The Plant Journal (2015) 82, 481–503 doi: 10.1111/tpj.12823 SI CHLAMYDOMONAS Algae after dark: mechanisms to cope with anoxic/hypoxic conditions Wenqiang Yang1,*, Claudia Catalanotti1, Tyler M. Wittkopp1,2, Matthew C. Posewitz3 and Arthur R. Grossman1 1Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, USA, 2Department of Biology, Stanford University, Stanford, CA 94305, USA, and 3Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO 80401, USA Received 18 November 2014; revised 28 February 2015; accepted 3 March 2015; published online 9 March 2015. *For correspondence (e-mail [email protected]) SUMMARY Chlamydomonas reinhardtii is a unicellular, soil-dwelling (and aquatic) green alga that has significant metabolic flexibility for balancing redox equivalents and generating ATP when it experiences hypoxic/ anoxic conditions. The diversity of pathways available to ferment sugars is often revealed in mutants in which the activities of specific branches of fermentative metabolism have been eliminated; compensatory pathways that have little activity in parental strains under standard laboratory fermentative conditions are often activated. The ways in which these pathways are regulated and integrated have not been extensively explored. In this review, we primarily discuss the intricacies of dark anoxic metabolism in Chlamydomonas, but also discuss aspects of dark oxic metabolism, the utilization of acetate, and the relatively uncharacter- ized but critical interactions that link chloroplastic and mitochondrial metabolic networks. Keywords: Chlamydomonas reinhardtii, dark growth, oxic conditions, anoxic conditions, fermentation, acetate metabolism. INTRODUCTION Chlamydomonas reinhardtii (referred to as Chlamydo- Ballester et al., 2010; Pootakham et al., 2010; Aksoy et al., monas throughout) is a soil-dwelling photosynthetic organ- 2013), phototaxis and photoperception (Nagel et al., 2002; ism with certain metabolic features that are similar to those Wagner et al., 2008), the characteristics of the carbon-con- associated with vascular plants (photosynthesis), and oth- centrating mechanism (Fang et al., 2012; Meyer and Grif- ers that were lost during vascular plant evolution (e.g. fla- fiths, 2013), and lipid biosynthesis for the potential gella biogenesis). This alga has been exploited as an production of biofuels (Li et al., 2012; Johnson and Alric, attractive reference system for several decades. As a result 2013). Moreover, Chlamydomonas synthesizes molecular of sequencing of the Chlamydomonas nuclear genome hydrogen (H2) when experiencing anoxia, which is likely a (Merchant et al., 2007), the development of sophisticated frequent occurrence during the evening in environments molecular techniques applicable to this alga (Harris, 2001; where there is limited aeration and active microbial respira- Grossman et al., 2007; Purton, 2007; Gonzalez-Ballester tion (Melis and Happe, 2001, 2004; Ghirardi et al., 2009; et al., 2011), and its ability to grow photoautotrophically, Grossman et al., 2011; Catalanotti et al., 2013; Yang et al., mixotrophically and heterotrophically, Chlamydomonas is 2014a). Finally, Chlamydomonas is a powerful model for ideal for dissecting a range of biological, cellular, molecular dissecting aspects of dark, oxic metabolism (Salinas et al., and physiological processes, including flagella/cilia func- 2014), for which little information is available. tion and assembly (Dutcher, 1995; Cao et al., 2013), the bio- genesis and activity of chloroplasts (Rochaix, 2001; DARK METABOLISM IN PHOTOSYNTHETIC ORGANISMS Duanmu et al., 2013; Heinnickel et al., 2013), acclimation of General aspects cells to changing nutrient conditions (macro- and micro- nutrients) (Merchant et al., 2006; Moseley et al., 2009; Page Photosynthetic microorganisms generate energy exclu- et al., 2009; Gonzalez- sively through dark metabolism for almost half of the day © 2015 The Authors 481 The Plant Journal © 2015 John Wiley & Sons Ltd 482 Wenqiang Yang et al. (Perez-Garcia et al., 2011). The availability of O2 during the have lesions in genes encoding proteins that function in dark phase of the diel cycle heavily influences the differen- mitochondria (see below), but the lesions may also affect tial activation of distinct metabolic processes. Many algae proteins located outside of the mitochondria. Several not only have extensive fermentation networks available to commonly used laboratory ‘wild-type’ strains, including + generate ATP when O2 is not available, but are also able to CC-4425 (D66 ), cw15 and CC–4619 (dw15), exhibit some respire intracellular energy stores (e.g. starch), as well as growth impairment in the dark (Table 1); this finding prob- assimilate extracellular organic substrates (e.g. acetate and ably reflects lesions that have accumulated during long- glucose) for growth/ATP generation when O2 becomes term growth of the cultures in continuous light, which may available. It is only by developing an understanding of the obscure features of these organisms that have evolved for metabolic circuits associated with dark, oxic and hypoxic fitness in the natural environment. metabolism and their integration over the diel cycle (with The mitochondrial electron transport chain (mETC) is the metabolism that dominates in the light) that we will obtain site of oxidative phosphorylation. It uses reductant gener- a comprehensive understanding of net carbon cycling and ated from glycolysis, the pyruvate dehydrogenase complex the overall energy budgets of photosynthetic organisms in and the tricarboxylic acid (TCA) cycle to establish an elec- the environment. Such studies may also provide valuable trochemical transmembrane gradient that drives ATP syn- information regarding specific roles of enzymes predicted thesis. Most Chlamydomonas mutants with compromised to be associated with dark metabolism and the diversity of mitochondrial function are unable to use acetate as a car- metabolic networks available to sustain ATP production in bon source for heterotrophic growth. ‘Dark-dying’ mutants the dark. To appreciate the variety of ways in which carbon include those that either lack or have defects in specific is cycled over the course of the day and the metabolic con- components associated with complexes I–IV of the mETC, sequences of this cycling, it is critical to understand fluctu- or that affect the proper assembly of these complexes. ations in aquatic and terrestrial O2 levels, the nature of The Chlamydomonas mitochondrial proteome includes catabolism in the dark, how much fixed carbon is directed approximately 350 proteins (Atteia et al., 2009), while the toward respiratory and fermentation processes daily, and mitochondrial genome contains only 12 genes, seven of the impact of catabolic processes on fixed carbon storage. which encode proteins that function in the mETC (Gray and Additionally, dark, anoxic metabolism in photosynthetic Boer, 1988; Michaelis et al., 1990). Therefore, the majority of microbes has important ecological consequences, as many proteins contributing to mitochondrial function, including algae and cyanobacteria excrete reduced energy carriers respiratory activity, are nucleus-encoded and imported into (e.g. organic acids/alcohols and H2) during the night when the organelle by the Transporter Inner Membrane and Trans- the environment becomes hypoxic or anoxic (Mus et al., porter Outer Membrane (TIM-TOM) for mitochondria protein 2007; Ananyev et al., 2008; Dubini et al., 2009; Carrieri et al., transport complex (Neupert, 1997). A number of Chlamydo- 2010). These excreted reducing equivalents and carbon sub- monas mutants that are defective for dark growth and are strates fuel the growth of an often diverse group of co-exist- disrupted for mitochondrial genes have been identified ing heterotrophic microbes. It is likely that the types and (known as dum, i.e. dark uniparental minus, indicating non- amounts of products secreted by specific photosynthetic Mendelian inheritance from the mtÀ parent), although most microorganisms markedly influence the types and densities Chlamydomonas mutants with dark-growth deficiencies of the biota present in a variety of aquatic and soil ecosys- have lesions in nuclear genes that encode mitochondria- tems (Hoehler et al., 2002; Spear et al., 2005). localized proteins that are not associated with a specific, In this review, we present current advances in our under- experimentally determined function (Table 1) (Salinas et al., standing of fermentation, and also describe older pioneer- 2014). ing studies, that demonstrate the fascinating mechanisms The first respiratory-deficient Chlamydomonas strains, used by algae, and particularly Chlamydomonas, to func- which were isolated by Wiseman et al. (1977), were gener- tion metabolically in the dark. We also briefly discuss ated by nitrosoguanidine mutagenesis followed by selec- aspects of metabolism in the light, trafficking of reductants tion for cells unable to grow in the dark. Several of these between chloroplasts and mitochondria, and chlororespira- nuclear mutants exhibited altered mitochondrial cyto- tion, as this information establishes a metabolic framework chrome c oxidase activity (Wiseman et al., 1977). through which to assess dark, oxic and anoxic metabolism. Subsequently, many mutants with defects in com- plex I (dum5, dum17, dum20, dum23, dum25), complex III Mitochondrial mutants defective for heterotrophic growth (dum1, dum11, dum15, dum22, dum24) or complex IV Chlamydomonas is capable of growing