DGAT1 + cysteine-oleosin expression in Lolium perenne leaves enhances photosynthetic efficiency, growth, and pasture energy density : A thesis submitted in partial fulfilment of the requirement for the Degree of Doctor of Philosophy at Lincoln University

Two compelling strategies for enhancing food security are increasing photosynthesis and engineering higher levels of valuable nutrients such as lipids into plant tissues. Manipulation of many gene combinations has increased vegetative lipid content, but the effect of introducing a new energy-dense C sink on plant growth is typically negative. Winichayakul et al. (2013) reported long-term lipid accumulation in the leaves and roots of Arabidopsis thaliana by co-expressing diacylglycerol O-acyltransferase [DGAT1; the final enzyme in the triacylglycerol (TAG) biosynthesis pathway] and a novel lipid droplet-encapsulating protein (cysteine-oleosin) designed to confer greater stability to lipid droplets in planta. Remarkably, increased rates of photosynthesis and shoot growth also occurred in DGAT1 + cysteine-oleosin (collectively ‘high metabolizable energy’ or ‘HME’) Arabidopsis. In this thesis, the effect of leaf HME expression on the growth and photosynthesis of the crop species Lolium perenne L. (perennial ryegrass) (PR) was studied. Leaf HME expression in PR increased leaf fatty acid content (FA) while simultaneously increasing growth. The primary objective of this thesis was to generate comprehensive evidence for this counterintuitive finding. The second objective was to investigate whole-plant, and especially leaf-level physiological and biochemical traits related to photosynthesis, under variable nitrogen (N) supply and both ambient and elevated atmospheric [CO2], that could account for increased HME PR growth. The final objective was to study the translation, from spaced pots indoors to field canopies, of the FA, energy, and growth enhancing traits associated with leaf HME expression in segregating PR populations, in order to quantify potential agronomic advantages of an HME cultivar. Leaf HME expression caused a shift in leaf C storage in PR leaves; away from water-soluble carbohydrates (WSC) and towards FA. HME expression induced a high specific leaf area (leaf area per unit of mass), and in some genetic backgrounds also increased net photosynthetic rate per unit leaf area, contributing to a greater photosynthetic rate per unit leaf mass (Amass) and per unit leaf nitrogen (PNUE). Under high N supply, total leaf area, relative growth rate, and total plant DW were enhanced for multiple independently-transformed HME lines/populations. The high HME PNUE was associated with enhanced mesophyll conductance to CO2, greater N investment in electron transport and ATP synthesis, and higher estimated light absorptance per chlorophyll. The correspondence between high leaf FA and increased Amass/PNUE/growth was found to depend upon a reduction in leaf WSC occurring. Further, HME expression made Amass more responsive to elevated atmospheric [CO2], suggesting that diverting a proportion of leaf WSC into FA may remove feedback inhibition of photosynthesis in some contexts. Indoor and field experiments with HME PR populations grown in small canopies under simulated grazing showed that increasing leaf FA content by at least 0.8 %DW increased herbage gross energy (GE) concentration by up to 0.5 kJ gDW-1. HME expression enhanced herbage DW accumulation (yield) in ‘mini swards’ arranged in spaced rows indoors but did not reliably enhance herbage production in dense swards indoors or field swards. Overall, this work supports the potential for using leaf lipids as alternative sinks for photosynthate to increase plant growth potential and for leaf lipid accumulation to enhance pasture energy density.