Carbon Supply and Photoacclimation Cross Talk in the Green Alga Chlamydomonas reinhardtii

Photosynthetic organisms are exposed to drastic changes in light conditions, which can affect their photosynthetic efficiency and induce photodamage. To face these changes, they have developed a series of acclimation mechanisms. In this work, we have studied the acclimation strategies of Chlamydomonas reinhardtii, a model green alga that can grow using various carbon sources and is thus an excellent system in which to study photosynthesis. Like other photosynthetic algae, it has evolved inducible mechanisms to adapt to conditions where carbon supply is limiting. We have analyzed how the carbon availability influences the composition and organization of the photosynthetic apparatus and the capacity of the cells to acclimate to different light conditions. Using electron microscopy, biochemical, and fluorescence measurements, we show that differences in CO₂ availability not only have a strong effect on the induction of the carbon-concentrating mechanisms but also change the acclimation strategy of the cells to light. For example, while cells in limiting CO₂ maintain a large antenna even in high light and switch on energy-dissipative mechanisms, cells in high CO₂ reduce the amount of pigments per cell and the antenna size. Our results show the high plasticity of the photosynthetic apparatus of C. reinhardtii. This alga is able to use various photoacclimation strategies, and the choice of which to activate strongly depends on the carbon availability.Light sustains virtually all life on Earth through the process of photosynthesis. However, light can be very harmful for oxygenic photosynthetic organisms, as excess absorption can lead to the production of reactive oxygen species. In order to survive and grow, these organisms have developed various photoacclimation mechanisms operating on different time scales that protect the cell from photodamage. In the green alga Chlamydomonas reinhardtii, these mechanisms vary from negative phototaxis and multicomponent nonphotochemical quenching (NPQ) to a number of physiological and biochemical changes (Erickson et al., 2015). C. reinhardtii cells are around 10 μm in diameter, and a large part of their total volume is occupied by a single horseshoe-shaped chloroplast (Sager and Palade, 1957). The photosynthetic machinery responsible for the light reactions is located in thylakoid membranes and contains four major components: PSII, cytochrome b₆f, PSI, and ATP synthase. Both photosystems bind chlorophyll (Chl) and carotenoid (Car) and are composed of a core and several outer antennae pigment-protein complexes, the main function of which is light harvesting and its conversion into chemical energy. The PSII core is composed of D1, D2, CP43, and CP47 pigment-protein complexes and several smaller subunits, the number of which varies between organisms (Shi et al., 2012). The outer antenna contains the light-harvesting complex II (LHCII), which in C. reinhardtii is encoded by nine LHCBM genes, and the minor antennae CP26 and CP29 (Nield et al., 2000; Teramoto et al., 2001; Natali and Croce, 2015). These complexes are assembled together to form PSII-LHCII supercomplexes (Tokutsu et al., 2012; Drop et al., 2014). The PSI core is composed of a PSAA-PSAB heterodimer and a number of smaller subunits (Jensen et al., 2007), and in C. reinhardtii the LHCI antenna consists of nine LHCA proteins (Mozzo et al., 2010) that are associated with the core to form the PSI-LHCI complex (Stauber et al., 2009; Drop et al., 2011).The composition and organization of the thylakoid membrane is light dependent. The gene expression of different LHCs has been reported to be affected by light acclimation (Teramoto et al., 2002; Durnford et al., 2003; Yamano et al., 2008) and to be NAB1 regulated (Mussgnug et al., 2005). It has been observed that long-term high-light exposure of C. reinhardtii cells leads to a 50% decrease of Chl content (Neale and Melis, 1986; Bonente et al., 2012) and to changes in Chl-to-Car ratio (Niyogi et al., 1997a; Baroli et al., 2003; Bonente et al., 2012), suggesting reduction of the antenna size (Neale and Melis, 1986), although, in a more recent report (Bonente et al., 2012), it was concluded that the antenna size is not modulated by light in this alga. Recently, a dependence of the antenna components on the carbon availability also was reported. It was shown that, when cells grown in acetate are shifted from high to low CO₂ concentration, the functional antenna size of PSII decreases and a down-regulation of LHCBM6/8 occurs (Berger et al., 2014).In the short term, the main response to high light is the dissipation of energy absorbed in excess heat in a process called qE, or energy-dependent quenching, which is the fastest component of NPQ. In land plants, the main player in this process is the protein PsbS (Li et al., 2002, 2004), while in C. reinhardtii, the process is centered around LHCSR1 and LHCSR3 (Peers et al., 2009; Dinc et al., 2016). LHCSR3, the most studied of the two, is a pigment-protein complex that is expressed within 1 h of high-light exposure (Allorent et al., 2013) in combination with CO₂ limitation (Yamano et al., 2008; Maruyama et al., 2014). The qE onset is triggered by lumen acidification sensed by LHCSR3/1 (Bonente et al., 2011; Liguori et al., 2013; Tokutsu and Minagawa, 2013; Dinc et al., 2016).Cars are well known to be involved in photoprotection. They quench triplet Chl and scavenge singlet oxygen (¹O₂; Frank and Cogdell, 1996). In C. reinhardtii, the antioxidant role of xanthophylls is well illustrated by the mutant npq1 lor1 lacking lutein and zeaxanthin (Niyogi et al., 1997b). This mutant is deficient in qE, but compared with other qE-deficient mutants like npq4 (Peers et al., 2009) and npq5 (Elrad et al., 2002), which are LHCSR3 and LHCBM1 knockouts, respectively, it is extremely light sensitive, due to the absence of quenching of triplet Chl and ¹O₂ by zeaxanthin and lutein.Aquatic oxygenic photosynthetic organisms meet several challenges in CO₂ fixation (Moroney and Ynalvez, 2007). First, the diffusion of CO₂ in water is 10,000 times slower than in air. Second, the CO₂-fixing enzyme Rubisco is not selective for CO₂ and also binds oxygen, resulting in the process of photorespiration. Third, the form of inorganic carbon depends on the pH (i.e. in alkaline pH, it is HCO₃⁻, while in acidic pH, it is CO₂; Beardall, 1981; Gehl et al., 1987). This diminishes even further the availability of CO₂ in the cell. In order to overcome these CO₂ fixation barriers, algae have developed carbon-concentrating mechanisms (CCMs; Moroney and Ynalvez, 2007). The essence of these processes lies in the active pumping of inorganic carbon in the cell via a number of transporters that concentrate it in the pyrenoid, a ball-like structure containing Rubisco, Rubisco activase, and intrapyrenoid thylakoids and surrounded by a starch sheath. In the pyrenoid, HCO₃⁻ is converted to CO₂ by CARBONIC ANHYDRASE3 (CAH3; Blanco-Rivero et al., 2012; Sinetova et al., 2012) and then fixed by Rubisco in the Calvin-Benson-Bassham cycle. CAH3 also is suggested to provide HCO₃⁻ in the proximity of the oxygen-evolving complex, where it may function as a proton carrier, removing H⁺ from water splitting to avoid photoinhibition (Villarejo et al., 2002; Shutova et al., 2008).C. reinhardtii also can grow mixotrophically using alternative organic carbon sources present in its environment. For example, it can take up acetate, which is then incorporated into the citric cycle, producing reducing equivalents and CO₂ (Johnson and Alric, 2012), and into the glyoxylate cycle, producing malate (Lauersen et al., 2016). In the presence of acetate, it has been reported that CO₂ uptake and oxygen evolution were decreased by half under saturating CO₂ and light intensities without affecting PSII efficiency, respiration, and cell growth (Heifetz et al., 2000). In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions (Chapman et al., 2015). Furthermore, acetate can replace PSII-associated HCO₃⁻, reducing ¹O₂ formation and, therefore, acting as a photoprotector during high-light acclimation (Roach et al., 2013).In short, high-light acclimation is a complex, multicomponent process that happens on different time scales. Furthermore, it is embedded in the overall metabolic network and is potentially influenced by different nutrients and metabolic states. A thorough understanding of this process and its regulation is crucial for fundamental research and applications. To determine if different carbon supply conditions trigger different light acclimation strategies and photoprotective responses, we systematically studied C. reinhardtii cells grown in mixotrophic, photoautotrophic, and high-CO₂ photoautotrophic conditions in different light intensities.We show that C. reinhardtii cells use different strategies to acclimate to high light depending on the carbon availability and trophic status. These results underline the strong connection between metabolism and light acclimation responses and reconcile the data from various reports. Furthermore, our study demonstrates how, in a dynamic system such as C. reinhardtii, a single change in growth conditions has large effects at multiple levels.