transferase, GST) pathways. In addition, 18 genes, including those encoding Δl-pyrroline-5-carboxylate synthetase (P5CS), protein phosphatase 2C-like protein (PP2C), and several chaperones, were differentially expressed in all genotypes under drought; thus they were more likely to be general drought-responsive genes in barley. These results could provide new insights into further understanding of drought-tolerance mechanisms in barley.

Variation of Enzyme Activities and Metabolite Levels in 24 Arabidopsis Accessions Growing in Carbon-Limited Conditions

Our understanding of the interaction of carbon (C) metabolism with nitrogen (N) metabolism and growth is based mainly on studies of responses to environmental treatments, and studies of mutants and transformants. Here, we investigate which metabolic parameters vary and which parameters change in a coordinated manner in 24 genetically diverse Arabidopsis (Arabidopsis thaliana) accessions, grown in C-limited conditions. The accessions were grown in short days, moderate light, and high nitrate, and analyzed for rosette biomass, levels of structural components (protein, chlorophyll), total phenols and major metabolic intermediates (sugars, starch, nitrate, amino acids), and the activities of seven representative enzymes from central C and N metabolism. The largest variation was found for plant weight, reducing sugars, starch at the end of the night, and several enzyme activities. High levels of one sugar correlated with high levels of other sugars and starch, and a trend to increased amino acids, slightly lower nitrate, and higher protein. The activities of enzymes at the interface of C and N metabolism correlated with each other, but were unrelated to carbohydrates, amino acid levels, and total protein. Rosette weight was unrelated or showed a weak negative trend to sugar and amino acid contents at the end of the day in most of the accessions, and was negatively correlated with starch at the end of the night. Rosette weight was positively correlated with several enzyme activities. We propose that growth is not related to the absolute levels of starch, sugars, and amino acids; instead, it is related to flux, which is indicated by the enzymatic capacity to use these central resources.

Association of barley photoperiod and vernalization genes with QTLs for flowering time and agronomic traits in a BC2DH population and a set of wild barley introgression lines

The control of flowering time has important impacts on crop yield. The variation in response to day length (photoperiod) and low temperature (vernalization) has been selected in barley to provide adaptation to different environments and farming practices. As a further step towards unraveling the genetic mechanisms underlying flowering time control in barley, we investigated the allelic variation of ten known or putative photoperiod and vernalization pathway genes between two genotypes, the spring barley elite cultivar ‘Scarlett’ (Hordeum vulgare ssp. vulgare) and the wild barley accession ‘ISR42-8’ (Hordeum vulgare ssp. spontaneum). The genes studied are Ppd-H1, VRN-H1, VRN-H2, VRN-H3, HvCO1, HvCO2, HvGI, HvFT2, HvFT3 and HvFT4. ‘Scarlett’ and ‘ISR42-8’ are the parents of the BC2DH advanced backcross population S42 and a set of wild barley introgression lines (S42ILs). The latter are derived from S42 after backcrossing and marker-assisted selection. The genotypes and phenotypes in S42 and S42ILs were utilized to determine the genetic map location of the candidate genes and to test if these genes may exert quantitative trait locus (QTL) effects on flowering time, yield and yield-related traits in the two populations studied. By sequencing the characteristic regions of the genes and genotyping with diagnostic markers, the contrasting allelic constitutions of four known flowering regulation genes were identified as ppd-H1, Vrn-H1, vrn-H2 and vrn-H3 in ‘Scarlett’ and as Ppd-H1, vrn-H1, Vrn-H2 and a novel allele of VRN-H3 in ‘ISR42-8’. All candidate genes could be placed on a barley simple sequence repeat (SSR) map. Seven candidate genes (Ppd-H1, VRN-H2, VRN-H3, HvGI, HvFT2, HvFT3 and HvFT4) were associated with flowering time QTLs in population S42. Four exotic alleles (Ppd-H1, Vrn-H2, vrn-H3 and HvCO1) possibly exhibited significant effects on flowering time in S42ILs. In both populations, the QTL showing the strongest effect corresponded to Ppd-H1. Here, the exotic allele was associated with a reduction of number of days until flowering by 8.0 and 12.7%, respectively. Our data suggest that Ppd-H1, Vrn-H2 and Vrn-H3 may also exert pleiotropic effects on yield and yield-related traits.

Heterosis manifestation during early Arabidopsis seedling development is characterized by intermediate gene expression and enhanced metabolic activity in the hybrids

Heterosis-associated cellular and molecular processes were analyzed in seeds and seedlings of Arabidopsis thaliana accessions Col-0 and C24 and their heterotic hybrids. Microscopic examination revealed no advantages in terms of hybrid mature embryo organ sizes or cell numbers. Increased cotyledon sizes were detectable 4 days after sowing. Growth heterosis results from elevated cell sizes and numbers, and is well established at 10 days after sowing. The relative growth rates of hybrid seedlings were most enhanced between 3 and 4 days after sowing. Global metabolite profiling and targeted fatty acid analysis revealed maternal inheritance patterns for a large proportion of metabolites in the very early stages. During developmental progression, the distribution shifts to dominant, intermediate and heterotic patterns, with most changes occurring between 4 and 6 days after sowing. The highest incidence of heterotic patterns coincides with establishment of size differences at 4 days after sowing. In contrast, overall transcript patterns at 4, 6 and 10 days after sowing are characterized by intermediate to dominant patterns, with parental transcript levels showing the largest differences. Overall, the results suggest that, during early developmental stages, intermediate gene expression and higher metabolic activity in the hybrids compared to the parents lead to better resource efficiency, and therefore enhanced performance in the hybrids.

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

BackgroundThe 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.ResultsBarley 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.ConclusionWe 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.

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.

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.

Asymmetric allele-specific expression in relation to developmental variation and drought stress in barley hybrids

In the present study, we analysed allele-specific expression (ASE) in the selfing species barley to assess the frequency of cis-acting regulatory variation and the effects of genetic background, developmental differences and drought stress on allelic expression levels. We measured ASE ratios in 30 genes putatively involved in stress responses in five hybrids and their reciprocals, namely Hordeum spontaneum 41-1/Alexis (HAl), Hordeum spontaneum 41-1/Arta (HAr), Sloop/WI3408 (SW), Tadmor/Sloop (TS) and Tadmor/WI3408 (TW). In order to detect cis-acting variation related to drought and developmental changes, the barley hybrids were grown under control and water-limited conditions, and leaf tissue was harvested at two developmental stages. The analysis demonstrated that more than half of the genes measured (63%) showed allelic differences in expression of up to 19-fold due to cis-regulatory variation in at least one cross by treatment/stage combination. Drought stress induced changes in allelic expression ratios, indicating differences between drought responsive cis-elements. In addition, ASE differences between developmental stages suggested the presence of cis-acting elements interacting with developmental cues. We were also able to demonstrate that the levels and frequency of allelic imbalance and hence differences in cis-regulatory elements are correlated with the genetic divergence between the parental lines, but may also arise as an adaptation to diverse habitats. Our findings suggest that cis-regulatory variation is a common phenomenon in barley, and may provide a molecular basis of transgression. Differential expression of near-isogenic members of the same gene family could potentially result in hybrid lines out performing their parents in terms of expression level, timing and response to developmental and environmental cues. Identification and targeted manipulation of cis-regulatory elements will assist in breeding improved crops with a better adaptation to changing environments.

Environmental Memory from a Circadian Oscillator: The Arabidopsis thaliana Clock Differentially Integrates Perception of Photic vs. Thermal Entrainment

The constraint of a rotating earth has led to the evolution of a circadian clock that drives anticipation of future environmental changes. During this daily rotation, the circadian clock of Arabidopsis thaliana (Arabidopsis) intersects with the diurnal environment to orchestrate virtually all transcriptional processes of the plant cell, presumably by detecting, interpreting, and anticipating the environmental alternations of light and temperature. To comparatively assess differential inputs toward phenotypic and physiological responses on a circadian parameter, we surveyed clock periodicity in a recombinant inbred population modified to allow for robust periodicity measurements after entrainment to respective photic vs. thermal cues, termed zeitgebers. Lines previously thermally entrained generally displayed reduced period length compared to those previously photically entrained. This differential zeitgeber response was also detected in a set of diverse Arabidopsis accessions. Thus, the zeitgebers of the preceding environment direct future behavior of the circadian oscillator. Allelic variation at quantitative trait loci generated significant differences in zeitgeber responses in the segregating population. These were important for periodicity variation dependent on the nature of the subsequent entrainment source. Collectively, our results provide a genetic paradigm for the basis of environmental memory of a preceding environment, which leads to the integrated coordination of circadian periodicity.