Algae After Dark: Mechanisms to Cope with Anoxic/Hypoxic Conditions

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 biogenesis 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

(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 complex 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 in the dark under (dum18, dum19) of the mitochondrial respiratory system oxic conditions, while at the same time maintaining photo- were identified after treatment of cells with the mutagenic synthetically competent thylakoid membranes, through dyes acriflavine and ethidium bromide (Matagne et al., assimilation and metabolism of acetate. Many Chlamydo- 1989; Dorthu et al., 1992; Colin et al., 1995; Duby and monas mutants deficient for dark heterotrophic growth Matagne, 1999; Remacle et al., 2001a,b; Cardol et al., 2002,

Table 1 Mutants that are unable to grow (or grow slowly) under dark oxic conditions, and mutants in genes encoding enzymes that function under dark anoxic conditions

Mutant name Protein encoded by mutated gene Method of creating mutation Publication

Dark growth deficiency CC–4425 (D66) Unknown Unknown W. Yang, unpublished CC–4619 (dw15) Unknown Unknown W. Yang, unpublished cw15 Unknown Unknown W. Yang, unpublished fdx5 Ferredoxin 5 Random insertional mutagenesis W. Yang, unpublished results ack1 Acetate kinase 1 Random insertional mutagenesis W. Yang, unpublished results dk series mutants Alterations in mitochondrial inner Nitrosoguanidine mutagenesis Wiseman et al., 1977 membranes and deficiencies in cytochrome oxidase activity dum series ‘Dark uniparental minus’, mutations Acriflavine-induced mutagenesis Reviewed by Salinas et al., 2014 in respiratory complexes I, III and IV nda1 Type II NAD(P)H dehydrogenase RNAi Lecler et al., 2012 atp2 CF1 b subunit RNAi Lapaille et al., 2010 icl1 Isocitrate lyase 1 Random insertional mutagenesis Plancke et al., 2014 y1 Protochlorophyllide oxidoreductase UV mutagenesis Sager, 1955 Dark anoxia pfl1 Pyruvate formate lyase 1 Random insertional mutagenesis Philipps et al., 2011; Catalanotti et al., 2012 amiPFL1 Pyruvate formate lyase 1 MicroRNA Burgess et al., 2012 pfr1 Pyruvate:ferredoxin oxidoreductase 1 TILLING C. Catalanotti, unpublished results adh1 Alcohol/aldehyde dehydrogenase 1 Random insertional mutagenesis Magneschi et al., 2012 ack1 Acetate kinase 1 Random insertional mutagenesis Yang et al., 2014b ack2 Acetate kinase 2 Random insertional mutagenesis Yang et al., 2014b pat2 Phosphate acetyltransferase 2 Random insertional mutagenesis Yang et al., 2014b ack1 ack2 Double mutant Cross between ack1 and ack2 Yang et al., 2014b pat2 ack2 Double mutant Cross between pat2 and ack2 Yang et al., 2014b hydEF Hydrogenase maturation factor EF Random insertional mutagenesis Posewitz et al., 2004a hydG Hydrogenase maturation factor G Random insertional mutagenesis M. C. Posewitz, unpublished results hydA1 Hydrogenase 1 Random insertional mutagenesis Meuser et al., 2012 hydA2 Hydrogenase 2 Random insertional mutagenesis Meuser et al., 2012 hydA1 hydA2 Double mutant Cross between hydA1 and hydA2 Meuser et al., 2012 amiTHB8 2–on–2 hemoglobin MicroRNA Hemschemeier et al., 2013b

2008). Chlamydomonas is unique among photosynthetic complex IV (cox1, encoding subunit 1 of cytochrome oxi- organisms in that its mitochondrial DNA may be targeted dase) retained some respiratory activity via the non-phos- for site-directed mutagenesis (via homologous recombina- phorylating alternative (salicylhydroxyamic acid-sensitive) tion) using biolistic transformation (Remacle et al., 2006). pathway, which transfers electrons from reduced ubiqui-

More recent approaches to identify mitochondrial mutants none to O2, but these strains only grew photoautotrophi- have exploited random insertional mutagenesis and RNA cally (Remacle et al., 2001b). Finally, all 17 subunits of interference to knockout or knockdown expression of Chlamydomonas complex V (mitochondrial ATP syn- nuclear genes important for mitochondrial function thase) are nucleus-encoded. Knockdown of ATP2 (encod-

(Table 1) (Cardol et al., 2006; Remacle et al., 2010; Barbieri ing the CF1 b subunit) resulted in decreased respiratory et al., 2011; Salinas et al., 2014). O2 consumption and obligate photoautotrophy as a con- While many mitochondrial mutants are disrupted for sequence of the loss of mitochondrial ATP synthesis (La- respiratory function and compromised for dark heterotro- paille et al., 2010). Interestingly, a decrease in ATP2 RNA phic growth, some exhibit less severe phenotypes. Some also affected photosynthetic activity, causing a shift into mutants defective for mitochondria- and nucleus-encoded state II [mobile light harvesting complex moves off of subunits of complex I grow slowly in the dark (Remacle photosystem II (PSII)], which may be part of a physiologi- et al., 2001a) and consume O2, generating a transmem- cal compensating response that favors cyclic electron brane proton gradient via a rotenone-resistant type II ﬂow (CEF) and increased ATP production in the light by NAD(P)H dehydrogenase (NDA1) coupled with electron the photosynthetic electron transport system (Lapaille transport to the alternative oxidase (Remacle et al., et al., 2010) and/or reduces the effect of a loss of mito- 2001a). nda1 RNAi lines showed abnormal growth pheno- chondrial respiration as an electron valve that oxidizes types when the knockdown lines were grown heterotro- chloroplast-generated reductant when the redox status of phically (Lecler et al., 2012). Some mutants affected in the plastid is increased (e.g. when there is excess excita- complex III (cob, encoding apocytochome b) and tion, such as under high light conditions).

chlorophyllide oxidoreductase, is yellow in the dark The glyoxylate cycle because of a decrease in chlorophyll production. Following The glyoxylate cycle, which occurs in the glyoxysome in this early work, the y–1 mutant was used for a series of many organisms, is important for acetate assimilation and studies on thylakoid membrane biosynthesis (Ohad et al., gluconeogenesis (Kornberg and Krebs, 1957). While 1967) and reconstitution of photosynthetic complexes (Hoo- Chlamydomonas does not have a visually apparent glyoxy- ber et al., 1991; White and Hoober, 1994). However, recent some, it does possess microbodies that appear to contain work has also identified Chlamydomonas mutants in path- components of the glyoxylate cycle (Hayashi and Shino- ways that are not directly associated with mitochondrial or zaki, 2012). Isocitrate lyase, a key enzyme of the glyoxylate glyoxysome function that are unable to grow in the dark. cycle encoded by the ICL gene, may be inactivated by glu- One of these strains is disrupted for the gene encoding the tathionylation and reactivated by glutaredoxin (Bedhomme chloroplast-targeted FDX5 protein (W. Yang, unpublished et al., 2009), possibly as a consequence of the chloroplast results). The FDX5 gene encodes one of six prototypical redox state and a shift in growth between heterotrophic plant-type ferredoxins (FDXs) in Chlamydomonas, five of and photoautotrophic activities. A recently characterized icl which (including FDX5) have been experimentally localized null mutant of Chlamydomonas was unable to grow het- to chloroplasts (Jacobs et al., 2009; Terauchi et al., 2009). erotrophically, and also did not grow robustly under mixo- When maintained in the dark, the fdx5 mutant has reduced trophic conditions (Plancke et al., 2014). The glyoxylate respiratory and photosynthetic electron transport rates, and cycle plays an essential role in heterotrophic growth by appears to be defective for desaturation of fatty acids and converting acetate to acetyl CoA, which then fuels gluco- maintenance of the proper ratio of monogalactosyldiacyl- neogenesis and other anabolic pathways. The icl mutant glycerol to digalactosyldiacylglycerol, which probably has also exhibited a highly pleiotropic phenotype, including extensive metabolic consequences (W. Yang, unpublished reduced acetate assimilation with a concomitant reduction results) (Table 1); additional work is necessary to determine in respiration, decreased b–oxidation activity and reduced the precise functions associated with FDX5 that are required levels of gluconeogenesis and glyoxylate cycle enzymes; for growth of Chlamydomonas in the dark. these changes cause an increased flow of carbon to amino Interaction between mitochondria and chloroplasts acid synthesis, which is reflected by increased levels of free amino acids in the mutant. The mutant strain also had Communication between organelles is critical for survival an increase in total fatty acid content, including neutral lip- of photosynthetic organisms. These inter-organelle interac- ids and free fatty acids, and increased oxidative stress tions may influence gene expression, the transfer of enzyme activities (e.g. superoxide dismutase and ascor- metabolites and reducing equivalents to various cellular bate peroxidase) (Plancke et al., 2014). compartments, and the production and utilization of ATP. At a basic level, photosynthesis provides sugars/polysac- Dark, heterotrophic growth and metabolism charides that are catabolized through glycolysis and the Many microalgae may be maintained in the dark when the TCA cycle to produce ATP by substrate-level phosphoryla- growth medium is supplemented with specific fixed tion, and reducing equivalents that may be used for respi- carbon sources. Heterotrophic growth may confer several ratory electron transport and ATP synthesis by oxidative advantageous features to microalgal cultivation (Perez- phosphorylation. Photosynthesis is also affected by a Garcia et al., 2011), allowing cost-effective scaling of algal range of compounds produced by respiration (including biomass production (Perez-Garcia et al., 2011). Under spe- ATP). Because of difficulties in assessing levels of respira- cific heterotrophic conditions, algae may grow faster than tory O2 consumption in the presence of light-driven photo- when they rely solely on sunlight to generate fixed carbon synthetic electron transport and the reduction of O2 and chemical bond energy, and the proportion of biomass through alternative oxidases (e.g. plastid terminal oxidase in lipid and nitrogen compounds may be increased. Thus, activity, photoreduction by PSI), the extent and signifi- heterotrophically grown microalgae often grow to higher cance of mitochondrial respiration in the light has been cell densities and produce lipids, polyunsaturated fatty long debated. Studies using inhibitors of mitochondrial acids, carotenoids, tocopherol, pigments and other high- oxidative phosphorylation (Kromer€ and Heldt, 1991) or value bioproducts at higher rates (Kobayashi et al., 1992; mutants defective for respiration (Cardol et al., 2009) have Ogbonna et al., 1998; Jiang et al., 1999; Wen and Chen, demonstrated that mitochondrial activity enhances the effi- 2003; Eriksen, 2008; Perez-Garcia et al., 2011). ciency of photosynthesis, and, in a more recent study In early studies, many Chlamydomonas mutants were (Dang et al., 2014), mitochondrial respiration was shown to identified that were defective for photosynthetic activity be critical for balancing cellular reducing equivalents with (Levine, 1969) and pigment biosynthesis (Wang et al., 1974). ATP production, especially when the availability of reduc- The Chlamydomonas y–1 mutant, first isolated by Sager tant for carbon fixation by ribulose-1,5–bisphosphate car- (1955) and found to be mutated in the gene encoding proto- boxylase (Rubisco) exceeds the rate at which ATP is

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 481–503 Dark hypoxic growth of algae 485 produced by photophosphorylation. Under such condi- dark proton gradient across thylakoid membranes may also tions, ATP may be acquired from mitochondria by traffick- function in controlling non-photochemical quenching upon ing electrons out of chloroplasts through the action of the the onset of light by modulating dark accumulation of xan- dihydroxyacetone 3–phosphate (DHAP)/3–phosphoglycer- thophyll cycle constituents (e.g. zeaxanthin, antheraxanthin ate (3–PGA) shuttle (Kromer,€ 1995; Boschetti and Schmid, and violaxanthin) (Gilmore and Bjorkman,€ 1995; Hoefnagel 1998). DHAP export coupled with its oxidation to 3–PGA and Wiskich, 1998). Finally, efficient photosynthetic electron generates NADH (or NADPH) for respiratory energy pro- flow after transfer of cells from the dark to the light requires duction. The rate of shuttling depends on the rate at which availability of electrons for PSI reduction (e.g. a partially chloroplastic 3–PGA is reduced and cytosolic DHAP is oxi- reduced PQ pool) following rapid photo-oxidation of the dized (Heineke et al., 1991). PSI reaction center chlorophyll special pair (P700 to P700+). The malate/oxaloacetate (OAA) shuttle is also central to The occurrence of a partially reduced PQ pool in the dark is inter-organelle communication. Chloroplast NADP+-depen- attributed to the transfer of reducing equivalents from mito- dent malate dehydrogenase (MDH) is activated in the light, chondria to chloroplasts, but also the degradation of chloro- and, like the DHAP/3–PGA shuttle, the malate/OAA shuttle plastic starch reserves (Bulte et al., 1990; Wieckowski and helps to adjust the cellular NADPH/NADP+ ratio and coordi- Wojtczak, 1997). The reduction state of the PQ pool also nate the availability of reducing equivalents with the syn- involves chlororespiration, a process that requires NAD(P)H thesis of ATP (Anderson and House, 1979; Scheibe, 1987; dehydrogenase (NDA2 in Chlamydomonas), which reduces Weber et al., 1995). When the redox state of chloroplasts is PQ, and plastid terminal oxidase 2, which regenerates oxi- + increased (high NADPH/NADP ), MDH uses NADPH to dized PQ through reduction of O2; this occurs in Chlamydo- reduce OAA to malate, which is then transported from chlo- monas and other photosynthetic organisms (Bennoun, roplasts to mitochondria where it is converted back to OAA 2002; Peltier and Cournac, 2002; Bailey-Serres and Voe- and NADH; the latter may then be re-oxidized through respi- senek, 2008; Jans et al., 2008; Houille-Vernes et al., 2011). ratory activity (Scheibe, 1987). This shuttle also affects the Although it was once thought that chlororespiration was NADPH/ATP ratio, which may be important for optimizing involved in dark ATP production in chloroplasts, there is carbon fixation in the light (Scheibe, 1987). While a malate/ now evidence suggesting that chlororespiration may not be

OAA transporter on the chloroplastic envelope of Chla- electrogenic (i.e. electron transfer from NAD(P)H to O2 is mydomonas has not been identified, the low CO2-inducible not coupled to the translocation of protons across thylakoid protein LCI20 is a candidate for this function (Terashima membranes) (Cournac et al., 2000; Johnson and Alric, et al., 2011; Johnson and Alric, 2013). The malate/OAA shut- 2013). tle may also potentially work in the opposite direction, Oxyhydrogen reaction transporting reducing equivalents from mitochondria to + chloroplasts when the stromal NADPH/NADP ratio is low, Although hydrogenases are sensitive to O2 (Ghirardi et al., or under conditions in which mitochondria are over- 1997), these enzymes are capable of extracting electrons reduced. Hence, this shuttle has an inter-organellar function from H2 in the dark when CO2 is also present in the sur- in the management of cellular reductant/energy demands in rounding atmosphere, under hypoxic conditions (e.g. both the light and the dark. approximately 1% O2); this reaction, called the oxyhydro- In the dark, mitochondrial respiration supplies most of gen reaction (Figure 1) may be observed in both whole the ATP for cell growth, which has also been linked to inter- algal cells (Gaffron, 1939, 1940, 1942a,b; Russell and Gibbs, actions between mitochondria and chloroplasts. For exam- 1968; Maione and Gibbs, 1986) and isolated chloroplasts ple, when oxidative phosphorylation is inhibited in (Chen and Gibbs, 1992). It has also been observed in the mitochondria in the dark, the chloroplastic plastoquinone cyanobacterium Anabaena sp. 7120 (Frenkel et al., 1949; (PQ) pool becomes reduced, and the photosynthetic appa- Houchins and Burris, 1981a,b). ratus transitions from state I (mobile antennae on photo- The oxyhydrogen reaction couples the uptake of H2 and system II) to state II (mobile antennae on photosystem I) O2 with the fixation of CO2 through the Calvin–Benson (Gans and Rebeille, 1990) by a mechanism that involves cycle/reductive pentose phosphate pathway (Gaffron, 1940; phosphorylation of the mobile light-harvesting antenna Badin and Calvin, 1950; Gingras et al., 1963; Russell and (Rochaix, 2007). Mitochondrial export of ATP and reduc- Gibbs, 1968). While it is likely that a significant amount of tants in the dark may also prime the chloroplast for efficient the O2 uptake associated with the oxyhydrogen reaction is photosynthetic activity upon the onset of light by maintain- the result of mitochondrial respiration (Allen and Horwitz, ing a proton gradient across the thylakoid membranes (Joli- 1957; Horwitz, 1957; Erbes and Gibbs, 1981; Chen and ot and Joliot, 1980); this transmembrane gradient is Gibbs, 1992), other electron transfer processes may also thought to be sustained by ATP imported from mitochon- supply the ATP required for CO2 fixation (Gaffron, 1942a,b; dria and hydrolyzed in chloroplasts via the ‘reverse’ activity Maione and Gibbs, 1986). These studies highlight that the of the thylakoid ATP synthase (Joliot and Joliot, 1980). A overall redox conditions of the environment may change

CO2 4 ATP NADH NADPH Calvin-benson e– 3 cycle H2 FNR NDH 1 2 mETC Hydrogenase

FDX 6 7 8 H+ – e CO2 Stroma PFR1 PQ Thylakoid

Lumen Pyruvate NADH PSII Cyt b6f PSI e– 9 5 e– Acetate Acetyl-CoA TCA ATP

cycle ? + + H O2 H H+ Glyoxylate H O 2 cycle

Gluconeogenesis

Figure 1. Possible electron transport during the oxyhydrogen reaction. The cells are grown with a gas mixture that contains oxygen (< 2%), carbon dioxide and hydrogen (approximately 3%). Lines and circles in various colors represent possible pathways occurring in the oxyhydrogen reactions. (1) Hydrogen may be oxidized by hydrogenases, and the electrons from H2 ultimately used to reduce O2 or CO2. Hydrogenases directly reduce ferredoxin (gray). (2) Reduced ferredoxin serves as substrate for (FNR), which forms NADPH (pink). (3) NADPH may be used for carbon ﬁxation (light blue). (4) NADPH may be converted to NADH and used in the mETC to produce ATP (green). (5) The TCA cycle produces NADH, which is also used by the mETC (dark blue). (6) Electrons from NADPH can also reduce the quinone pool, and this electron may be accepted by O2 or other unknown components in the lumen (purple). (7) Electrons can also be donated by FDX to the cytochrome b6f complex (orange). Coupled to proton transfer, this process may facilitate generation of a pH gradient. (8) FDX may reduce PFR1 to form pyruvate, which could be directed to gluconeogenesis (red). (9) Acetate can be converted to acetyl CoA for the glyoxylate cycle to produce organic compounds for gluconeogenesis. NDH, NAD(P)H dehydrogenase. dramatically over the course of the day. These changes, NADH that accumulates during anoxic glycolysis must then plus the availability of various substrates such as H2 and be recycled by alternative mechanisms, which, in the case

O2, and light intensities, may trigger alternative electron of Chlamydomonas, typically involves metabolizing pyru- flow involving both mitochondrial and chloroplastic elec- vate to a variety of reduced, fermentative end-products tron carriers, although many of these pathways are still that are secreted from cells, e.g. formate, acetate, lactate, very poorly defined. The use of alternative pathways for succinate, glycerol, ethanol and H2 (Gfeller and Gibbs, the generation of energy/reductant also highlights the met- 1984, 1985; Mus et al., 2007; Dubini et al., 2009; Philipps abolic flexibility of at least some soil-inhabiting algae, et al., 2011; Catalanotti et al., 2012, 2013; Magneschi et al., organisms that must rapidly respond to dramatic changes 2012; Yang et al., 2014a). in redox conditions with changing temperature, light, O2 In both soil and aquatic environments, Chlamydomonas and nutrient levels. may experience hypoxic/anoxic conditions, especially at

night when photosynthetic O2 evolution ceases and envi- DISRUPTION OF CHLAMYDOMONAS FERMENTATION ronmental O2 levels dramatically decrease because of PATHWAYS respiratory activity. Fermentation metabolism may also occur in the light when cells experience anoxic conditions Anoxia and fermentation (i.e. when respiratory consumption of O2 by the microbial

The energetics of an ecosystem may be markedly affected community exceeds photosynthetic O2 production). Chla- by O2 levels, which continually ﬂuctuate over the course of mydomonas may rapidly acclimatize to anoxia (Gfeller and a day. Algal cells experiencing hypoxic/anoxic conditions Gibbs, 1984, 1985; Kreuzberg, 1984; Gibbs et al., 1986; typically generate energy by substrate-level phosphoryla- Ohta et al., 1987) by activating a variety of metabolic/fer- tion, which requires glycolytic catabolism of ﬁxed carbon mentation pathways (Tsygankov et al., 2002; Hemscheme-

(polysaccharides/sugars). If O2 cannot be used as a termi- ier and Happe, 2005; Atteia et al., 2006; Mus et al., 2007; nal electron acceptor to re-oxidize the NADH generated by Dubini et al., 2009; Timmins et al., 2009) and regulating the the glycolytic degradation of carbohydrates and TCA cycle expression of genes encoding activities integral to those activity, the cells decrease the rate of glycolytic metabo- pathways (Grossman et al., 2007; Merchant et al., 2007). lism, causing decreased rates of ATP production. The Furthermore, aspects of Chlamydomonas fermentation

metabolism appear to be highly ﬂexible based on physio- tion of H2 and CO2 (Gfeller and Gibbs, 1984; Kreuzberg, logical/metabolic studies using wild-type and mutant 1984; Ohta et al., 1987; Mus et al., 2007; Dubini et al., 2009; strains (Gfeller and Gibbs, 1984, 1985; Kreuzberg, 1984; Catalanotti et al., 2012; Magneschi et al., 2012). In the envi- Gibbs et al., 1986; Ohta et al., 1987; Hemschemeier and ronment, these released fermentation products probably Happe, 2005; Atteia et al., 2006; Mus et al., 2007; Dubini supply heterotrophic microbes with organic compounds et al., 2009; Timmins et al., 2009; Grossman et al., 2011; and reductants for growth and development. Philipps et al., 2011; Burgess et al., 2012; Catalanotti et al., Two major pyruvate-metabolizing enzymes of Chla- 2012, 2013; Magneschi et al., 2012; Meuser et al., 2012; mydomonas include the pyruvate formate lyase PFL1 and Yang et al., 2014b), global examination of gene expression the pyruvate:ferredoxin oxidoreductase PFR1 (the latter is as cells acclimatize to anoxic conditions (Mus et al., 2007; sometimes designated PFOR). PFL1 was localized to both Hemschemeier et al., 2013a), and analysis of the Chla- mitochondria and chloroplasts based on measurements of mydomonas genome through homology searches (Gross- activity, proteomic data and immunological analyses man et al., 2007, 2011; Merchant et al., 2007). (Kreuzberg et al., 1987; Atteia et al., 2006; Terashima et al., During dark fermentation, cellular carbohydrate reserves 2010), whereas PFR1 was localized exclusively to chlorop- are metabolized through glycolysis to generate ATP; the lasts (Terashima et al., 2010; van Lis et al., 2013). The PFL1 NADH that is co-produced must be re-oxidized to sustain reaction catalyzes the conversion of pyruvate to acetyl CoA energy production through the glycolytic breakdown of and formate, and this appears to be the dominant enzyme sugars. Pyruvate, the end-product of glycolysis, is a sub- in pyruvate metabolism after Chlamydomonas acclimatizes strate for many Chlamydomonas fermentation pathways to dark anoxia (Mus et al., 2007; Philipps et al., 2011; Catal- (Figure 2). The activities of these circuits are reﬂected by anotti et al., 2012), while in the PFR1 reaction, pyruvate is the secretion of organic acids and alcohols, and the evolu- converted to acetyl CoA, CO2 and reduced FDX. PFR1 was

Starch Glycolysis Feedback regulation of glycolysis ATP NAD + NADH + H+ Pi ADP Phosphoenol PEPC GAP pyruvate Oxaloacetate DHAP CO ADP ATP 2 ADP NADH + H+ NAD(P)H + H+ ADP ATP NAD + CO PYK 2 PYC MDH ATP Glycerol NAD(P)+ α-ketoglutarate Glutamate + CO 2 NAD(P)H + H Alanine FUM ALAAT Pyruvate Malate + MME4 NAD(P)+ NADH + H CoASH NAD+ Fumarate + PDC3 PFR1ox FDXred 2H LDH NADH + H+ PFL1 PFR1 HYDA1/2 Lactate CO2 FMR PFR1 FDX + red ox H2 NAD Formate CO Succinate Acetaldehyde 2 Acetyl-CoA HYDEF/HYDG + NADH + H+ 2NADH + 2H P i PAT2/PAT1 3-hydroxybutyrate ADH1 ADH1 CoASH + NAD Acetyl-P 2NAD+ + NAD CoASH ADP Ethanol ALDH ACK1/ACK2 ATP NADH + H+ Acetate

Figure 2. Fermentative metabolism. Glycolysis (highlighted with a blue background and white lines) degrades photosynthetic hexoses (often from starch) to pyruvate. In wild-type cells, under anoxic conditions, pyruvate can be used as a substrate by several enzymes, including PFL1 and PFR1 to form acetyl CoA, which is the substrate for an acetate-producing pathway catalyzed by PAT1/2 and ACK1/2, highlighted with an orange background, or the ethanol-producing pathway catalyzed by ADH1. Pyruvate can also be used as a substrate to produce ethanol via the PDC3/ADH1 pathway, in which acetaldehyde serves as an inter- mediate. PFR1 is an oxidoreductase that can reduce FDX during the conversion of pyruvate to CO2 and acetyl CoA. This reduced FDX can be used by HYDA1 and HYDA2 to generate H2. The compounds highlighted with a yellow background represent the major external metabolites excreted by anoxic wild-type cells, while the compounds highlighted with a green background represent the metabolites that accumulate (both externally and internally) in various mutant strains under anoxic conditions. The colors used to represent the enzymes indicate the subcellular localizations of the various proteins: purple, dual localization in chloroplasts and mitochondria; blue, chloroplast; red, mitochondria; black, cytosol or unknown. ALAAT, alanine aminotransferase; FMR, fumarate reductase; FUM, fumarase; HYDEF, hydrogenase assembly factor EF; HYDG, hydrogenase assembly factor G; LDH, lactate dehydrogenase; MME4, malic enzyme 4; PEPC, phos- phoenolpyruvate carboxylase; PYC, pyruvate carboxylase; PYK, pyruvate kinase. The black lines and arrows represent pathways occurring in wild-type cells, while the red lines and arrows represent pathways occurring in various mutants. The pink line represents possible feedback regulation of the acetate-producing pathway on glycolysis.

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 481–503 488 Wenqiang Yang et al. shown to efﬁciently interact with both FDX1 and FDX2 (low hydrogenase activity, and consequently does not produce micromolar Km values) (Noth et al., 2013), but not the other H2 under anoxic conditions (Posewitz et al., 2004a). Chlamydomonas FDXs (ferredoxins) (van Lis et al., 2013). Analyses of metabolites synthesized by this mutant

Pyruvate oxidation by PFR1 is coupled to the generation of under anoxic conditions revealed lower levels of CO2, two molecules of reduced FDX, which may be used by the extracellular formate, acetate and ethanol relative to wild- hydrogenases HYDA1 and HYDA2 (Mus et al., 2007; Dubini type cells, but increased carboxylation of pyruvate to et al., 2009; Meuser et al., 2012; Noth et al., 2013) to cata- generate extracellular succinate, which sustains the recy- lyze H2 production. This pathway has been reconstructed cling of NADH (Dubini et al., 2009). Transcript and metab- in vitro using biochemically purified constituents (Chla- olite analyses both strongly suggest that carboxylation of mydomonas HYDA1, FDX1 and PFR1), with robust H2 pro- pyruvate in the hydEF–1 mutant leads to generation of duction being observed in the presence of pyruvate. either malate or OAA, which is subsequently converted Intriguingly, these in vitro reconstitution experiments dem- to succinate by reverse TCA cycle reactions; the succinate onstrated that PFR1 also oxidizes oxaloacetate (Noth et al., is excreted from the cells (Figure 2) (Dubini et al., 2009). 2013), which, if relevant in vivo, would have profound In addition, by studying the hydEF–1 mutant, hydroge- implications regarding the ability of amino acid and lipid nase function was shown to be important for facilitating catabolic pathways (and acetate assimilation to C4) to feed photosynthetic processes under anoxic conditions (Ghy- into H2 production via PFR1 reduction of FDX. Reduced sels et al., 2013). FDX may be re-oxidized by several redox enzymes in addition to hydrogenases, including nitrite and sulfate/sulfite pfl1 mutants. Several independent pfl1 mutants have reductases. PFL1 and PFR1 activities appear to occur simul- been isolated and analyzed (Philipps et al., 2011; Burgess taneously, with both enzymes acting on the same sub- et al., 2012; Catalanotti et al., 2012). Under dark anoxic con- strate (Mus et al., 2007; Atteia et al., 2013; van Lis et al., ditions, the mutants exhibited increases in pyruvate decar- 2013; Noth et al., 2013). This finding suggests the potential boxylation and accumulation of extracellular ethanol and for re-routing fermentative electron flow in Chlamydo- lactate, as well as increased intracellular levels of alanine, monas toward PFR1-dependent production of H2 (poten- succinate, malate and fumarate relative to wild-type cells tially a sustainable, clean fuel). Such a possibility has been (Figure 2) (Philipps et al., 2011; Catalanotti et al., 2012). tested by disrupting specific fermentation pathways (e.g. Dark H2 production in the pfl-1 mutant isolated by Philips eliminating PFL1) to potentially boost the rate of H2 gener- et al. (2011) was either similar to or somewhat higher than ation (see below). The acetyl CoA produced as a conse- the level observed in wild-type cells, while the pfl1 mutants quence of PFL1 and PFR1 activities (Figure 2) is either characterized under the conditions used by Catalanotti et al. reduced to ethanol by the alcohol/acetaldehyde dehydro- (2012) exhibited lower H2 accumulation and in vitro activity genase ADH1 (Hemschemeier and Happe, 2005; Atteia than wild-type cells; these differences may be a conse- et al., 2006; Dubini et al., 2009) or metabolized to acetate quence of differences in the parental strains used to gener- by the phosphate acetyltransferase (PAT) and acetate ate the mutants or in the assay/induction conditions for kinase (ACK) reactions (Atteia et al., 2006; Yang et al., dark anoxic H2 production. Interestingly, increased amounts 2014a,b); these latter reactions occur in both Chlamydo- of 3–hydroxybutyrate were excreted into the medium in monas mitochondria (PAT1 and ACK2) and chloroplasts pfl1–KD1 and pfl1–KD2 knockdown lines, suggesting the (PAT2 and ACK1) (Mus et al., 2007; Grossman et al., 2011; build-up of acetyl CoA, which, as suggested by the authors, Catalanotti et al., 2013; Yang et al., 2014a,b). An alternative may be the consequence of increased b-oxidation of fatty pathway for ethanol production may involve direct decar- acids or inhibition of the TCA cycle and/or the glyoxylate boxylation of pyruvate to CO2 and acetaldehyde through shunt (Burgess et al., 2012). the action of pyruvate decarboxylase (PDC3). The acetaldehyde generated in this reaction may be reduced to ethanol The adh1 mutant. The Chlamydomonas alcohol/acetalde- by alcohol dehydrogenase activity, with a recent study sug- hyde dehydrogenase ADH1 is highly similar to the Escheri- gesting that Chlamydomonas ADH1 is able to generate eth- chia coli AdhE enzyme. Immunoblot analyses showed anol from both acetyl CoA and acetaldehyde (Magneschi similar levels of pyruvate formate lyase, acetate kinase and et al., 2012). While the ADH1 reaction using acetyl CoA as hydrogenase in wild-type cells and the adh1 mutant, and, a substrate oxidizes two NADH molecules, only a single although the mutant appeared to express more PFR1, there

NADH is oxidized in the reaction using acetaldehyde. was no increase in H2 production. Furthermore, although the adh1 mutant was unable to synthesize any ethanol or Mutants affected in fermentation metabolism CO2, it accumulated lower levels of formate and higher lev- The hydEF mutant. The hydEF–1 mutant has been char- els of acetate, lactate and especially glycerol relative to acterized in some detail over the last 10 years (Posewitz wild-type cells, allowing effective re-oxidation of NADH et al., 2004a; Dubini et al., 2009). This mutant has no (Figure 2) (Magneschi et al., 2012).

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 481–503 Dark hypoxic growth of algae 489 sta mutants. Hydrogenase activity was reduced in two chloroplasts contributes more than that in mitochondria to Chlamydomonas mutants that are unable to accumulate the health of cells experiencing hypoxic/anoxic conditions. starch, sta6 (Zabawinski et al., 2001; Chochois et al., 2009) In these mutants, the block in acetate metabolism appears and sta7 (Posewitz et al., 2004b), under dark anaerobic to occur too far down the central metabolic pathway to conditions, and HYDA1 and HYDA2 transcript levels were readily allow re-direction of metabolites to other pathways, decreased in these strains (Posewitz et al., 2004b). This while the inability to sustain acetate and ATP production indicates that signals other than simply the lack of O2 slows down glycolytic metabolism (Figures 2 and 3) (Yang (potentially cellular redox status) are involved in activating et al., 2014b). Furthermore, acetate may be synthesized HYDA transcription. In contrast, under conditions of sulfur under anoxic conditions even when both the chloroplastic starvation in the light, the sta6 mutant has hydrogenase and mitochondrial PAT/ACK pathways are disrupted, sug- activity similar to that of wild-type cells (Chochois et al., gesting that the cells have other metabolic routes for gen- 2009). In addition, analysis of the sta6 mutant showed that erating acetate, as discussed below. starch breakdown contributes to H2 production via dona- tion of electrons to the PQ pool, and contribution of elec- ACETATE METABOLISM/FERMENTATION trons from the oxidation of H O by photosystem II also 2 General aspects occurs (Chochois et al., 2009). Acetate may be used as the sole energy source for growth

The stm6 mutant. Disruption of the gene encoding a of Chlamydomonas when O2 is used as the terminal elec- homolog of the human mitochondrial transcription termi- tron acceptor. Upon uptake (Figure 3), acetate is converted nation factor state transition mutant (STM6) in Chlamydo- to acetyl CoA via one of two pathways, both of which con- monas led to various phenotypes including inhibition of sume ATP. One pathway involves direct conversion of ace- CEF under anaerobic conditions (eliminating competition tate to acetyl CoA by acetyl CoA synthetase (ACS), while between the hydrogenase and PSI-dependent CEF), the second requires a two-step reaction catalyzed by ACK increased starch accumulation (providing additional reduc- and PAT, acting in the reverse direction to that of acetate tant for PQ reduction), a decrease in the number of active production during fermentation. Acetyl CoA then enters photosystem II reaction centers, an increased rate of respi- the metabolic networks of the cell through the glyoxylate ration, and an elevated rate of sustained H2 photoproduc- cycle, combining with glyoxylate (to form malate) or with tion during sulfur deprivation relative to wild-type cells OAA (to form citrate); the output for one ‘turn’ of the cycle (Kruse et al., 2005a,b; Rupprecht, 2009). Additional genetic is a molecule of succinate. modiﬁcations generated in the stm6 genetic background Under anoxic/hypoxic conditions, photophosphorylation have also been examined. When this mutant is trans- appears to be necessary for sustained acetate assimilation formed with a gene encoding a glucose transporter, the (Wiessner, 1965; Gibbs et al., 1986). The presence of ace- resulting stm6 Glc4 strain exhibits increased glucose tate also helps to maintain cells in an anoxic state in the uptake and improved H2 photoproduction (Doebbe et al., light and under certain conditions of nutrient deprivation

2007). Furthermore, the stm6 Glc4 strain, which has a because it promotes rapid catabolic consumption of O2 reduced antenna size, exhibits an additional increase in the (Kosourov and Seibert, 2009; Morsy, 2011). This has been level of H2 production (Doebbe et al., 2010). This mutant demonstrated for sta mutants, in which acetate putatively has a complex pleiotropic phenotype that may be the supports high respiratory rates, which show more rapid result of multiple primary and secondary defects. anaerobiosis and H2 generation (Chochois et al., 2009); a similar result was obtained for immobilized wild-type cells The 2–on–2 hemoglobin mutant. Twelve hemoglobin ho- (Kosourov and Seibert, 2009). Acetate is also a building mologs are encoded on the Chlamydomonas genome. A block used for storage of reduced carbon in the form of 2–on–2 hemoglobin, designated THB8, was shown to be triacylglycerides (Johnson and Alric, 2013). Finally, when required for normal hypoxic growth and expression of Chlamydomonas experiences anoxic/hypoxic conditions, it genes controlled by anoxia (Hemschemeier et al., 2013b). produces acetate (which is excreted) through the PAT/ACK

This mutant is discussed further below with respect to O2 pathway. This pathway recycles CoASH from acetyl CoA, sensing. and, at the same time, generates ATP (one molecule per acetate generated), which contributes to cell maintenance pat/ack mutants. The PAT/ACK pathway promotes cellular (Mus et al., 2007; Tielens et al., 2010; Atteia et al., 2013; ﬁtness during dark, anoxic acclimation, coupling the pro- Yang et al., 2014a,b). duction of acetate to ATP synthesis (Atteia et al., 2006; Chlamydomonas has two parallel PAT/ACK pathways Yang et al., 2014b). Characterization of ack and pat involving four proteins: PAT1, PAT2, ACK1 and ACK2. Sev- mutants (three single mutants and two double mutants) in eral reports have shown that PAT1/ACK2 are localized to Chlamydomonas showed that the PAT/ACK pathway in mitochondria while PAT2/ACK1 are located in chloroplasts

Acetate Acetate Cytosol

YaaH Acetate AMT

AMT Acetate ATP ATP ADP Acetate ? Acetate PPi

ACK AMP-ACS Acetyl-CoA Acetyl- Acetyl- phosphate AMP AMP PAT AMP-ACS AMP-ACS CoASH CoASH CoASH Pi Acetate O2 Acetyl- Acetyl-CoA AMP AMP Pi –O2 CoASH Acetate PPi AMP-ACS PAT Acetyl- ASCT ATP ACT1 phosphate /SCL CGLD2 Acetate Acetate ACK

YaaH ADP ALDH ATP Acetate Acetaldehyde

? NADH NAD+ Acetate Mito or chloro Acetate

Figure 3. Acetate metabolism under dark oxic and dark anoxic conditions. Various potential routes for acetate metabolism in Chlamydomonas are presented. The outer black rectangle represents the plasma membrane, while the inner yellow rectangle represents the chloroplastic or mitochondrial membranes; acetate metabolism occurs within these organelles. The pink line is used to separate oxic (top) and anoxic (bottom) conditions inside mitochondria and chloroplasts. Double bars in various colors represent putative acetate transporters (the different colors were used to indicate that there may be different transporter types) localized on the plasma membrane, chloroplastic and mitochondrial membranes. The enzymes shown in red are encoded by high-conﬁdence gene models present in the Chlamydomonas genome, while the enzymes shown in gray represent gene models for which the function is not absolutely clear. Solid lines represent conﬁrmed Chlamydomonas reactions, while dashed lines indicate proposed reactions based on gene model analyses and homology searches using Phytozome 9.0 (http://www.phytozome.net/). ACT1, acyl CoA thioesterase; CGLD2, acyl CoA thioesterase; AMT, ammonium transporters; YaaH, members of the GPR1/FUN34/YaaH family (putative acetate transporters).

(Atteia et al., 2006, 2009; Terashima et al., 2010; Yang complicated than expected. The ack1 and pat2 strains et al., 2014b). PAT/ACK activities of Chlamydomonas typi- exhibited a more pronounced decrease in acetate secretion cally constitute the dominant pathways for acetate synthe- under dark anoxic conditions compared with the ack2 sis under dark anoxic conditions. The activities of these strain (Yang et al., 2014b), suggesting a dominant role for pathways and the accumulation of acetate in cells experi- the chloroplast in acetate production. Among the chloro- encing anoxia may be affected by altering various plastic enzyme mutants, the pat2–1 mutant consistently branches of fermentation metabolism through generation produced less acetate than the ack1 mutant, as expected if of mutants. For example, accumulation of extracellular ace- non-enzymatic hydrolysis of acetyl-P produced by PAT tate during anoxia was diminished in pfl1 mutants (Bur- contributes to the observed levels of secreted acetate in gess et al., 2012; Catalanotti et al., 2012) and by treating the mutant strains. Two double mutants, ack1 ack2 and anoxic cultures with a PFL inhibitor (Philipps et al., 2011). pat2–1 ack2, were also generated; both chloroplastic and However, acetate production increased in the adh1 single mitochondrial PAT/ACK pathways are blocked in each of and pfl1–1 adh1 double mutants (Catalanotti et al., 2012; these strains. Increases in lactate production were Magneschi et al., 2012). In the hydEF–1 mutant, acetate observed in the pat2–1 and pat2–1 ack2 mutants, suggest- production was reduced to half of that of wild-type cells as ing differences in regulation of fermentation metabolism in much of the pyruvate was no longer converted to ace- the pat2 genetic backgrounds; re-routing of metabolites tyl CoA but was carboxylated and then reduced to succi- was always observed in the pat2–1 genetic background. nate by reverse TCA reactions (Dubini et al., 2009). This may be expected as the metabolic block in the chloroplast in pat2–1 strains occurs at the level of acetyl CoA, Fermentative pathways in ack and pat mutants contributing to increased pyruvate accumulation. This Insertional mutants of Chlamydomonas disrupted for pyruvate may be readily re-directed toward lactate produc- genes encoding the chloroplastic and mitochondrial ace- tion for redox balancing, as observed in the adh1 and pfl1– tate kinases ACK1 and ACK2 and the chloroplastic phos- 1 mutants (Figure 3) (Catalanotti et al., 2012; Magneschi phate acetyltransferase PAT2 were recently isolated and et al., 2012). Double mutants that have neither the chloro- characterized (Yang et al., 2014b), revealing that fermenta- plastic nor mitochondrial PAT/ACK acetate-producing path- tive acetate metabolism in Chlamydomonas was more ways (ack1 ack2 and pat2–1 ack2 double mutants) still

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 481–503 Dark hypoxic growth of algae 491 accumulated acetate in the medium during exposure to significant changes in the levels of any transcript encoding anoxic conditions, albeit at lower levels than in wild-type this enzyme in any of the mutants following exposure to cells (approximately 50% relative to wild-type cells). These dark anoxic conditions; however, this activity has been results suggest that routes other than the PAT/ACK path- reported to be under post-translational control (Takasaki way function in acetate generation in these mutants (Yang et al., 2004). The AMP-ACS pathway is functionally equiva- et al., 2014b). Interestingly, acetate production is also lent to the PAT/ACK pathway in that ATP production is retained through undetermined activities in mutants of retained, and decreased glycolysis and acetate excretion Clostridium species lacking ACK activity (Sillers et al., are not expected if this compensatory mechanism is trig- 2008; Kuit et al., 2012). There are a number of potential gered in pat ack mutants. Finally, acetate:succinate CoA routes that may account for acetate production in the Chla- transferase (ASCT) and succinyl CoA ligase (SCL) (van mydomonas mutants blocked in both the chloroplastic and Grinsven et al., 2008; Millerioux et al., 2012) may be mitochondrial PAT/ACK pathways. First, there may be involved in acetate accumulation. ASCT transfers the CoA spontaneous hydrolysis of acetyl-P to acetate and Pi (Kosh- moiety of acetyl CoA to succinate, and SCL converts succi- land, 1952; Di Sabato and Jencks, 1961). This reaction nyl CoA back to succinate. Although SCL homologs are denies the cell the ATP that is generated by the action of encoded on the Chlamydomonas genome, no ASCT homo- ACK, which may potentially result in a diminished rate of logs have been identified, and it is therefore unlikely that glycolysis and concomitant reduction of secretion of fer- this pathway represents a viable alternative for acetate pro- mentation metabolites (Figure 3). Second, acetyl CoA duction in Chlamydomonas (Atteia et al., 2013). hydrolase activity (Tielens et al., 2010) may release acetate PAT and ACK activities are key enzymes of acetate-pro- and CoASH from acetyl CoA without ATP production. ducing pathways in Chlamydomonas during hypoxia/ Genes encoding homologs of acetyl CoA hydrolase are anoxia. Mutants defective for the genes encoding these present on the Chlamydomonas genome. This activity may enzymes exhibit a reduction in the rate of glycolysis be important when acetyl CoA accumulates in cells and is (Yang et al., 2014b). Current data do not allow unambigu- not rapidly metabolized by alternative. Third, aldehyde ous conclusions regarding the origins of acetate produc- dehydrogenase (ALDH) activity may oxidize acetaldehyde tion in the double mutants. However, the data do (from pyruvate decarboxylation) to acetate (Kirch et al., suggest that acetate is formed without production of 2004, 2005; Brocker et al., 2013). In Chlamydomonas, pyru- ATP, as the rates of accumulation of all fermentation vate may be decarboxylated by PDC3 to generate acetalde- products are attenuated. Two favored hypotheses consis- hyde, which may then be oxidized to acetate by ALDH tent with the lack of ATP production during acetate syn- activity, similar to the reaction used by some yeast (Remize thesis include the possibility that acetyl-P is hydrolyzed et al., 2000). However, this reaction involves formation of non-enzymatically in aqueous medium to acetate and NADH, and accumulation of NADH would reduce the rate phosphate, as acetyl-P is not easily re-directed to other of glycolysis unless the cells were able to rapidly re-oxidize metabolic pathways, or that acetyl CoA is hydrolyzed to it by production and excretion of a reduced organic com- acetate and CoASH, which may be catalyzed by acyl/ace- pound. The excretion of metabolites that serve as this tyl CoA hydrolases; other pathways for acetate produc- ‘reductant sink’ was not observed, even though some tion may also exist (Yang et al., 2014b). ALDH transcripts (e.g. ALDH3) show significant accumula- Acetate transport and assimilation tion in the various pat ack mutant strains (Yang et al., 2014b). However, no significant ALDH activity was detected AcpA, a member of the GPR1/FUN34/YaaH membrane pro- among mutant and wild-type strains at various times after tein family, is essential for acetate permease activity in the imposition of dark anoxic conditions despite transcript hyphal fungus Aspergillus nidulans (Robellet et al., 2008). increases (Yang et al., 2014b). Fourth, acetate may be gen- In Saccharomyces cerevisiae, the ortholog of AcpA is Ady2 erated by acetyl CoA synthetases functioning in the (Paiva et al., 2004). Based on homology to AcpA, five reverse direction. Many organisms are able to catalyze this genes encoding putative members of GPR1/FUN34/YaaH reaction using ADP-forming acetyl CoA synthetase (ADP- family were identified on the Chlamydomonas genome ACS) (Tielens et al., 2010), and a few reports have even (Table 2 and Figure 3). The level of transcripts for two of suggested that acetate production may also be achieved these putative acetate transporters increased during dark by AMP-forming acetyl CoA synthetases (AMP-ACS) (Taka- to light transition (Duanmu et al., 2013). Ady2 was shown saki et al., 2004; Yoshii et al., 2009). However, AMP-ACS to be important for the periodic ammonium export from enzymes typically function exclusively in the direction of S. cerevisiae colonies observed during late development acetyl CoA synthesis (Tielens et al., 2010). No gene model (Palkova et al., 2002), indicating a potential relationship for an ADP-ACS was identified on the Chlamydomonas between acetate and ammonium uptake/metabolism; these genome. If AMP-ACS were used for acetate production, relationships have not been explored in algae. Active ace- ATP production would be retained, but we did not observe tate transport requires ATP, which has been used to

Table 2 Enzymes potentially involved in acetate assimilation

Name Phytozome9.0 ID NCBI number Annotation Localization predication TM domain number

ACK1 Cre09.g396700 XP_001694505 Acetate kinase C 0 ACK2 Cre17.g709850 XP_001691682 Acetate kinase M 0 PAT1 Cre09.g396650 XP_001691787 Phosphate acetyltransferase C 0 PAT2 Cre17.g699000 XP_001694504 Phosphate acetyltransferase M 0 ACS1 g1290.t1 XP_001700210 Acetyl CoA synthetase O 0 ACS2 g1224.t1 XP_001700230 Acetyl CoA synthetase C 0 ACS3 Cre07.g353450 XP_001702039 Acetyl CoA synthetase O 0 ACS4 Cre01.g055500 XP_001700230 Acetyl CoA synthetase M or SP 0 ALDH1 g13400.t1 XP_001694180 Aldehyde dehydrogenase O 0 ALDH2 Cre16.g675650 XP_001695943 Aldehyde dehydrogenase M 0 ALDH3 Cre12.g500150 XP_001690955 Aldehyde dehydrogenase M 0 ALDH4 Cre12.g520350 XP_001696928 Aldehyde dehydrogenase M 0 ALDH5 Cre01.g033350 XP_001690075 Aldehyde dehydrogenase SP 0 ALDH6 G8982.t1 XP_001694332 Aldehyde dehydrogenase M 0 ALDH7 g16809 XP_001698924 Aldehyde dehydrogenase C 0 ALDH8 Cre13.g605650 XP_001699134 Aldehyde dehydrogenase O or SP 0 SCLA1 Cre03.g193850 XP_001693108 Succinate CoA ligase M 0 SCLB1a g17060.t1 XP_001691581 Succinate CoA ligase M 0 ACT1 g16435.t1 XP_001692073 Acyl CoA thioesterase O 0 CGLD2 g837.t1 XP_001690113 Acyl CoA thioesterase M 0 YaaH-1 Cre17.g700750 XP_001691606 GPR1/FUN34/yaaH family Membrane 6 YaaH-2 Cre17.g702900 XP_001691586.1 GPR1/FUN34/yaaH family Membrane 6 yaaH-3 Cre17.g702950 XP_001691752 GPR1/FUN34/yaaH family Membrane 6 yaaH-4 Cre17.g700450 XP_001691608 GPR1/FUN34/yaaH family Membrane 6 yaaH-5 Cre17.g700650 XP_001691772 GPR1/FUN34/yaaH family Membrane 6 AMT1.1 Cre03.g159254 AF479643 Ammonium transporter Membrane 11 AMT1.2 Cre06.g293051 AF530051 Ammonium transporter Membrane 11 AMT1.3 Cre14.g629920 AF509497 Ammonium transporter Membrane 10 AMT1.4 Cre13.g569850 AY542491 Ammonium transporter Membrane 11 AMT1.5 Cre09.g400750 AY542492 Ammonium transporter Membrane 10 AMT1.6 Cre07.g355650 AY548756 Ammonium transporter Membrane 11 AMT1.7a Cre02.g111050 AY588244 Ammonium transporter Membrane 11 AMT1.7b Cre02.g111050 AY548755 Ammonium transporter Membrane 11 AMT1.8 Cre12.g531000 AY548754 Ammonium transporter Membrane 10

The localization of ACS, ALDH, ammonium transporters (Fernandez and Galvan, 2007), the GPR1/FUN34/yaaH family of acetate transporters and other possible acetate metabolism-related proteins were predicted using PredAlgo software (http://omictools.com/predalgo-s8353.html). C, chloroplast; M, mitochondrion; SP, secretory pathway; O, other. The transmembrane (TM) domain number was predicted using the TMHMM server v2.0 (http://www.cbs.dtu.dk/services/TMHMM/). ACS3 (Atteia et al., 2009; Terashima et al., 2010), ALD1/ALD2 (also named ALDH3/ALDH8, Yang et al., 2014b) and SCLA1/SCLB1a are all localized to mitochondria (Atteia et al., 2009). explain the low rates of Chlamydomonas acetate uptake or anoxic, the pyruvate may be converted to metabolites during anoxia in the dark (Gibbs et al., 1986). In the light, that serve as electron acceptors, allowing re-oxidization of

CEF triggered by low O2 levels (Alric, 2010, 2014) maintains NADH formed as a consequence of glycolysis. These path- cyclic photophosphorylation (ATP production) under hyp- ways for recycling electron carriers under hypoxic/anoxic oxic/anoxic conditions (Klob et al., 1973; Alric, 2014), and conditions (fermentation metabolism) may occur in differ- the occurrence of cyclic photophosphorylation during ent cellular compartments. For some eukaryotic organ- anoxia helps sustain acetate assimilation in Chlamydo- isms, including the protistan parasites such as Giardia and monas mundana (Russell and Gibbs, 1968) and other Entamoeba species (Muller€ et al., 2012), fermentation green algae (Wiessner, 1965), including Chlamydomonas occurs entirely in the cytosol. Fermentation may also occur reinhardtii (Gibbs et al., 1986). partly in hydrogenosomes, as is the case for Trichomonas vaginalis (Muller,€ 1993). In algae, end-products of glycoly- SUBCELLULAR LOCALIZATION AND sis may be metabolized in the cytosol, chloroplasts and COMPARTMENTATION OF METABOLIC PATHWAYS mitochondria. Furthermore, a number of metabolic reac- As discussed above, glycolysis is a conduit for eukaryotic tions may occur in more than one cellular compartment, carbon and energy metabolism, leading to production of with some enzymes routed to multiple locations or differ- pyruvate, ATP and NADH. When the cells become hypoxic ent isoforms of the enzyme targeted to different compart-

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 481–503 Dark hypoxic growth of algae 493 ments. For example, PFL1 is localized to both chloroplasts have fermentation pathways evolved in the algae? How and mitochondria in Chlamydomonas; dual localization of diverse are they among algae? How are they tailored to dif- proteins is not uncommon in eukaryotes (Atteia et al., ferent environments? How are they regulated? 2006; Martin, 2010; Muller€ et al., 2012). Furthermore, Regulation of genes encoding fermentative enzymes enzymes associated with glycolysis, the oxidative pentose phosphate pathway and gluconeogenesis are located in Transcripts encoding many enzymes involved in fermenta- both the cytosol and chloroplasts. While the glycolytic and tion accumulate in Chlamydomonas during anoxia (Mus oxidative pentose phosphate pathways in plants are local- et al., 2007; Hemschemeier et al., 2013a) but others do not. ized in both the cytosol and chloroplasts (Plaxton, 1996; Expression of genes encoding the fermentative proteins Joyard et al., 2010), the glycolytic enzymes appear to be PDC3, lactate dehydrogenase and ADH2 was shown to be differentially partitioned in Chlamydomonas; enzymes that primarily controlled by diurnal rhythms (Whitney et al., catalyze the formation of glyceraldehyde 3–phosphate 2011), while transcripts of genes encoding other proteins from glucose are located in chloroplasts, while enzymes such as PFR1 and HYDA1 exhibit marked accumulation at that transform 3–phosphoglycerate to pyruvate are located the onset of anoxia (Mus et al., 2007). Furthermore, while in the cytosol (Ball, 1998; Johnson and Alric, 2013). Fur- the level of PFR1 transcript increases at the onset of anoxia thermore, while enzymes involved in acetate assimilation (Mus et al., 2007), anoxia elicits an increase in PFL1 mRNA and production are located in both mitochondria and chlo- levels, with no increase in PFL1 protein levels (Atteia et al., roplasts, some Chlamydomonas TCA cycle enzymes are 2006; Philipps et al., 2011; Catalanotti et al., 2012). These localized to both mitochondria and to microbodies that results suggest that differences in the regulation of fermen- may represent glyoxysomes (Hayashi and Shinozaki, 2012; tation genes occur at both the transcriptional and transla- Johnson and Alric, 2013); those present in the microbodies tional levels. Interestingly, the molecular mass of PFL1 probably participate in the glyoxylate cycle. Also, most cel- from anoxic cells was shown to be slightly less than the lular compartments require mechanisms for redox balanc- molecular mass of the protein from cells maintained under ing and ATP synthesis, and some enzymes associated with oxic conditions, suggesting that anoxic conditions trigger a these activities may be shared among compartments, e.g. post-translational modification that may activate the the transhydrogenase is present in both mitochondria and enzyme (Atteia et al., 2006; Catalanotti et al., 2012). chloroplasts (Atteia et al., 2009; Terashima et al., 2011). Several other factors also affect the transcriptional activ- These considerations raise the fundamental question of ity of fermentation genes. The rate of starch degradation what events lead to the transfer of partial or entire meta- under anoxic conditions modulates intracellular NAD(P)H bolic pathways to new compartments. This issue is far levels and/or the oxidation state of the PQ pool, both of from resolved, and we are still uncertain of the localization which elicit changes in transcriptional activity of numerous of many proteins in the cell. Furthermore, it is becoming genes (Escoubas et al., 1995; Rutter et al., 2001; Pfannsch- evident that, over evolutionary time, enzymes and path- midt and Liere, 2005), while the production and detoxifica- ways readily undergo re-compartmentation to the mito- tion of reactive oxygen species are probably also chondria, cytosol, hydrogenosomes or chloroplasts. In important for controlling hypoxic responses (Antal et al., some cases, gene duplications may lead to specialization 2003; Bailey-Serres and Chang, 2005; Guzy and Schumack- of the duplicated proteins, with each of the two paralogous er, 2006). For example, hydrogen peroxide (H2O2) synthe- proteins targeted to a specific compartment and tailored sized by a NADPH oxidase is required for induction of ADH for function therein. Small changes in targeting sequences in Arabidopsis (Baxter-Burrell et al., 2002). resulting in mis-targeting may explain how individual Although few molecular studies have been performed to activities and even entire pathways become resident in elucidate the regulatory features controlling the genes and more than one cellular compartment (Martin, 2010). In proteins responsive to hypoxia/anoxia, exciting details con- Chlamydomonas, the confirmed localization of PAT2 and cerning this regulation are beginning to emerge. A 21– ACK1 in chloroplasts and PAT1 and ACK2 in mitochondria, 128 bp region upstream of the HYDA1 gene transcription and the phenotypic consequences of lesions in genes start site was initially shown to be involved in controlling encoding each of these components, are helping to estab- the expression of HYDA1 (Stirnberg and Happe, 2004). lish the functional importance of subcellular localization Reporter gene analysis and electrophoretic mobility shift and isoform specialization (Yang et al., 2014b). assays demonstrated that CRR1, the copper-responsive regulatory factor (Sommer et al., 2010), plays a role in CONTROL OF FERMENTATION METABOLISM HYDA1 transcriptional control through its squamosa pro- While progress has been made in defining algal fermenta- moter-binding protein domain (Pape et al., 2012). Two con- tion pathways and elucidating their effect on cellular sensus CRR1-binding GTAC motifs are present in the metabolism, there are still numerous questions associated HYDA1 promoter, and are necessary for full promoter with fermentation and anoxic/hypoxic metabolism. How activity under hypoxic conditions; CRR1 binds to one of

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 481–503 494 Wenqiang Yang et al. these GTAC cores in vitro (Pape et al., 2012). The same half-life of the protein. In Arabidopsis, hypoxia-responsive GTAC motifs are present in the promoter of FDX5, which is transcription factors are targeted for N–end degradation; also regulated by CRR1 in response to copper levels and substrates for the pathway include ethylene response fac-

O2 conditions (Lambertz et al., 2010). CRR1 plays an impor- tor group VII transcription factors, which are susceptible tant role in regulating several genes encoding key proteins through a motif at their N-terminus, starting with Met-Cys. (e.g. HYDA1 and PRF1) that are involved in dark, hypoxic In some plants, the regulators are not susceptible to this metabolism in Chlamydomonas, and, in particular, inﬂu- degradation, and such plants are generally more tolerant ences a subset of proteins that are also regulated under to hypoxic conditions. In rice, the dominant regulator of conditions of copper deﬁciency (Hemschemeier et al., hypoxia, SUB1A–1, is not a substrate for the N–end rule 2013a). The importance of CRR1 in hypoxic metabolism is degradation pathway (Gibbs et al., 2011; Licausi, 2011; Sa- underscored by the observation that crr1 mutants exhibit a sidharan and Mustroph, 2011). severe growth attenuation phenotype during hypoxia in In E. coli, there are two pathways that function in O2 the light (Hemschemeier et al., 2013a). However, additional sensing. One pathway is through Fnr (Bunn and Poyton, hypoxia/anoxia regulatory strategies must exist that are 1996), a global regulator of a large number of E. coli genes independent of CRR1, as the majority of the mRNAs that that acts as either a transcriptional activator or repressor differentially accumulate after dark, hypoxic acclimation (Spiro and Guest, 1991). The second pathway involves the appear to be relatively insensitive to CRR1. For example, ArcA/ArcB two-component regulators (Bunn and Poyton, approximately 1400 transcripts differentially accumulated 1996). The response regulator ArcA may be phosphory- after acclimation of Chlamydomonas cells to dark, hypoxic lated by the sensor protein ArcB (Iuchi and Lin, 1992); the conditions, but only approximately 40 of these were aber- latter is a histidine kinase that undergoes autophosphoryla- rantly regulated in crr1 mutants under the same conditions tion under anoxic conditions (Kato et al., 1997). This phos-

(Hemschemeier et al., 2013a). Moreover, HYDA1 transcript phorylation cascade promotes acclimation to low O2 accumulation is still observed in the crr1 mutant in conditions by activating or repressing speciﬁc genes. In response to anoxia, albeit at attenuated levels (Pape et al., Rhizobium meliloti, FixL and FixJ are two-component regu-

2012; Hemschemeier et al., 2013a). Overall, these data indi- lators that mediate the bacterium’s response to O2 condi- cate that CRR1 has an important role in regulating the tran- tions; these have been extensively studied with respect to script levels of a subset of hypoxia-/anoxia-responsive induction of nitrogen fixation genes under anaerobic con- genes, but additional regulatory factors that have yet to be ditions. The FixL protein is an O2 sensor (membrane pro- identified must also play a significant role in transcriptional tein) that behaves like ArcB in E. coli, phosphorylating the responses to O2 availability. response regulator FixJ (Monson et al., 1992). Phosphory- lated FixJ activates nifA and fixK (Gilles-Gonzalez et al., O sensing/regulation in various organisms 2 1994), which encode two regulatory elements. NifA is

In animals, prolyl 4–hydroxylases directly sense O2 and are involved in expression of genes encoding subunits of involved in controlling responses to anoxia (Guzy and Schu- nitrogenase (and factors required to synthesize active macker, 2006). A constitutively expressed hypoxia-inducing nitrogenase), while FixK controls expression of genes factor is hydroxylated on conserved proline residues in the required for microaerobic growth (Dixon and Kahn, 2004). presence of O2. This modification targets the hypoxia-induc- Yeast has more complicated O2 sensing regulatory mecha- ing factor for ubiquitin-dependent degradation. In the nisms, involving the regulatory elements Hap1–5p, Mot3p, absence of O2, hydroxylation of the hypoxia-inducing factor Rox1p, Upc2p and Ecm22p (Kwast et al., 1998; Poyton, ceases, and the protein accumulates and triggers expression 1999; Hughes et al., 2005; Davies and Rine, 2006; Todd of several target genes. Prolyl 4–hydroxylases may have et al., 2006; Hughes and Espenshade, 2008; Grahl and Cra- other protein targets that accumulate under anoxic condi- mer, 2010); a detailed description of the intricacies of this tions (not necessarily transcription/regulatory factors) and system is beyond the scope of this review. are rapidly degraded as cells transition from anoxic to oxic O sensing/regulation in Chlamydomonas conditions (Semenza, 2011). In Arabidopsis and rice (Oryza 2 sativa), the levels of prolyl 4–hydroxylase transcripts are In Chlamydomonas,noO2 sensing regulatory factors anal- strongly induced by O2 deprivation (Lasanthi-Kudahettige ogous to mammalian hypoxia-inducing factors have been et al., 2007; Vlad et al., 2007), raising the possibility that their identified. However, several of the 22 prolyl 4–hydroxylas- role as sensing elements may be conserved in plants. es encoded on the Chlamydomonas genome are signifi- There are also clear examples of the involvement of pro- cantly up-regulated in response to anoxia (Mus et al., 2007; tein degradation in responses to anoxia in plants. The Hemschemeier et al., 2013a), and a subset of these are reg- N–end rule reflects an evolutionarily conserved mechanism ulated to a degree by CRR1 (Hemschemeier et al., 2013a). for eliciting protein degradation, whereby the N-terminal Although still highly speculative, determination of the amino acid is an important factor in determining the activity of one or more of these prolyl 4–hydroxylases in

Chlamydomonas may provide insight into how this alga fermentative processes in photosynthetic organisms. The senses O2, maintains genes in an inactive state when O2 is use of multiple mechanisms may enable metabolic versa- present, and targets key proteins involved in fermentation tility and fine tuning of the responses to dynamic environ- metabolism for destruction as algal cells transition from mental conditions. Full clarification of the regulatory anoxic to oxic conditions. However, many (or all) of these pathways, especially for Chlamydomonas, requires signifi- hydroxylases may not function in regulating hypoxic/ cantly more work in order to elucidate modes of sensing anoxic responses, but instead may modify the cell-wall an oxic/anoxic environment, and the diversity of transcrip- structure through proline hydroxylation; Chlamydomonas tional and post-transcriptional processes responsible for has a proteinaceous, hydroxyproline-rich cell wall. eliciting acclimation responses. Murthy et al. (2012) recently used Chlamydomonas gen- Other metabolic strategies to cope with hypoxia/anoxia ome inspection to identify nine proteins with homology to the O2 sensing, Per-Arnt-Sim (PAS)-heme domains present Many photosynthetic organisms have evolved a set of in the FixL proteins of rhizobia (Murthy et al., 2012). Tran- pathways, some of which generate a modest amount of script levels for most of these proteins increase during energy, that function during exposure to anoxic conditions anoxia (Mus et al., 2007; Hemschemeier et al., 2013a), and (Muller€ et al., 2012; Catalanotti et al., 2013). In addition, the PAS domains of two FixL-like proteins (FXL1 and FXL5) microbes and plants have also evolved a set of specific were heterologously expressed in E. coli and shown to strategies that they use to cope with hypoxia/anoxia. Dur- bind heme and O2 at physiologically relevant concentra- ing starch breakdown, amylase levels increase in some tions. Although the FXL proteins, which are large proteins species to satisfy the increased carbon demand under hyp- (>1000 amino acids) with multiple predicted transmem- oxic/anoxic conditions (Weigelt et al., 2009). In some spe- brane domains, are candidate O2 sensors, experiments cies, to increase energy use efficiency, sucrose directly linking them to physiological roles in O2 sensing degradation shifts from invertase to sucrose synthase to and signal transduction have yet to be reported. form UDP-glucose, which uses pyrophosphate as the sub- As mentioned above, it has also been demonstrated that strate to synthesize UTP/ATP; this shift helps to increase a2–on–2 hemoglobin designated THB8 has a critical role net ATP production (Zeng et al., 1999). Pyruvate and gluta- in the Chlamydomonas anaerobic response (Hemscheme- mate may be converted to alanine and 2–oxoglutarate by ier et al., 2013b). Silencing of the THB8 gene causes both a the alanine/2-oxoglutarate shunt, which prevents the loss growth defect under anoxic conditions in the light and of carbon through fermentation pathways and yields ATP mis-regulation of several genes that respond to hypoxic through substrate level phosphorylation (Araujo et al., conditions, including HYDA2 and CYG2 (encoding an aden- 2012). Additionally, glutamic acid decarboxylase uses pro- ylate/guanylate cyclase). The growth defect is exacerbated tons as its substrate and may help stabilize cytosolic pH by an NO scavenger, suggesting that the hypoxic/anoxic via the c–aminobutyric acid (GABA) shunt (Miyashita and responses in Chlamydomonas are at least partially con- Good, 2008; Bailey-Serres et al., 2012), while a reduction in trolled by both the 2–on–2 hemoglobin and an NO-depen- the respiratory rate results from down-regulation of net dent signaling pathway (Hemschemeier et al., 2013b). A NADH production via the TCA cycle, reduced mETC activity role for nitric oxide in O2 sensing has also been reported and/or triggering of mechanisms associated with O2 con- for pea (Pisum sativum) (Borisjuk et al., 2007), and may servation (Chang et al., 2012). also be involved in the degradation of photosynthetic pro- Many plants produce ethanol as well as lactate during teins in N-deprived Chlamydomonas cells (Wei et al., hypoxia. The regulation of these pathways appears to be 2014). However, it is still not clear whether the THB8 pro- under pH control; ethanol production appears to be critical tein is part of the sensing mechanism, and more work is for the maintenance of cytosolic pH, as supported by data required to determine whether the other 2–on–2 hemoglo- demonstrating that a decrease in cytosolic pH of approxi- bins in Chlamydomonas function in anoxic/hypoxic accli- mately 0.6 units favors ethanol production (Roberts et al., mation and/or sensing of O2 levels. 1989; Bailey-Serres and Voesenek, 2008; Catalanotti et al., Finally, there are also some studies showing that the 2013). A less common fermentation process largely occur- acclimation of plants to anoxic conditions may involve eth- ring in marine environments involves cytosolic opine for- ylene response factor transcriptional elements (Bailey-Ser- mation. In this redox reaction, a pyruvate–amino acid res and Voesenek, 2010; Gibbs et al., 2011; Licausi, 2011 ). condensation regenerates NAD+. Possible advantages of Chlamydomonas has putative ethylene response factor this reaction are redox balancing, cytosolic pH control, and transcription factors (Merchant et al., 2007), but none have maintenance of osmotic equilibrium (Ballantyne, 2004). the cysteine at the N–terminus that has been associated Increased de-nitrification may also occur when cells experi- with O2 sensing in plants. ence anoxia. In fungi and other eukaryotic organisms, Together, these results suggest that there are a number there are two de-nitrification pathways; one is typically of different factors and mechanisms involved in regulating localized to mitochondria and usually occurs under low O2

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 481–503 496 Wenqiang Yang et al. conditions, while the other, often referred to as ammonia (in the cytosol), the TCA cycle (in the mitochondrion) and fermentation, is localized in the cytosol (Takasaki et al., chloroplast metabolism, may also contribute to cellular 2004) and is activated under strict anoxic conditions. The redox conditions, although, in the absence of net acetate latter pathway involves reduction of nitrate to ammonium assimilation (cells maintained in minimal medium), the using reductant generated by the catabolic oxidation of production of NAD(P)H by MDH in the dark is negligible. ethanol (the donor of electrons) and concomitant acetate Redox regulation synthesis, coupled to substrate-level phosphorylation (Zhou et al., 2002). Nitrate respiration has been reported in The redox conditions of photosynthetic organisms have diatoms as a mechanism to survive dark, anoxic conditions profound effects on their physiological and metabolic pro-

(Kamp et al., 2011). Finally, the generation of H2 in algal cesses. Changes in activities of catalytic processes as well chloroplasts may serve as a redox valve, although H2 pro- as the organization of macromolecular complexes in mem- duction may also occur in mitochondria-like organelles in branes may accompany redox changes associated with the the stramenopiles and in hydrogenosomes in the amoebo- dark and hypoxic/anoxic and high-light conditions. Anoxia zoa (Muller€ et al., 2012 and Catalanotti et al., 2013). creates a more reduced stromal redox poise, which has been shown to enhance CEF measured in the presence of Mitochondrial respiration and chlororespiration 3–(3,4-dichlorophenyl)-1,1–dimethylurea (DCMU) in wild- Inhibition of mitochondrial respiration appears to have at type Chlamydomonas cells. This CEF enhancement was least two major metabolic consequences. First, the flow of not observed in a pgrl1 mutant (Tolleter et al., 2011). The electrons to O2 is blocked, leading to NADH accumulation association of PGRL1 with the PSI–light-harvesting com- in the mitochondrion and cytosol, which probably results plex I supercomplex, which is involved in CEF, was favored in inhibition of the TCA cycle and glycolysis (e.g. at the in Chlamydomonas cells maintained under anoxic condi- level of the pyruvate dehydrogenase complex). Second, tions in the light (Iwai et al., 2010; Takahashi et al., 2013). depletion of ATP during dark maintenance may promote In the moss Physcomitrella patens, quantitative proteomics the glycolytic breakdown of starch/sugars, as ATP is an demonstrated severe down-regulation of the photosystems allosteric inhibitor of hexokinase and phosphofructokinase but up-regulation of the chloroplastic NADH dehydroge- (Klock and Kreuzberg, 1991); when mitochondria are per- nase complex, plastocyanin, and Ca2+ sensors in the pgrl1 forming aerobic respiration, the substrate of phosphofruc- mutant, indicating that, in the absence of PGRL1, a set of tokinase, fructose-6–phosphate, is more abundant than its metabolic reactions may be elicited to compensate for product, fructose-1,6–bisphosphate, while this equilibrium decreased CEF under anoxic light conditions (Kukuczka is reversed under anaerobic conditions when NAD(P)H is et al., 2014). Furthermore, Ca2+ sensor (CAS) and Anaero- not readily recycled (Klock and Kreuzberg, 1991). The con- bic Response 1 (ANR1) proteins showed increased abun- sequences of the simultaneous slowing of respiratory NAD dance under anoxic conditions, associate with each other (P)H oxidation and stimulation of upstream glycolytic steps and with PGRL1, and all become part of a large active PSI– has an additive effect resulting in an elevated cellular cytochrome b6f complex performing CEF (Terashima et al., redox state. Furthermore, inhibition of both mitochondrial 2012). Furthermore, pgrl1 knockdown lines exhibited respiration and chlororespiration leads to reduction of hypersensitivity to iron deficiency, linking Fe limitation to chloroplastic electron carriers, including the PQ pool; the formation/remodeling of the supercomplex associated either of these respiratory processes appears to be suffi- with CEF (Petroutsos et al., 2009). It was also shown that cient to re-oxidize most NAD(P)H produced by glycolysis conformational changes in the PGRL1 protein are linked to (Alric, 2010, 2014). In addition, another enzyme that is the cellular redox state (Johnson et al., 2014). Phenotypic likely to have a major effect on NAD(P)H accumulation in comparative analyses have demonstrated that PGRL1 is the cytosol and chloroplasts during anoxic growth is the crucial for acclimation of Chlamydomonas cells to high glycolytic enzyme glyceraldehyde phosphate dehydroge- light and anoxia; analyses of the double mutant pgrl1 npq4 nase. Under dark aerobic conditions, a downstream prod- (where the gene disrupted in npq4 encodes LHCSR3) con- uct of this reaction, 3–phosphoglycerate, is more abundant firmed a complementary role of PGRL1 and LHCSR3 in than glyceraldehyde-3-phosphate (the substrate of glycer- managing excess absorbed excitation energy (Kukuczka aldehyde phosphate dehydrogenase), suggesting rapid et al., 2014). In addition, both proteins are required for oxidation of NADPH in the presence of O2. This equilibrium photoprotection and for survival of the cells under low O2 is reversed under anaerobic conditions (Klock and Kreuz- (Kukuczka et al., 2014). The integrated interactions between berg, 1991), when cellular NAD(P)H levels increase. redox, high light and anoxia are still being decoded; how- Another reaction that is likely to affect cellular redox to ever, it is becoming clear that the overlapping features of some extent is catalyzed by glucose-6–phosphate dehydro- these conditions elicit overlapping regulatory processes genase and inhibited by NADPH (Lendzian and Bassham, and use of at least some shared regulatory elements to tai- 1975). MDH, which is associated with the glyoxylate cycle lor the activities of the metabolic machinery to cellular con-

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 481–503 Dark hypoxic growth of algae 497 ditions. There are several other energetic and redox con- into compensatory responses that allow sustained ATP siderations that distinguish light from dark growth in pho- production while eliminating reducing equivalents tosynthetic organisms. NADPH plays a critical role in through generation of reduced carbon compounds that driving anabolic processes, including the synthesis of lip- are excreted from cells. Initial characterizations of Chla- ids, amino acids and nucleotides, and is directly produced mydomonas have demonstrated that this alga has flexi- by the activity of FDX:NADP+ oxidoreductase. During dark ble, mixed-acid fermentation pathways, with features metabolism, many reactions including those of the TCA common to bacterial-, plant- and yeast-type fermentation. cycle and glycolysis generate NADH; the oxidative pentose Most enzymes for fermentative metabolism in the algae, phosphate pathway produces NADPH. Interconversion inferred from genomic and metabolic studies, have not between NADH and NADPH may be achieved by the pyri- been biochemically characterized. Expression patterns of dine nucleotide transhydrogenase (Agledal et al., 2010; genes encoding these enzymes, the biochemical proper- Holm et al., 2010). This enzyme regulates the NAD(H)/ ties of these enzymes (including potential interactions NADP(H) ratio through a reversible hydride transfer that with each other), and the diversity of fermentation path- occurs in either an energy-dependent or energy-indepen- ways plus the extent to which they are used under vari- dent manner (Olausson et al., 1992; Pedersen et al., 2008); ous conditions, require further examination in a broader the NAD(H)/NADP(H) ratio helps to control the extent to spectrum of algal systems. Additionally, the diversity of which the cells perform catabolic and anabolic processes. external and internal end-products accumulated by vari- Some bacteria, including E. coli, rely heavily on pyridine ous algae during fermentation is still mostly unknown. nucleotide transhydrogenase activity to modulate metabo- Such information is critical for developing a clear under- lism (Sauer et al., 2004; Fuhrer and Sauer, 2009). standing of metabolic diversity both within and among Other electron carriers, such as the FDXs and thioredox- the various algal groups, and the ways in which fermenta- ins, are small redox carriers that supply electrons to a range tion pathways have been shaped by environmental condi- of cellular processes, as previously discussed. Furthermore, tions. Furthermore, there are many technologies, while some of the FDX proteins may be efficiently reduced including flux balance analysis, mass flux analysis, time- by NADH or NADPH, FDXs with a very negative redox resolved fluorescence measurements and the use of O2 potential may only be able to acquire electrons through PSI, microsensors that may help to evaluate the redox condi- which suggests tailoring of redox components in the light tions of cells and correlate those conditions with the activ- and the dark. As mentioned above, we isolated a mutant of ities of both oxic and anoxic metabolisms. An Chlamydomonas that does not grow in the dark (but does understanding of the various pathways critical for dark grow in the light) and is null for FDX5 (W. Yang, unpub- metabolism and the ways in which these pathways are lished results). This result supports the concept that the controlled constitutes a domain of metabolism that must FDX family in Chlamydomonas represents a group of pro- be fully described if we are to understand the energy bud- teins with a specialized function as electron carriers, but get of photosynthetic microbes in the environment and their functions may only be possible in the light (when PSI potential ways to manipulate carbon cycling. Finally, fer- through FDX1 supplies much of the reductant) or dark mentation metabolism in algae appears to represent a sig- (where NADH supplies most of the reductant). More infor- nificant ecological component of carbon flux in soils (and mation is required with respect to the redox potential of the sediments) that strongly affects its content of organic various FDXs and the affinities with which they interact with acids, alcohols and H2, which in turn affects the biotic their specific target proteins. Other redox carriers such as composition of the ecosystem. thioredoxins may also be critical for ‘dark’ metabolism. ACKNOWLEDGEMENTS PERSPECTIVES The work performed in our laboratories and described here was Chlamydomonas is a metabolically versatile organism that supported by grants from the US the Department of Energy (num- bers DE-FG02-12ER16338 and DE-FG02-12ER16339). Aspects of the performs photosynthetic CO fixation, aerobic respiration 2 work were also funded by US National Science Foundation grants and anaerobic fermentation. This alga is a model for to A.R.G. (MCB0824469 and MCB-0951094). examining many aspects of photosynthetic metabolism, and has been the subject of numerous metabolic studies. REFERENCES

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