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.

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.

Storing carbon in leaf lipid sinks enhances perennial ryegrass carbon capture especially under high N and elevated CO2.

By modifying two genes involved in lipid biosynthesis and storage [cysteine oleosin (cys-OLE)/diacylglycerol O-acyltransferase (DGAT)], the accumulation of stable lipid droplets in perennial ryegrass (Lolium perenne) leaves was achieved. Growth, biomass allocation, leaf structure, gas exchange parameters, fatty acids, and water-soluble carbohydrates were quantified for a high-expressing cys-OLE/DGAT ryegrass transformant (HL) and a wild-type (WT) control grown under controlled conditions with 1-10 mM nitrogen (N) supply at ambient and elevated atmospheric CO2. A dramatic shift in leaf carbon (C) storage occurred in HL leaves, away from readily mobilizable carbohydrates and towards stable lipid droplets. HL exhibited an increased growth rate, mainly in non-photosynthetic organs, leading to a decreased leaf mass fraction. HL leaves, however, displayed an increased specific leaf area and photosynthetic rate per unit leaf area, delivering greater overall C capture and leaf growth at high N supply. HL also exhibited a greater photosynthesis response to elevated atmospheric CO2. We speculate that by behaving as uniquely stable microsinks for C, cys-OLE-encapsulated lipid droplets can reduce feedback inhibition of photosynthesis and drive greater C capture. Manipulation of many genes and gene combinations has been used to increase non-seed lipid content. However, the cys-OLE/DGAT technology remains the only reported case that increases plant biomass. We contrast cys-OLE/DGAT with other lipid accumulation strategies and discuss the implications of introducing lipid sinks into non-seed organs for plant energy homeostasis and growth.