AtLEC2 upregulated the photosynthetic elements and improved the photon capture capacity of C. sorokiniana-AtLEC2
In recent years, microalgal genomics has made significant advances, and the lipid yield of microalgae has been improved through genetic engineering technologies (Talebi et al. 2013, Cecchin et al. 2019, Gao et al. 2014, Fan et al. 2015, Shang et al. 2016). However, lipid content is regulated by operating a single gene of key enzymes involved in lipid metabolism (Williams and Laurens 2010, Xue et al. 2016, Fan et al. 2014). In this study, AtLEC2 expression promoted the accumulation of lipid in C. sorokiniana, which increased from 23–51%. Growth curves and photosynthetic parameters showed that growth cycles and Fv/Fm values had no significant changes, while light response curves of ETR changed significantly.
ETR is an essential parameter of photosynthetic efficiency(Conde-álvarez et al. 2002). In this study, the ETR of C. sorokiniana-AtLEC2 and WT were similar when light intensity was under 50 µmol·m− 2·s− 1. However, when light intensity was increased to 100 µmol·m− 2·s− 1, the ETR of C. sorokiniana-AtLEC2 was significantly higher than WT (Fig. 2C). Thus, C. sorokiniana-AtLEC2 could have higher photosynthetic efficiency. Furthermore, under low light intensities with limited photons, the harvest of photons between C. sorokiniana-AtLEC2 and WT was consistent. However, with increasing light intensities, C. sorokiniana-AtLEC2 showed a higher capture capacity of photons.
The changes in ETR were consistent with protein expression in photosynthesis (Fig. 5). ATP synthase F0, Photosystem II (psbC, psbB, psbH), Photosystem I (psaA, psaB), Cytochrome b6 (petB), Cytochrome b6-f (petD), Chlorophyll a-b binding protein (LHCB2), and Photosystem I assembly proteins were upregulated in C. sorokiniana-AtLEC2. Moreover, Oxygen-evolving enhancer protein 3 (psbQ) downregulated 1.3 folds but other detected Oxygen-evolving enhancer proteins were upregulated 2.6 folds. According to Thomas et al.(Thomas et al. 2001), the psaE− mutant did not affect the rates of PSI cyclic electron transport despite significant psaE downregulation. In general, the AtLEC2 improved the light capture and the electron transport of C. sorokiniana-AtLEC2.
AtLEC2 improved the TAG accumulation in C. sorokiniana-AtLEC2 by upregulating the expression of G3PDH
Based on the proteomic analysis, enzymes involved in carbon fixation like TPI chloroplast type and ribulose bisphosphate carboxylase/oxygenase activase were downregulated without any decline in photosynthetic efficiency. Maybe it was due to the upregulation of G3PDH. G3PDH catalyzes the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate (G3P), which provides the glycerol backbone for TAG synthesis (Herrera-Valencia et al. 2012, Yao et al. 2014). Thus, G3PDH, a significant intermediary, connects glycolysis and TAG synthesis (Wang et al. 2018, Zhao et al. 2019).
In this study, G3PDH expression was upregulated by 8.8 folds in C. sorokiniana-AtLEC2, with enhanced conversion capacity of G3P from dihydroxyacetone phosphate. Dihydroxyacetone phosphate was consumed in large quantities. TPI catalyzed the isomerization between dihydroxyacetone phosphate and glyceraldehyde-3-phosphate(Beck 1956). The rapid nature results in the maintenance of equilibrium between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. At the equilibrium state, the concentration of dihydroxyacetone phosphate is much higher than glyceraldehyde 3-phosphate. Glyceraldehyde-3-phosphate produced during photosynthesis in chloroplasts was directly released into the cytoplasm by Triose Phosphate/Phosphate Translocator (Flügge and Heldt 1984) and was further used in the synthesis of TAG (Fig. 6). The down-regulated expression of TPI chloroplast type (10.6 folds) and upregulated cytoplasmic type (1.6 folds) verified it. The consumption of glyceraldehyde-3-phosphate affected the replenishment of ribulose-1,5-biphosphate (RuBP) in the Calvin cycle. However, RuBP is more likely to bind to rubisco in its inactive state. RuBP is a potent inhibitor of rubisco(Spreitzer and Salvucci 2002, Jr 2003). The reduction of RuBP increased the activity of rubisco indirectly (Streusand and Portis 1987). Therefore, the total activity of rubisco and the photosynthetic efficiency was not decreased despite the downregulation of Ribulose bisphosphate carboxylase/oxygenase activase and rubisco, which was consistent with the test results of photosynthetic efficiency.
Upregulated proteins are illustrated in red; downregulated proteins are in blue. RUBP: ribulose-1,5-bisphosphate; Rubisco: ribulose bisphosphate carboxylase/oxygenase; PDK: phosphoglyceric kinase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; TPT: Triose Phosphate/Phosphate Translocator; TPI: triose phosphate isomerase; G3PDH: glycerol-3-phosphate dehydrogenase; ACS: long-chain acyl-CoA synthetase; GPAT: glycerol-3-phosphate O-acyltransferase; LPAAT: lysophosphatidate acyltransferase; PAP: phosphatidate phosphatase; DGAT: diacylglycerol O-acyltransferase.