Overexpression of ubiquitin-like LpHUB1 gene confers drought tolerance in perennial ryegrass.

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin-like post-translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate-controlled growth chambers. Here, pasture-type and turf-type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.

Giving drought the cold shoulder: a relationship between drought tolerance and fall dormancy in an agriculturally important crop.

The growth of fall dormant/freezing tolerant plants often surpasses the growth of non-fall dormant/non-freezing tolerant types of the same species under water-limited conditions, while under irrigated conditions non-fall dormant types exhibit superior yield performance. To investigate the mechanism behind this phenomenon, we exposed seven diverse alfalfa (Medicago sativa) cultivars to water-limited and fully watered conditions and measured their shoot growth, shoot water potential and gas exchange parameters and the relative abundance of taproot RNA transcripts associated with chilling stress/freezing tolerance. Fall dormant cultivars had greater shoot growth relative to the fully watered controls under a mild water deficit (a cumulative water deficit of 625 mL pot(-1)) and did not close their stomata until lower shoot water potentials compared with the more non-fall dormant cultivars. Several gene transcripts previously associated with freezing tolerance increased in abundance when plants were exposed to a mild water deficit. Two transcripts, corF (encodes galactinol synthase) and cas18 (encodes a dehydrin-like protein), increased in abundance in fall dormant cultivars only. Once water deficit stress became severe (a cumulative water deficit of 2530 mL pot(-1)), the difference between fall dormancy groups disappeared with the exception of the expression of a type 1 sucrose synthase gene, which decreased in fall dormant cultivars. The specific adaptation of fall dormant cultivars to mild water deficit conditions and the increase in abundance of specific genes typically associated with freezing tolerance in these cultivars is further evidence of a link between freezing tolerance/fall dormancy and adaption to drought conditions in this species.

Impact of defoliation severity on photosynthesis, carbon metabolism and transport gene expression in perennial ryegrass.

Defoliation severity affects grass regrowth. The changes to biological processes affecting regrowth induced by severe defoliation are not fully understood, nor have they been investigated at a molecular level in field-grown plants. Field-grown perennial ryegrass (Lolium perenne L.) plants were defoliated to 20, 40 or 60mm during winter. Throughout regrowth, transcript profiles of 17 genes involved in photosynthesis and carbon metabolism or transport were characterised in stubble and lamina tissue. Although defoliation to 20mm reduced residual lamina area and stubble water-soluble carbohydrate reserves compared with plants defoliated to 40 or 60mm, net herbage regrowth was not reduced. Transcript profiles indicated a potential compensatory mechanism that may have facilitated regrowth. At the one-leaf regrowth stage, plants defoliated to 20mm had greater abundance of photosynthesis-related gene transcripts (rca, rbcS1, rbcS2, fba, fbp and fnr) and 20% greater stubble total nitrogen than plants defoliated to 60mm. A greater capacity for photosynthesis in outer leaf sheaths may be one potential mechanism used by severely defoliated plants to compensate for the reduced residual lamina area; however, this premise requires further investigation.

Plants modify biological processes to ensure survival following carbon depletion: a Lolium perenne model.

BACKGROUND: Plants, due to their immobility, have evolved mechanisms allowing them to adapt to multiple environmental and management conditions. Short-term undesirable conditions (e.g. moisture deficit, cold temperatures) generally reduce photosynthetic carbon supply while increasing soluble carbohydrate accumulation. It is not known, however, what strategies plants may use in the long-term to adapt to situations resulting in net carbon depletion (i.e. reduced photosynthetic carbon supply and carbohydrate accumulation). In addition, many transcriptomic experiments have typically been undertaken under laboratory conditions; therefore, long-term acclimation strategies that plants use in natural environments are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Perennial ryegrass (Lolium perenne L.) was used as a model plant to define whether plants adapt to repetitive carbon depletion and to further elucidate their long-term acclimation mechanisms. Transcriptome changes in both lamina and stubble tissues of field-grown plants with depleted carbon reserves were characterised using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR data for select key genes indicated that plants reduced fructan degradation, and increased photosynthesis and fructan synthesis capacities following carbon depletion. This acclimatory response was not sufficient to prevent a reduction (P<0.001) in net biomass accumulation, but ensured that the plant survived. CONCLUSIONS: Adaptations of plants with depleted carbon reserves resulted in reduced post-defoliation carbon mobilization and earlier replenishment of carbon reserves, thereby ensuring survival and continued growth. These findings will help pave the way to improve plant biomass production, for either grazing livestock or biofuel purposes.

Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.).

BACKGROUND: Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. RESULTS: Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. CONCLUSIONS: This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here.

Transcriptome analysis reveals season-specific rbcS gene expression profiles in diploid perennial ryegrass (Lolium perenne L.).

Perennial ryegrass (Lolium perenne L.) is a major grass species used for forage and turf throughout the world, and gains by conventional breeding have reached a plateau. Perennial ryegrass is an outcrossing, self-incompatible diploid (2n = 2x = 14) with a relatively large genome (4067 Mbp/diploid genome; Evans, G.M., Rees, H., Snell, C.L. and Sun, S. (1972) The relation between nuclear DNA amount and the duration of the mitotic cycle. Chrom. Today, 3, 24-31). Using tissues sourced from active pastures during the peak of the autumn, winter, spring and summer seasons, we analysed the ryegrass transcriptome employing a Serial Analysis of Gene Expression (SAGE) protocol, with the dual goals of understanding the seasonal changes in perennial ryegrass gene expression and enhancing our ability to select genes for genetic manipulation. A total of 159,002 14-mer SAGE tags was sequenced and mapped to the perennial ryegrass DNA database, comprising methyl-filtered (GeneThresher) and expressed sequence tag (EST) sequences. The analysis of 14,559 unique SAGE tags, which were present more than once in our SAGE library, revealed 964, 1331, 346 and 131 exclusive transcripts to autumn, winter, spring and summer, respectively. Intriguingly, our analysis of the SAGE tags revealed season-specific expression profiles for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco), LprbcS. The transcript level for LprbcS was highest in spring, and then decreased gradually between summer and winter. Five different copies of LprbcS were revealed in ryegrass, with one possibly producing splice variant transcripts. Two highly expressed LprbcS genes were reported, one of which was not active in autumn. Another LprbcS gene showed an inverse expression profile to the autumn inactive LprbcS in a manner to compensate the expression level.

Cloning of the promoter for a novel barley gene, Lem1, and its organ-specific promotion of Gfp expression in lemma and palea.

The differential display method was used to identify a novel barley gene, Lem1, expressed primarily in the outer organs (lemma and palea) that enclose developing florets and seeds. The promoter was isolated from a BAC genomic clone and used in a translational fusion with a green fluorescent protein gene (Gfp) to produce a transient expression vector. After particle bombardment, Gfp was expressed only in lemmas, paleas and awns of developing spikelets. Lem1 did not promote Gfp expression in vegetative leaves or in mature spikes, although expression of co-bombarded uidA (GUS) occurred under the regulation of a ubiquitin promoter. This reproduced the developmentally regulated pattern of mRNA accumulation. Deletion studies showed that the promoter activity is confined to a cis element within 80 bp of the transcription start site. Upstream from this, the promoter contains putative auxin-, ethylene- and gibberellin-responsive elements or homologues. Lem1 was found to be a single intronless gene encoding an acidic 102 amino acid protein, possibly associated with membranes. In a two-rowed barley, Lem1 mRNA was absent in the lateral spikelets, which fail to develop, and present only in the developing median spikelets. This suggests that Lem1 may play a role in organ development.