Linking Dynamic Phenotyping with Metabolite Analysis to Study Natural Variation in Drought Responses of Brachypodium distachyon.

Drought is an important environmental stress limiting the productivity of major crops worldwide. Understanding drought tolerance and possible mechanisms for improving drought resistance is therefore a prerequisite to develop drought-tolerant crops that produce significant yields with reduced amounts of water. Brachypodium distachyon (Brachypodium) is a key model species for cereals, forage grasses, and energy grasses. In this study, initial screening of a Brachypodium germplasm collection consisting of 138 different ecotypes exposed to progressive drought, highlighted the natural variation in morphology, biomass accumulation, and responses to drought stress. A core set of ten ecotypes, classified as being either tolerant, susceptible or intermediate, in response to drought stress, were exposed to mild or severe (respectively, 15 and 0% soil water content) drought stress and phenomic parameters linked to growth and color changes were assessed. When exposed to severe drought stress, phenotypic data and metabolite profiling combined with multivariate analysis revealed a remarkable consistency in separating the selected ecotypes into their different pre-defined drought tolerance groups. Increases in several metabolites, including for the phytohormones jasmonic acid and salicylic acid, and TCA-cycle intermediates, were positively correlated with biomass yield and with reduced yellow pixel counts; suggestive of delayed senescence, both key target traits for crop improvement to drought stress. While metabolite analysis also separated ecotypes into the distinct tolerance groupings after exposure to mild drought stress, similar analysis of the phenotypic data failed to do so, confirming the value of metabolomics to investigate early responses to drought stress. The results highlight the potential of combining the analyses of phenotypic and metabolic responses to identify key mechanisms and markers associated with drought tolerance in both the Brachypodium model plant as well as agronomically important crops.

Comparison between Agrobacterium-mediated and direct gene transfer using the gene gun.

Agrobacterium-mediated transformation and direct gene transfer using the gene gun (microparticle -bombardment) are the two most widely used methods for plant genetic modification. The Agrobacterium method has been successfully practiced in dicots for many years, but only recently have efficient protocols been developed for grasses. Microparticle bombardment has evolved as a method delivering exogenous nucleic acids into plant genome and is a commonly employed technique in plant science. Here these two systems are compared for transformation efficiency, transgene integration, and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The tall fescue transformation protocols lead to the production of large numbers of fertile, independent transgenic lines.

Nucleotide diversity and linkage disequilibrium of nine genes with putative effects on flowering time in perennial ryegrass (Lolium perenne L.).

Optimization of flowering is an important breeding goal in forage and turf grasses, such as perennial ryegrass (Lolium perenne L.). Nine floral control genes including Lolium perenne CONSTANS (LpCO), SISTER OF FLOWERING LOCUS T (LpSFT), TERMINAL FLOWER1 (LpTFL1), VERNALIZATION1 (LpVRN1, identical to LpMADS1) and five additional MADS-box genes, were analyzed for nucleotide diversity and linkage disequilibrium (LD). For each gene, about 1 kb genomic fragments were isolated from 10 to 20 genotypes of perennial ryegrass of diverse origin. Four to twelve haplotypes per gene were observed. On average, one single nucleotide polymorphism (SNP) was present per 127 bp between two randomly sampled sequences for the nine genes (π = 0.00790). Two MADS-box genes, LpMADS1 and LpMADS10, involved in timing of flowering showed high nucleotide diversity and rapid LD decay, whereas MADS-box genes involved in floral organ identity were found to be highly conserved and showed extended LD. For LpMADS4, LpMADS5, LpCO, LpSFT and LpTFL1, LD extended over the entire region analyzed. The results are compared to previously published results on resistance genes within the same collection of genotypes and the prospects for association mapping of floral control in perennial ryegrass are discussed.

Cloning, characterization and expression analysis of tonoplast intrinsic proteins and glutamine synthetase in ryegrass (Lolium perenne L.).

Perennial ryegrass (Lolium perenne L.) is the most important turf and forage grass species of the temperate regions. It requires substantial input of nitrogen fertilizer for optimum yield. Improved nitrogen use efficiency (NUE) is therefore one of the main breeding targets. However, limited knowledge is currently available on the genes controlling NUE in perennial ryegrass. The aim of the present study was to isolate genes involved in ammonium transport and assimilation. In silico screening of a Lolium EST-library using known sequences of tonoplast intrinsic proteins (TIPs) and cytosolic glutamine synthetase (GS1) revealed a number of homologous sequences. Using these sequences, primers were designed to obtain the full-length sequences by RACE-PCR. Three TIP genes (LpTIP1;1, LpTIP1;2 and LpTIP2;1) and two GS genes (LpGS1a and LpGS1b) were isolated. Characterization in S. cerevisiae confirmed a function in ammonium transport for LpTIP1;1 and LpTIP2;1 and in synthesis of glutamine for LpGS1a and LpGS1b. Cytoimmunochemical studies showed that GS protein was present in the chloroplasts and cytosol of leaf cells, while TIP1 proteins localized to the tonoplast. At the expression level, Lolium GS1 genes responded to N starvation and re-supply in a manner consistent with functions in primary N assimilation and N remobilization. Similarly, the expression of LpTIPs complied with a role in vacuolar ammonium storage. Together, the reported results provide new understanding of the genetic basis for N assimilation and storage in ryegrass.

Improved fructan accumulation in perennial ryegrass transformed with the onion fructosyltransferase genes 1-SST and 6G-FFT.

Carbohydrate limitation has been identified as a main cause of inefficient nitrogen use in ruminant animals, which feed mainly on fresh forage, hay and silage. This inefficiency results in suboptimal meat and milk productivity. One important molecular breeding strategy is to improve the nutritional value of ryegrass (Lolium perenne) by increasing the fructan content through expression of heterologous fructan biosynthetic genes. We developed perennial ryegrass lines expressing sucrose:sucrose 1-fructosyltransferase and fructan:fructan 6G-fructosyltransferase genes from onion (Allium cepa) which exhibited up to a 3-fold increased fructan content. Further, the high fructan content was stable during the growth period, whereas the fructan content in an elite variety, marketed as a high sugar variety, dropped rapidly after reaching its maximum and subsequently remained low.

Frequency, type, and distribution of EST-SSRs from three genotypes of Lolium perenne, and their conservation across orthologous sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa.

BACKGROUND: Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies in several plant species. They are used for cultivar identification, variety protection, as anchor markers in genetic mapping, and in marker-assisted breeding. Currently, a limited number of SSR markers are publicly available for perennial ryegrass (Lolium perenne). We report on the exploitation of a comprehensive EST collection in L. perenne for SSR identification. The objectives of this study were 1) to analyse the frequency, type, and distribution of SSR motifs in ESTs derived from three genotypes of L. perenne, 2) to perform a comparative analysis of SSR motif polymorphisms between allelic sequences, 3) to conduct a comparative analysis of SSR motif polymorphisms between orthologous sequences of L. perenne, Festuca arundinacea, Brachypodium distachyon, and O. sativa, 4) to identify functionally associated EST-SSR markers for application in comparative genomics and breeding. RESULTS: From 25,744 ESTs, representing 8.53 megabases of nucleotide information from three genotypes of L. perenne, 1,458 ESTs (5.7%) contained one or more SSRs. Of these SSRs, 955 (3.7%) were non-redundant. Tri-nucleotide repeats were the most abundant type of repeats followed by di- and tetra-nucleotide repeats. The EST-SSRs from the three genotypes were analysed for allelic- and/or genotypic SSR motif polymorphisms. Most of the SSR motifs (97.7%) showed no polymorphisms, whereas 22 EST-SSRs showed allelic- and/or genotypic polymorphisms. All polymorphisms identified were changes in the number of repeat units. Comparative analysis of the L. perenne EST-SSRs with sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa identified 19 clusters of orthologous sequences between these four species. Analysis of the clusters showed that the SSR motif generally is conserved in the closely related species F. arundinacea, but often differs in length of the SSR motif. In contrast, SSR motifs are often lost in the more distant related species B. distachyon and O. sativa. CONCLUSION: The results indicate that the L. perenne EST-SSR markers are a valuable resource for genetic mapping, as well as evaluation of co-location between QTLs and functionally associated markers.

Protein interactions of MADS box transcription factors involved in flowering in Lolium perenne.

Regulation of flowering time is best understood in the dicot model species Arabidopsis thaliana. Molecular analyses revealed that genes belonging to the MADS box transcription factor family play pivotal regulatory roles in both the vernalization- and photoperiod-regulated flowering pathways. Here the analysis of three APETALA1 (AP1)-like MADS box proteins (LpMADS1-3) and a SHORT VEGETATIVE PHASE (SVP)-like MADS box protein (LpMADS10) from the monocot perennial grass species Lolium perenne is reported. Features of these MADS box proteins were studied by yeast two-hybrid assays. Protein-protein interactions among the Lolium proteins and with members of the Arabidopsis MADS box family have been studied. The expression pattern for LpMADS1 and the protein properties suggest that not the Arabidopsis AP1 gene, but the SUPPRESSOR OF CONSTANS1 (SOC1) gene, is the functional equivalent of LpMADS1. To obtain insight into the molecular mechanism underlying the regulation of LpMADS1 gene expression in vernalization-sensitive and -insensitive Lolium accessions, the upstream sequences of this gene from a winter and spring growth habit variety were compared with respect to MADS box protein binding. In both promoter elements, a putative MADS box transcription factor-binding site (CArG-box) is present; however, the putative spring promoter has a short deletion adjacent to this DNA motif. Experiments using yeast one-hybrid and gel retardation assays demonstrated that the promoter element is bound by an LpMADS1-LpMADS10 higher order protein complex and, furthermore, that this complex binds efficiently to the promoter element from the winter variety only. This strongly supports the model that LpMADS1 together with LpMADS10 controls the vernalization-dependent regulation of the LpMADS1 gene, which is part of the vernalization-induced flowering process in Lolium.

Photoperiodic regulation of flowering in perennial ryegrass involving a CONSTANS -like homolog.

Photoperiod and vernalization are the two key environmental factors of the floral induction of perennial ryegrass (Lolium perenne L.). Transition from vegetative to reproductive growth will only occur after an extended vernalization period, followed by an increase in day length and temperature. Here we report on the isolation and characterization of a L. perenne gene (LpCO ) that is homologous to CONSTANS , and which is tightly coupled to the floral inductive long day signal. Like other monocot CO-like proteins, the LpCO contains a zinc finger domain with a non-conserved B-Box2. Although the B-Box2 has been demonstrated to be essential for the function of the Arabidopsis CO (AtCO), LpCO is able to complement the Arabidopsis co-2 mutant, and ectopic expression in Arabidopsis wild type leads to early flowering. The LpCO transcript exhibits diurnal oscillations and is expressed at higher levels during long days.

MADS-box genes from perennial ryegrass differentially expressed during transition from vegetative to reproductive growth.

In contrast to well-studied dicot plants like Arabidopsis and Antirrhinum, relatively few genes controlling the transition to flowering and flower development of agronomically important monocot species have been identified. In perennial ryegrass (Lolium perenne) the transition from vegetative to reproductive growth is triggered by an obligate vernalization period (primary induction) of at least 12 weeks at temperatures below 5 degrees C under short days, followed by increased temperature and day length (secondary induction). Here we report the isolation of nine ryegrass MADS-box (LpMADS) genes by a differential display method specific to this family of transcription factors. Three of the nine MADS-box genes show homology to the APETALA 1 (AP1) subfamily, two to the SEPALLATA (SEP) subfamily, one to the AGAMOUS-LIKE 6 (AGL6) subfamily, and three show homology to the newly identified OsMADS1 subfamily. The three AP1 homologues are up-regulated, both in the shoot apex and in leaves, in response to vernalization, while expression of the other six are increased by secondary induction during inflorescence development, although not in leaves. Differences in the sequence and hierarchy of flowering gene expression patterns indicate that the Arabidopsis-based flowering model is not completely applicable to explain the molecular events leading to the floral transition in grasses.