Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Insight into the regulatory networks underlying the high lipid perennial ryegrass growth under different irradiances.

Under favourable conditions, perennial ryegrass (Lolium perenne) engineered to accumulated high lipid (HL) carbon sink in their leaves was previously shown to also enhance photosynthesis and growth. The greater aboveground biomass was found to be diminished in a dense canopy compared to spaced pots. Besides, the underlying genetic regulatory network linking between leaf lipid sinks and these physiological changes remains unknown. In this study, we demonstrated that the growth advantage was not displayed in HL Lolium grown in spaced pots under low lights. Under standard lights, analysis of differentiating transcripts in HL Lolium reveals that the plants had elevated transcripts involved in lipid metabolism, light capturing, photosynthesis, and sugar signalling while reduced expression of genes participating in sugar biosynthesis and transportation. The plants also had altered several transcripts involved in mitochondrial oxidative respiration and redox potential. Many of the above upregulated or downregulated transcript levels were found to be complemented by growing the plants under low light. Overall, this study emphasizes the importance of carbon and energy homeostatic regulatory mechanisms to overall productivity of the HL Lolium through photosynthesis, most of which are significantly impacted by low irradiances.

Evolution and conserved functionality of organ size and shape regulator PEAPOD.

Transcriptional regulator PEAPOD (PPD) and its binding partners comprise a complex that is conserved throughout many core eudicot plants with regard to protein domain sequence and the function of controlling organ size and shape. Orthologues of PPD also exist in the basal angiosperm Amborella trichopoda, various gymnosperm species, the lycophyte Selaginella moellendorffii and several monocot genera, although until now it was not known if these are functional sequences. Here we report constitutive expression of orthologues from species representing diverse taxa of plant phylogeny in the Arabidopsis Δppd mutant. PPD orthologues from S. moellendorffii, gymnosperm Picea abies, A. trichopoda, monocot Musa acuminata, and dicot Trifolium repens were able to complement the mutant and return it to the wild-type phenotype, demonstrating the conserved functionality of PPD throughout vascular plants. In addition, analysis of bryophyte genomes revealed potential PPD orthologues in model liverwort and moss species, suggesting a more primitive lineage for this conserved regulator. The Poaceae (grasses) lack the genes for the PPD module and the reason for loss of the complex from this economically significant family is unclear, given that grasses were the last of the flowering plants to evolve. Bioinformatic analyses identified putative PPD orthologues in close relatives of the Poaceae, indicating that the explanation for absence of PPD in the grasses may be more complex than previously considered. Understanding the mechanisms which led to loss of PPD from the grasses will provide insight into evolution of the Poaceae.

Changes in Leaf-Level Nitrogen Partitioning and Mesophyll Conductance Deliver Increased Photosynthesis for Lolium perenne Leaves Engineered to Accumulate Lipid Carbon Sinks.

Diacylglycerol acyl-transferase (DGAT) and cysteine oleosin (CO) expression confers a novel carbon sink (of encapsulated lipid droplets) in leaves of Lolium perenne and has been shown to increase photosynthesis and biomass. However, the physiological mechanism by which DGAT + CO increases photosynthesis remains unresolved. To evaluate the relationship between sink strength and photosynthesis, we examined fatty acids (FA), water-soluble carbohydrates (WSC), gas exchange parameters and leaf nitrogen for multiple DGAT + CO lines varying in transgene accumulation. To identify the physiological traits which deliver increased photosynthesis, we assessed two important determinants of photosynthetic efficiency, CO2 conductance from atmosphere to chloroplast, and nitrogen partitioning between different photosynthetic and non-photosynthetic pools. We found that DGAT + CO accumulation increased FA at the expense of WSC in leaves of L. perenne and for those lines with a significant reduction in WSC, we also observed an increase in photosynthesis and photosynthetic nitrogen use efficiency. DGAT + CO L. perenne displayed no change in rubisco content or Vcmax but did exhibit a significant increase in specific leaf area (SLA), stomatal and mesophyll conductance, and leaf nitrogen allocated to photosynthetic electron transport. Collectively, we showed that increased carbon demand via DGAT+CO lipid sink accumulation can induce leaf-level changes in L. perenne which deliver increased rates of photosynthesis and growth. Carbon sinks engineered within photosynthetic cells provide a promising new strategy for increasing photosynthesis and crop productivity.

Condensed Tannins in White Clover (Trifolium repens) Foliar Tissues Expressing the Transcription Factor TaMYB14-1 Bind to Forage Protein and Reduce Ammonia and Methane Emissions in vitro.

Grazing ruminants contribute to global climate change through enteric methane and nitrous oxide emissions. However, animal consumption of the plant polyphenolics, proanthocyanidins, or condensed tannins (CTs) can decrease both methane emissions and urine nitrogen levels, leading to reduced nitrous oxide emissions, and concomitantly increase animal health and production. CTs are largely absent in the foliage of important temperate pasture legumes, such as white clover (Trifolium repens), but found in flowers and seed coats. Attempts at enhancing levels of CT expression in white clover leaves by mutagenesis and breeding have not been successful. However, the transformation of white clover with the TaMYB14-1 transcription factor from Trifolium arvense has resulted in the production of CTs in leaves up to 1.2% of dry matter (DM). In this study, two generations of breeding elevated foliar CTs to >2% of DM. The CTs consisted predominantly of prodelphinidins (PD, 75-93%) and procyanidins (PC, 17-25%) and had a mean degree of polymerization (mDP) of approximately 10 flavan-3-ol subunits. In vitro studies showed that foliar CTs were bound to bovine serum albumin and white clover proteins at pH 6.5 and were released at pH 2.-2.5. Using rumen in vitro assays, white clover leaves containing soluble CTs of 1.6-2.4% of DM significantly reduced methane production by 19% (p ≤0.01) and ammonia production by 60% (p ≤ 0.01) relative to non-transformed wild type (WT) controls after 6 h of incubation. These results provide valuable information for further studies using CT expressing white clover leaves for bloat prevention and reduced greenhouse gas emissions in vivo.

Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function.

The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis.

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.

Genetic transformation of western clover (Trifolium occidentale D. E. Coombe.) as a model for functional genomics and transgene introgression in clonal pasture legume species.

BACKGROUND: Western clover (Trifolium occidentale) is a perennial herb with characteristics compatible for its development as an attractive model species for genomics studies relating to the forage legume, white clover (Trifolium repens). Its characteristics such as a small diploid genome, self-fertility and ancestral contribution of one of the genomes of T. repens, facilitates its use as a model for genetic analysis of plants transformed with legume or novel genes. RESULTS: In this study, a reproducible transformation protocol was established following screening of T. occidentale accessions originating from England, Ireland, France, Spain and Portugal. The protocol is based upon infection of cotyledonary explants dissected from mature seed with the Agrobacterium tumefaciens strain GV3101 carrying vectors which contain the bar selection marker gene. Transformation frequencies of up to 7.5% were achieved in 9 of the 17 accessions tested. Transformed plants were verified by PCR and expression of the gusA reporter gene, while integration of the T-DNA was confirmed by Southern blot hybridisation and segregation of progeny in the T1 generation. CONCLUSIONS: Development of this protocol provides a valuable contribution toward establishing T. occidentale as a model species for white clover. This presents opportunities for further improvement in white clover through the application of biotechnology.

Expression of the R2R3-MYB transcription factor TaMYB14 from Trifolium arvense activates proanthocyanidin biosynthesis in the legumes Trifolium repens and Medicago sativa.

Proanthocyanidins (PAs) are oligomeric flavonoids and one group of end products of the phenylpropanoid pathway. PAs have been reported to be beneficial for human and animal health and are particularly important in pastoral agricultural systems for improved animal production and reduced greenhouse gas emissions. However, the main forage legumes grown in these systems, such as Trifolium repens and Medicago sativa, do not contain any substantial amounts of PAs in leaves. We have identified from the foliar PA-accumulating legume Trifolium arvense an R2R3-MYB transcription factor, TaMYB14, and provide evidence that this transcription factor is involved in the regulation of PA biosynthesis in legumes. TaMYB14 expression is necessary and sufficient to up-regulate late steps of the phenylpropanoid pathway and to induce PA biosynthesis. RNA interference silencing of TaMYB14 resulted in almost complete cessation of PA biosynthesis in T. arvense, whereas Nicotiana tabacum, M. sativa, and T. repens plants constitutively expressing TaMYB14 synthesized and accumulated PAs in leaves up to 1.8% dry matter. Targeted liquid chromatography-multistage tandem mass spectrometry analysis identified foliar PAs up to degree of polymerization 6 in leaf extracts. Hence, genetically modified M. sativa and T. repens plants expressing TaMYB14 provide a viable option for improving animal health and mitigating the negative environmental impacts of pastoral animal production systems.

Mapping, complementation, and targets of the cysteine protease actinidin in kiwifruit.

Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.

High levels of a fungal superoxide dismutase and increased concentration of a PR-10 plant protein in associations between the endophytic fungus Neotyphodium lolii and ryegrass.

Neotyphodium lolii is a fungal endosymbiont of the ryegrass Lolium perenne. Its growth is tightly controlled and synchronized with that of the plant. How the symbionts communicate is largely unknown but failure of the endophyte to elicit a defense response is considered crucial for successful symbiosis. In silver-stained two-dimensional gels of protein extracts from endophyte-infected ryegrass, a fungal Cu/Zn superoxide dismutase was detectable, even though the fungus accounts for only <¹/500 of the biomass, indicating that it is an abundant fungal protein and that the fungus needs protection against reactive oxygen species. The plant increased levels of a pathogenesis-related class 10 (PR-10) protein; when equal amounts of protein from infected and uninfected plants were loaded, PR-10 was only detectable in extracts from infected plants. Presence of the endophyte did not lead to a significant increase in PR10 transcript levels. In protein extracts from a symbiosis containing an N. lolii variant with an abnormal in planta growth pattern, the fungal Cu/Zn superoxide dismutase but not PR-10 protein was detectable. The correlation between increased PR-10 levels and presence of a normally growing endophyte is suggestive of a role of a very limited host defense in the interaction between grass and endophyte.