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

Abstract

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 V_cmax 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.

Keywords: Lolium perenne; cysteine oleosin; diacylglycerol acyl-transferase; lipid; photosynthesis; sink strength.

Figure 1

Percent difference (±SE) in leaf fatty acids compared to respective NT (A), recombinant protein contents for diacylglycerol acyl-transferase (DGAT; B) and cysteine-oleosin (C), and stain free gel showing equal protein loading for each cell (D), for five DGAT + CO lines and three respective NT controls. Matching genetic backgrounds are grouped together. ^*p < 0.05.

Figure 2

Stacked means (±SE) of high molecular weight carbohydrates (shaded grey, ) and low molecular weight carbohydrates (shaded white, ) in the leaves of five DGAT + CO transformed L. perenne lines and respective non-transformed controls. Matching genetic backgrounds are grouped together. n = 10. **Statistically differs from NT, p < 0.01.

Figure 3

Net photosynthesis (A) and whole-plant relative growth rate (B) for five DGAT + CO lines and three NT lines. Means ± SE. ^*Statistically differs from NT, p < 0.05; n = 10. Matching genetic backgrounds are shaded together.

Figure 4

Leaf N concentration (N_mass; A), N per unit leaf area (N_area; B) and photosynthetic nitrogen use efficiency measured at 600 μmol photons m⁻² s⁻¹ (PNUE_amb; C) for five independently transformed DGAT + CO L. perenne lines and respective NT controls. Means ± SE. ^*Statistically differs from NT, p < 0.05; n = 10. Matching genetic backgrounds are shaded together.

Figure 5

Photosynthesis vs. foliar carbohydrates for DGAT + CO and NT Lolium perenne. Lines from each genetic background are shaded together irrespective of DGAT + CO or NT; NT1 and DGAT + CO1-2 (), NT2 and DGAT + CO3-4 () and NT3 and DGAT + CO5 (). Trendline represents NT2 and NT3 derived lines. Photosynthesis measured at 600 μmol photons m^-2 s^-1.

Figure 6

Specific leaf area (SLA; A), photosynthesis at 1500 μmol photons m^-2 s^-1 (A_sat; B), leaf N concentration (N_mass; C), N per unit leaf area (N_area; D), stomatal conductance (g_s; E) and photosynthetic nitrogen use efficiency (PNUE_sat; F) for L. perenne DGAT + CO5 (open circles ) and NT control (NT3; closed circles ) grown under 1-7 mM NO₃^- supply. Means ± SE; n = 3 for 1, 2, and 3 mM treated plants, n = 5 for 5 and 7.5 mM treated plants.

Figure 7

Photosynthesis vs. leaf N, expressed on a mass (A) and area (B) basis for Lolium perenne DGAT + CO5 (open circles; ) and NT control NT3 (closed circles; ) grown under 1–7.5 mM NO₃⁻ supply. Photosynthesis measurements were made at 1500 μmol photons m⁻² s⁻¹.

Figure 8

Within-leaf N partitioning for L. perenne DGAT + CO5 and NT control NT3 grown under 5 mM NO₃⁻ supply. N_R (N invested in rubisco), N_S-R (N invested in non-rubisco soluble protein), N_P (N invested in pigment protein complexes), N_E (N invested in “bioenergetics”) and N_O (“other” N) as a proportion of total leaf N. Means ± SE, ^*p < 0.05, ^**p < 0.01, n = 6–8.