A Metabolomic Approach to Study Major Metabolite Changes during Acclimation to Limiting CO2 in Chlamydomonas reinhardtii

Using a gas chromatography-mass spectrometry-time of flight technique, we determined major metabolite changes during induction of the carbon-concentrating mechanism in the unicellular green alga Chlamydomonas reinhardtii. In total, 128 metabolites with significant differences between high- and low-CO₂-grown cells were detected, of which 82 were wholly or partially identified, including amino acids, lipids, and carbohydrates. In a 24-h time course experiment, we show that the amino acids serine and phenylalanine increase transiently while aspartate and glutamate decrease after transfer to low CO₂. The biggest differences were typically observed 3 h after transfer to low-CO₂ conditions. Therefore, we made a careful metabolomic examination at the 3-h time point, comparing low-CO₂ treatment to high-CO₂ control. Five metabolites involved in photorespiration, 11 amino acids, and one lipid were increased, while six amino acids and, interestingly, 21 lipids were significantly lower. Our conclusion is that the metabolic pattern during early induction of the carbon-concentrating mechanism fit a model where photorespiration is increasing.Most microalgae express a carbon-concentrating mechanism (CCM; Raven et al., 2005). The green alga Chlamydomonas reinhardtii is one of the species with a CCM (Giordano et al., 2005). The CCM in C. reinhardtii is typically induced when the concentration of CO₂ in the air bubbled through the cultures is decreased to around 0.5% or lower (Vance and Spalding, 2005). Within the first few hours after starting the induction, numerous genes are either up- or down-regulated (Miura et al., 2004; Yamano et al., 2008; Yamano and Fukuzawa, 2009). However, the change in gene expression is only manifested as rather limited detectable changes in the abundance of proteins (Manuel and Moroney, 1988; Spalding and Jeffrey, 1989). Even though the response to decreased concentrations of inorganic carbon (C_i) is fast (Eriksson et al., 1998), the algal cells go through a transient phase before the CCM is fully operational.Many genes coding for enzymes of the photorespiratory pathway are up-regulated (Marek and Spalding, 1991; Miura et al., 2002, 2004) within 20 min and show a transient expression pattern (Tural and Moroney, 2005; Yamano et al., 2008). A decline in starch content is also detectable within 30 min after transfer to low CO₂ (Kuchitsu et al., 1988). This decline in starch is followed, after about 2 h, by a net synthesis of starch (Thyssen et al., 2001) that is mainly deposited around the pyrenoid, rather than as the starch grains distributed in the stroma normally found in high-CO₂-grown cells. When the CCM is fully induced, the alga can concentrate C_i inside the cell/chloroplast against a free-energy gradient. Accumulation of C_i increases the CO₂-oxygen ratio at the site of Rubisco (Giordano et al., 2005), with a corresponding increase in photosynthesis, a decrease in photorespiration, and a greater capacity for net organic carbon production at low external C_i (Giordano et al., 2003).Induction of the CCM is thus known to affect genes of many different pathways, and especially the photorespiratory pathway has been studied extensively (Moroney et al., 1986; Tural and Moroney, 2005). The aim of this work was to screen for metabolic changes in order to find key metabolites that could trigger the expression of genes that regulate the CCM. We have extended the analyses by using metabolomics to detect changes in major metabolites, particularly in the beginning of the induction period but also in preliminary experiments, over a 24-h time period. The resulting metabolic changes have enabled us to propose a working model for the coordinated regulation of cellular metabolism during the induction of the CCM in C. reinhardtii.     

Responses of Arabidopsis and Wheat to Rising CO2 Depend on Nitrogen Source and Nighttime CO2 Levels

A major contributor to the global carbon cycle is plant respiration. Elevated atmospheric CO₂ concentrations may either accelerate or decelerate plant respiration for reasons that have been uncertain. We recently established that elevated CO₂ during the daytime decreases plant mitochondrial respiration in the light and protein concentration because CO₂ slows the daytime conversion of nitrate (NO₃−) into protein. This derives in part from the inhibitory effect of CO₂ on photorespiration and the dependence of shoot NO₃− assimilation on photorespiration. Elevated CO₂ also inhibits the translocation of nitrite into the chloroplast, a response that influences shoot NO₃− assimilation during both day and night. Here, we exposed Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum) plants to daytime or nighttime elevated CO₂ and supplied them with NO₃− or ammonium as a sole nitrogen (N) source. Six independent measures (plant biomass, shoot NO₃−, shoot organic N, ¹⁵N isotope fractionation, ¹⁵NO₃⁻ assimilation, and the ratio of shoot CO₂ evolution to O₂ consumption) indicated that elevated CO₂ at night slowed NO₃− assimilation and thus decreased dark respiration in the plants reliant on NO₃−. These results provide a straightforward explanation for the diverse responses of plants to elevated CO₂ at night and suggest that soil N source will have an increasing influence on the capacity of plants to mitigate human greenhouse gas emissions.The CO₂ concentration in Earth’s atmosphere has increased from about 270 to 400 µmol mol^–1 since 1800, and may double before the end of the century (Intergovernmental Panel on Climate Change, 2013). Plant responses to such increases are highly variable, but plant nitrogen (N) concentrations generally decline under elevated CO₂ (Cotrufo et al., 1998; Long et al., 2004). One explanation for this decline is that CO₂ inhibits nitrate (NO₃−) assimilation into protein in the shoots of C₃ plants during the daytime (Bloom et al., 2002, 2010, 2012, 2014; Cheng et al., 2012; Pleijel and Uddling, 2012; Myers et al., 2014; Easlon et al., 2015; Pleijel and Högy, 2015). This derives in part from the inhibitory effect of CO₂ on photorespiration (Foyer et al., 2009) and the dependence of shoot NO₃− assimilation on photorespiration (Rachmilevitch et al., 2004; Bloom, 2015).A key factor in global carbon budgets is plant respiration at night (Amthor, 1991; Farrar and Williams, 1991; Drake et al., 1999; Leakey et al., 2009). Nighttime elevated CO₂ may inhibit, have a negligible effect on, or stimulate dark respiration, depending on the plant species (Bunce, 2001, 2003; Wang and Curtis, 2002), plant development stage (Wang et al., 2001; Li et al., 2013), experimental approach (Griffin et al., 1999; Baker et al., 2000; Hamilton et al., 2001; Bruhn et al., 2002; Jahnke and Krewitt, 2002; Bunce, 2004), and total N supply (Markelz et al., 2014). The current study is, to our knowledge, the first to examine the influence of N source, NO₃− versus ammonium (NH₄+), on plant dark respiration at elevated CO₂ during the night.Plant organic N compounds account for less than 5% of the total dry weight of a plant, but conversion of NO₃− into organic N expends about 25% of the total energy in shoots (Bloom et al., 1989) and roots (Bloom et al., 1992). During the day, photorespiration supplies a portion of the energy (Rachmilevitch et al., 2004; Foyer et al., 2009), but at night, this energetic cost is borne entirely by the respiration of C substrates (Amthor, 1995) and may divert a substantial amount of reductant from the mitochondrial electron transport chain (Cousins and Bloom, 2004). The relative importance of NO₃− assimilation at night versus the day, however, is still a matter of intense debate (Nunes-Nesi et al., 2010). Here, we estimated NO₃− assimilation using several independent methods and show in Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum), two diverse C₃ plants, that NO₃− assimilation at night can be substantial, and that elevated CO₂ at night inhibits this process.     

Elevated Temperature and CO2 Stimulate Late-Season Photosynthesis But Impair Cold Hardening in Pine

Rising global temperature and CO₂ levels may sustain late-season net photosynthesis of evergreen conifers but could also impair the development of cold hardiness. Our study investigated how elevated temperature, and the combination of elevated temperature with elevated CO₂, affected photosynthetic rates, leaf carbohydrates, freezing tolerance, and proteins involved in photosynthesis and cold hardening in Eastern white pine (Pinus strobus). We designed an experiment where control seedlings were acclimated to long photoperiod (day/night 14/10 h), warm temperature (22°C/15°C), and either ambient (400 μL L⁻¹) or elevated (800 μmol mol⁻¹) CO₂, and then shifted seedlings to growth conditions with short photoperiod (8/16 h) and low temperature/ambient CO₂ (LTAC), elevated temperature/ambient CO₂ (ETAC), or elevated temperature/elevated CO₂ (ETEC). Exposure to LTAC induced down-regulation of photosynthesis, development of sustained nonphotochemical quenching, accumulation of soluble carbohydrates, expression of a 16-kD dehydrin absent under long photoperiod, and increased freezing tolerance. In ETAC seedlings, photosynthesis was not down-regulated, while accumulation of soluble carbohydrates, dehydrin expression, and freezing tolerance were impaired. ETEC seedlings revealed increased photosynthesis and improved water use efficiency but impaired dehydrin expression and freezing tolerance similar to ETAC seedlings. Sixteen-kilodalton dehydrin expression strongly correlated with increases in freezing tolerance, suggesting its involvement in the development of cold hardiness in P. strobus. Our findings suggest that exposure to elevated temperature and CO₂ during autumn can delay down-regulation of photosynthesis and stimulate late-season net photosynthesis in P. strobus seedlings. However, this comes at the cost of impaired freezing tolerance. Elevated temperature and CO₂ also impaired freezing tolerance. However, unless the frequency and timing of extreme low-temperature events changes, this is unlikely to increase risk of freezing damage in P. strobus seedlings.Land surface temperature is increasing, particularly in the northern hemisphere (IPCC, 2014), which is dominated by boreal and temperate forests. At higher latitudes, trees rely on temperature and photoperiod cues to detect changing seasons and to trigger cessation of growth and cold hardening during the autumn (Ensminger et al., 2015). For boreal and temperate evergreen conifers, cold hardening involves changes in carbohydrate metabolism, down-regulation of photosynthesis, accumulation of cryoprotective metabolites, and development of freezing tolerance (Crosatti et al., 2013; Ensminger et al., 2015). These processes minimize freezing damage and enable conifers to endure winter stresses. However, rising temperatures result in asynchronous phasing of temperature and photoperiod characterized by delayed arrival of first frosts (McMahon et al., 2010), which may impact the onset and development of cold hardening during autumn.Short photoperiod induces the cessation of growth in many tree species (Downs and Borthwick, 1956; Heide, 1974; Repo et al., 2000; Böhlenius et al., 2006). As a consequence, carbon demand in sink tissue decreases toward the end of the growing season, and the bulk of photoassimilate is translocated from source tissues to storage tissues (Hansen and Beck, 1994; Oleksyn et al., 2000). In addition, cryoprotective soluble sugars, including sucrose, raffinose, and pinitol, accumulate in leaf tissues to enhance freezing tolerance (Strimbeck et al., 2008; Angelcheva et al., 2014). Thus, by winter, leaf nonstructural carbohydrates are mainly comprised of mono- and oligosaccharides, and only minimal levels of starch remain (Hansen and Beck, 1994; Strimbeck et al., 2008). The concurrent decrease of photoassimilate and demand for metabolites that occur during the cessation of growth also impacts the citric acid cycle that mediates between photosynthesis, respiration, and protein synthesis. The citric acid cycle generates NADH to fuel ATP synthesis via mitochondrial electron transport, as well as amino acid precursors (Shi et al., 2015). In C3 plants, the enzyme phosphoenolpyruvate carboxylase (PEPC) converts phosphoenolpyruvate to oxaloacetic acid in order to supplement the flow of metabolites to the citric acid cycle and thus controls the regulation of respiration and photosynthate partitioning (O’Leary et al., 2011).Cessation of growth, low temperature, and presumably short photoperiod decrease the metabolic sink for photoassimilates, resulting in harmful excess light energy (Öquist and Huner, 2003; Ensminger et al., 2006) and increased generation of reactive oxygen species (Adams et al., 2004). During autumn and the development of cold hardiness, conifers reconfigure the photosynthetic apparatus in order to avoid formation of excess light and reactive oxygen species. This involves a decrease in chlorophylls and PSII reaction center core protein D1 (Ottander et al., 1995; Ensminger et al., 2004; Verhoeven et al., 2009), as well as aggregation of light-harvesting complex proteins (Ottander et al., 1995; Busch et al., 2007). Additionally, photoprotective carotenoid pigments accumulate in leaves, especially the xanthophylls, zeaxanthin, and lutein that contribute to nonphotochemical quenching (NPQ) via thermal dissipation of excess light energy (Busch et al., 2007; Verhoeven et al., 2009; Demmig-Adams et al., 2012). Prolonged exposure to low temperature induces sustained nonphotochemical quenching (NPQ_S), where zeaxanthin constitutively dissipates excess light energy (Ensminger et al., 2004; Demmig-Adams et al., 2012; Fréchette et al., 2015).In conifers, freezing tolerance is initiated during early autumn in response to decreasing photoperiod (Rostad et al., 2006; Chang et al., 2015) and continues to develop through late autumn in response to the combination of short photoperiod and low temperature (Strimbeck and Schaberg, 2009; Chang et al., 2015). In addition to changes in carbohydrate content, freezing tolerance also involves the expression of specific dehydrins (Close, 1997; Kjellsen et al., 2013). Members of the dehydrin protein family are involved in responses to osmotic, salt, and freezing stress (Close, 1996). Dehydrins have been associated with improved freezing tolerance in many species including spinach (Kaye et al., 1998), strawberry (Houde et al., 2004), cucumber (Yin et al., 2006), peach (Wisniewski et al., 1999), birch (Puhakainen et al., 2004), and spruce (Kjellsen et al., 2013). In angiosperms, a characteristic Lys-rich dehydrin motif known as the K-segment interacts with lipids to facilitate membrane binding (Koag et al., 2003; Eriksson et al., 2011). Several in vitro studies have demonstrated dehydrin functions including prevention of aggregation and unfolding of enzymes (using Vitis riparia; Hughes and Graether, 2011), radical scavenging (using Citrus unshiu; Hara et al., 2004), and suppression of ice crystal formation (using Prunus persica; Wisniewski et al., 1999). To date, dehydrin functions have not been demonstrated in planta.Rising temperatures since the mid-twentieth century have delayed the onset of autumn dormancy and increased length of the growing season in forests across the northern hemisphere (Boisvenue and Running, 2006; Piao et al., 2007; McMahon et al., 2010). Studies have shown that elevated temperatures ranging from +4°C to +20°C above ambient can delay down-regulation of photosynthesis in several evergreen conifers. Consistent findings were apparent among climate-controlled chamber studies exposing Pinus strobus seedlings to a sudden shift in temperature and/or photoperiod (Fréchette et al., 2016), as well as chamber studies exposing Picea abies seedlings to simulated autumn conditions using a gradient of decreasing temperature and photoperiod (Stinziano et al., 2015). Similar findings were also demonstrated in open-top chamber experiments exposing mature Pinus sylvestris to a gradient of decreasing temperature and natural photoperiod (Wang, 1996). Elevated temperature (+4°C above ambient) also impaired cold hardening in Pseudotsuga menziesii seedlings (Guak et al., 1998) and mature P. sylvestris (Repo et al., 1996) exposed to a decreasing gradient of temperature and natural photoperiod using open-top chambers. In contrast, a recent study showed that smaller temperature increments (+1.5°C to +3°C) applied using infrared heaters did not delay down-regulation of photosynthesis or impair freezing tolerance in field-grown P. strobus seedlings that were acclimated to larger diurnal and seasonal temperature variations (Chang et al., 2015). For many tree species, photoperiod determines cessation of growth (Tanino et al., 2010; Petterle et al., 2013), length of the growing season (Bauerle et al., 2012), and development of cold hardiness (Welling et al., 1997; Li et al., 2003; Rostad et al., 2006). However, the effects of climate warming on tree phenology are complex and can be unpredictable due to species- and provenance-specific differences in sensitivity to photoperiod and temperature cues (Körner and Basler, 2010; Basler and Körner, 2012; Basler and Körner, 2014).The effect of elevated CO₂ further increases uncertainties in the response of trees to warmer climate. Similar to warmer temperature, elevated CO₂ may also delay the down-regulation of photosynthesis in evergreens and extend the length of the growing season, as demonstrated in mature P. sylvestris (Wang, 1996). Elevated CO₂ increases carbon assimilation (Curtis and Wang, 1998; Ainsworth and Long, 2005) and biomass production (Ainsworth and Long, 2005) during the growing season. The effects could continue during the autumn if dormancy or growth cessation is delayed, which suggests that elevated CO₂ may increase annual carbon uptake. However, long-term exposure to elevated CO₂ can also down-regulate photosynthesis during the growing season (Ainsworth and Long, 2005). Prior studies that have attempted to determine the impact of a combination of elevated CO₂ and/or temperature on cold hardening in evergreens have largely focused on freezing tolerance, with contrasting results. Open-top chamber experiments showed that a combination of elevated temperature and CO₂ both delayed and impaired freezing tolerance of P. menziesii seedlings (Guak et al., 1998) and evergreen broadleaf Eucalyptus pauciflora seedlings (Loveys et al., 2006) but did not affect freezing tolerance of mature P. sylvestris (Repo et al., 1996). A recent field experiment examining mature trees revealed that Larix decidua, but not Pinus mugo, exhibited enhanced freezing damage following six years of exposure to combined soil warming and elevated CO₂ (Rixen et al., 2012). In contrast, a climate-controlled study showed that exposure to elevated CO₂ advanced the date of bud set and improved freezing tolerance in Picea mariana seedlings (Bigras and Bertrand, 2006). In a second study on similar seedlings conducted by the same authors, exposure of trees to elevated CO₂ also enhanced freezing tolerance but impaired the accumulation of sucrose and raffinose (Bertrand and Bigras, 2006). These previous experiments used experimental conditions where temperature and photoperiod gradually decreased. While this approach aims to mimic natural conditions, it is difficult to distinguish specific responses to either photoperiod or temperature. Because of the contrasting findings from previous studies, we designed an experiment aiming to separate the effects of photoperiod, temperature, and CO₂ on a wide range of parameters that are involved in cold hardening in conifers.Our study aimed to determine (1) how induction and development of the cold hardening process is affected by a shift from long to short photoperiod under warm conditions and (2) how the combination of warm air temperature and elevated CO₂ affects photoperiod-induced cold hardening processes in Eastern white pine (P. strobus). To assess the development of cold hardening, we measured photosynthetic rates, changes in leaf carbohydrates, freezing tolerance, and proteins involved in photosynthesis and cold hardening over 36 d. Assuming that both low temperature and short photoperiod cues are required to induce cold hardening in conifers, we hypothesized that warm temperature and the combination of warm temperature and elevated CO₂ would prevent seedlings growing under autumn photoperiod from down-regulating photosynthesis. We further hypothesized that warm temperature and the combination of warm temperature and elevated CO₂ would impair the development of freezing tolerance, due to a lack of adequate phasing of the low temperature and short photoperiod signals.     

Effect of Rubisco Activase Deficiency on the Temperature Response of CO2 Assimilation Rate and Rubisco Activation State: Insights from Transgenic Tobacco with Reduced Amounts of Rubisco Activase

The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. To elucidate its role in maintaining CO₂ assimilation rate at high temperature, we examined the temperature response of CO₂ assimilation rate at 380 μL L⁻¹ CO₂ concentration (A₃₈₀) and Rubisco activation state in wild-type and transgenic tobacco (Nicotiana tabacum) with reduced Rubisco activase content grown at either 20°C or 30°C. Analyses of gas exchange and chlorophyll fluorescence showed that in the wild type, A₃₈₀ was limited by ribulose 1,5-bisphosphate regeneration at lower temperatures, whereas at higher temperatures, A₃₈₀ was limited by ribulose 1,5-bisphosphate carboxylation irrespective of growth temperatures. Growth temperature induced modest differences in Rubisco activation state that declined with measuring temperature, from mean values of 76% at 15°C to 63% at 40°C in wild-type plants. At measuring temperatures of 25°C and below, an 80% reduction in Rubisco activase content was required before Rubisco activation state was decreased. Above 35°C, Rubisco activation state decreased slightly with more modest decreases in Rubisco activase content, but the extent of the reductions in Rubisco activation state were small, such that a 55% reduction in Rubisco activase content did not alter the temperature sensitivity of Rubisco activation and had no effect on in vivo catalytic turnover rates of Rubisco. There was a strong correlation between Rubisco activase content and Rubisco activation state once Rubisco activase content was less that 20% of wild type at all measuring temperatures. We conclude that reduction in Rubisco activase content does not lead to an increase in the temperature sensitivity of Rubisco activation state in tobacco.The catalytic sites of Rubisco must be activated for CO₂ fixation to take place. This requires the carbamylation of a Lys residue at the catalytic sites to allow the binding of Mg²⁺ and ribulose 1,5-bisphosphate (RuBP; Andrews and Lorimer, 1987). Rubisco activase facilitates carbamylation and the maintenance of Rubisco activity by removing inhibitors such as tight-binding sugar phosphates from Rubisco catalytic sites in an ATP-dependent manner (Andrews, 1996; Spreitzer and Salvucci, 2002; Portis, 2003; Parry et al., 2008). The activity of Rubisco activase is regulated by the ATP/ADP ratio and redox state in the chloroplast (Zhang and Portis, 1999; Zhang et al., 2002; Portis, 2003).In many plant species, Rubisco activation state decreases at high temperature in vivo (Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004b; Cen and Sage, 2005; Yamori et al., 2006b; Makino and Sage, 2007). However, it is unclear what the primary mechanisms underlying the inhibition of Rubisco activation are and whether Rubisco deactivation limits CO₂ assimilation rate at high temperature. It has been proposed that Rubisco activation state decreases at high temperature, because the activity of Rubisco activase is insufficient to keep pace with the faster rates of Rubisco inactivation at high temperatures (Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004a, 2004c; Kim and Portis, 2006). In in vitro assays using purified Rubisco and Rubisco activase, the activity of Rubisco activase was sufficient for the activation of Rubisco at the optimum temperature but not at high temperatures (Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004a, 2004c). ATP hydrolysis activity of Rubisco activase in vitro has varying temperature optima among species (e.g. 25°C in Antarctic hairgrass [Deschampsia antarctica] and spinach [Spinacia oleracea] but 35°C in tobacco [Nicotiana tabacum] and cotton [Gossypium hirsutum]), and Rubisco activase more readily dissociates into inactive forms at high temperature, causing a loss of Rubisco activase capacity (Crafts-Brandner and Law, 2000; Salvucci and Crafts-Brandner, 2004b). Moreover, the rates of inhibitor formation by misprotonation of RuBP during catalysis increased at higher temperatures (Salvucci and Crafts-Brandner, 2004c; Kim and Portis, 2006). CO₂ assimilation rates and plant growth were improved under heat stress in transgenic Arabidopsis expressing thermotolerant Rubisco activase isoforms generated by either gene-shuffling technology (Kurek et al., 2007) or chimeric Rubisco activase constructs (Kumar et al., 2009). These results support the view that the reduction of Rubisco activase activity limits the Rubisco activation and, therefore, the CO₂ assimilation rates at high temperatures.It has also been suggested that the decrease in CO₂ assimilation rate at high temperatures is caused by a limitation of RuBP regeneration capacity (e.g. electron transport capacity) rather than by Rubisco deactivation per se (Schrader et al., 2004; Wise et al., 2004; Cen and Sage, 2005; Makino and Sage, 2007; Kubien and Sage, 2008). These groups suggest that Rubisco deactivation at high temperature may be a regulatory response to the limitation of one of the processes contributing to electron transport capacities. For example, at high temperature, protons can leak through the thylakoid membrane, impairing the coupling of ATP synthesis to electron transport (Pastenes and Horton, 1996; Bukhov et al., 1999, 2000). As the electron transport capacity becomes limiting, ATP/ADP ratios and the redox potential of the chloroplast decline, causing a loss of Rubisco activase activity and, in turn, a reduction in the Rubisco activation state (Zhang and Portis, 1999; Zhang et al., 2002; Sage and Kubien, 2007). Based on this understanding, the decline in the Rubisco activation state at high temperature may be a regulated response to a limitation in electron transport capacity rather than a consequence of a direct effect of heat on the integrity of Rubisco activase.Temperature dependence of CO₂ assimilation rate shows a considerable variation with growth temperature (Berry and Björkman, 1980; Hikosaka et al., 2006; Sage and Kubien, 2007). Plants grown at low temperature generally exhibit higher CO₂ assimilation rates at low temperatures compared with plants grown at high temperature, but they exhibit lower rates at high temperature. Furthermore, both the temperature response of Rubisco activation state and the limiting step of CO₂ assimilation rate (a Rubisco versus RuBP regeneration limitation) have been shown to differ depending on growth temperature (Hikosaka et al., 1999; Onoda et al., 2005; Yamori et al., 2005, 2006a, 2006b, 2008). This suggests that the regulation of Rubisco activation state could also differ in plants grown at different growth temperatures. Here, we analyzed the effects of Rubisco activase content on Rubisco activation state and CO₂ assimilation rate at leaf temperatures ranging from 15°C to 40°C in tobacco grown under two different temperature regimes (day/night temperatures of 20°C/15°C or 30°C/25°C). We used wild-type and transgenic tobacco with a range of reductions in Rubisco activase content to examine the dependence of Rubisco activation on Rubisco activase content over the range of leaf temperatures (Mate et al., 1993, 1996).     

NdhV Is a Subunit of NADPH Dehydrogenase Essential for Cyclic Electron Transport in Synechocystis sp. Strain PCC 6803

Two mutants sensitive to heat stress for growth and impaired in NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET) were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in the same sll0272 gene, encoding a protein highly homologous to NdhV identified in Arabidopsis (Arabidopsis thaliana). Deletion of the sll0272 gene (ndhV) did not influence the assembly of NDH-1 complexes and the activities of CO₂ uptake and respiration but reduced the activity of NDH-CET. NdhV interacted with NdhS, a ferredoxin-binding subunit of cyanobacterial NDH-1 complex. Deletion of NdhS completely abolished NdhV, but deletion of NdhV had no effect on the amount of NdhS. Reduction of NDH-CET activity was more significant in ΔndhS than in ΔndhV. We therefore propose that NdhV cooperates with NdhS to accept electrons from reduced ferredoxin.Cyanobacterial NADPH dehydrogenase (NDH-1) complexes are localized in the thylakoid membrane (Ohkawa et al., 2001, 2002; Zhang et al., 2004; Xu et al., 2008; Battchikova et al., 2011b) and participate in a variety of bioenergetic reactions, such as respiration, cyclic electron transport around photosystem I (NDH-CET), and CO₂ uptake (Ogawa, 1991; Mi et al., 1992; Ohkawa et al., 2000). Structurally, the cyanobacterial NDH-1 complexes closely resemble energy-converting complex I in eubacteria and the mitochondrial respiratory chain regardless of the absence of homologs of three subunits in cyanobacterial genomes that constitute the catalytically active core of complex I (Friedrich et al., 1995; Friedrich and Scheide, 2000; Arteni et al., 2006). Over the past decade, new subunits of NDH-1 complexes specific to oxygenic photosynthesis have been identified in several cyanobacterial strains. They are NdhM to NdhQ and NdhS (Prommeenate et al., 2004; Battchikova et al., 2005, 2011b; Nowaczyk et al., 2011; Wulfhorst et al., 2014; Zhang et al., 2014; Zhao et al., 2014b, 2015), in addition to NdhL first identified in the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803) about 20 years ago (Ogawa, 1992). Among them, NdhS possesses a ferredoxin (Fd)-binding motif and was shown to bind Fd, which suggested that Fd is one of the electron donors to NDH-1 complexes (Mi et al., 1995; Battchikova et al., 2011b; Ma and Ogawa, 2015). Deletion of NdhS strongly reduced the activity of NDH-CET but had no effect on respiration and CO₂ uptake (Battchikova et al., 2011b; Ma and Ogawa, 2015). The NDH-CET plays an important role in coping with various environmental stresses regardless of its elusive mechanism. For example, this function can greatly alleviate heat-sensitive growth phenotypes (Wang et al., 2006a; Zhao et al., 2014a). Thus, heat treatment strategy can help in identifying the proteins essential to NDH-CET.Here, a new oxygenic photosynthesis-specific (OPS) subunit NdhV was identified in Synechocystis 6803 with the help of heat treatment strategy, and its deletion did not influence the assembly of NDH-1L and NDH-1MS complexes and the activities of CO₂ uptake and respiration but impaired the NDH-CET activity. We give evidence that NdhV interacts with NdhS and is another component of Fd-binding domain of cyanobacterial NDH-1 complex. A possible role of NdhV on the NDH-CET activity is discussed.     

Enhanced Photosynthesis and Growth in atquac1 Knockout Mutants Are Due to Altered Organic Acid Accumulation and an Increase in Both Stomatal and Mesophyll Conductance

Stomata control the exchange of CO₂ and water vapor in land plants. Thus, whereas a constant supply of CO₂ is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO₂ concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions.Stomata are functionally specialized microscopic pores that control the essential exchange of CO₂ and H₂O with the environment in land plants. Stomata are found on the surfaces of the majority of the aerial parts of plants, rendering them as the main control point regulating the flow of gases between plants and their surrounding atmosphere. Accordingly, the majority of water loss from plants occurs through stomatal pores, allowing plant transpiration and CO₂ absorption for the photosynthetic process (Bergmann and Sack, 2007; Kim et al., 2010). The maintenance of an adequate water balance through stomatal control is crucial to plants because cell expansion and growth require tissues to remain turgid (Sablowski and Carnier Dornelas, 2014), and minor reductions in cell water volume and turgor pressure will therefore compromise both processes (Thompson, 2005). As a result, the high sensitivity of plant tissues to turgor has prompted the use of reverse genetic studies in attempt to engineer plants with improved performance (Cowan and Troughton, 1971; Xiong et al., 2009; Borland et al., 2014; Franks et al., 2015).In most land plants, not only redox signals invoked by shifts in light quality (Busch, 2014) but also the transport of inorganic ions (e.g. K⁺, Cl⁻, and NO⁻₃) as well as metabolites such as the phytohormone abscisic acid (ABA), Suc, and malate, are important players controlling stomatal movements (Hetherington, 2001; Roelfsema and Hedrich, 2005; Pandey et al., 2007; Blatt et al., 2014; Kollist et al., 2014). In this context, although organic acids in plants is known to support numerous and diverse functions both within and beyond cellular metabolism, only recently have we obtained genetic evidence to support that modulation of guard cell malate and fumarate concentration can greatly influence stomatal movements (Nunes-Nesi et al., 2007; Araújo et al., 2011b; Penfield et al., 2012; Medeiros et al., 2015). Notably malate, in particular, has been considered as a key metabolite and one of the most important organic metabolites involved in guard cell movements (Hedrich and Marten, 1993; Fernie and Martinoia, 2009; Meyer et al., 2010). During stomatal aperture, the flux of malate into guard cells coupled with hexoses generated on starch breakdown lead to decreases in the water potential, and consequently, water uptake by the guard cells ultimately opens the stomata pore (Roelfsema and Hedrich, 2005; Vavasseur and Raghavendra, 2005; Lee et al., 2008). On the other hand, during stomatal closure, malate is believed to be converted into starch, which has no osmotic activity (Penfield et al., 2012) or, alternatively, is released from the guard cells to the surrounding apoplastic space (Lee et al., 2008; Negi et al., 2008; Vahisalu et al., 2008; Meyer et al., 2010).The role of organic acids on the stomatal movements has been largely demonstrated by studies related to malate transport (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In the last decade, two protein families were identified and functionally characterized to be directly involved with organic acid transport at the guard cell plasma membrane and to be required for stomatal functioning (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In summary, AtABCB14, a member of the ABC (ATP binding cassette) family, which is involved in malate transport from apoplast to guard cells, was described as a negative modulator of stomatal closure induced by high CO₂ concentration; notably, exogenous application of malate minimizes this response (Lee et al., 2008). In addition, members of a small gene family, which encode the anion channels SLAC1 (slow anion channel 1) and four SLAC1-homologs (SLAHs) in Arabidopsis (Arabidopsis thaliana), have been described to be involved in stomatal movements. SLAC1 is a well-documented S-type anion channel that preferentially transports chloride and nitrate as opposed to malate (Vahisalu et al., 2008, 2010; Geiger et al., 2010; Du et al., 2011; Brandt et al., 2012; Kusumi et al., 2012). Lack of SLAC1 in Arabidopsis and rice (Oryza sativa) culminated in a failure in stomatal closure in response to high CO₂ levels, low relative humidity, and dark conditions (Negi et al., 2008; Vahisalu et al., 2008; Kusumi et al., 2012). Although mutations in AtSLAC1 impair S-type anion channel functions as a whole, the R-type anion channel remained functional (Vahisalu et al., 2008). Indeed, a member of the aluminum-activated malate transporter (ALMT) family, AtALMT12, an R-type anion channel, has been demonstrated to be involved in malate transport, particularly at the plasma membrane of guard cells (Meyer et al., 2010; Sasaki et al., 2010). Although AtALMT12 is a member of ALMT family, it is not activated by aluminum, and therefore Meyer et al. (2010) proposed to rename it as AtQUAC1 (quick-activating anion channel 1; Imes et al., 2013; Mumm et al., 2013). Hereafter, we will follow this nomenclature. Deficiency of a functional AtQUAC1 has been documented to lead to changes in stomatal closure in response to high levels of CO₂, dark, and ABA (Meyer et al., 2010). Taken together, these studies have clearly demonstrated that both S- and R-type anion channels are key modulators of stomatal movements in response to several environmental factors.Despite a vast number of studies involving the above-mentioned anion channels, little information concerning the metabolic changes caused by their impairment is currently available. Such information is important to understand stomatal movements, mainly considering that organic acids, especially the levels of malate in apoplastic/mesophyll cells, have been highlighted as of key importance in leaf metabolism (Fernie and Martinoia, 2009; Araújo et al., 2011a, 2011b; Lawson et al., 2014; Medeiros et al., 2015). Here, we demonstrate that a disruption in the expression of AtQUAC1, which leads to impaired stomatal closure (Meyer et al., 2010), was accompanied by increases in mesophyll conductance (g_m), which is defined as the conductance for the transfer of CO₂ from the intercellular airspaces (C_i) to the sites of carboxylation in the chloroplastic stroma (C_c). By further characterization of atquac1 knockout plants, we demonstrated that reduced diffusive limitations resulted in higher photosynthetic rates and altered respiration that, in turn, led to enhanced biomass accumulation. Overall, the results obtained are discussed both in terms of the importance of organic acid transport in plant cell metabolism and with regard to the contribution that it plays in the regulation of both stomatal function and growth.     

Crystal Structure and Functional Characterization of Photosystem II-Associated Carbonic Anhydrase CAH3 in Chlamydomonas reinhardtii

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO₂ and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO₂ fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO₃^– increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO₃^– from CO₂. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO₂/HCO₃^– on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO₂ concentration.Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes, which catalyze the interconversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻), a reaction that otherwise proceeds slowly at physiological pH. CAs belong to three evolutionary distinct classes, α, β, and γ, which share no significant amino acid sequence identity and are thought to be the result of convergent evolution (Hewett-Emmett and Tashian, 1996; Supuran, 2008; Ferry, 2010; Rowlett, 2010). Animals have only the α-CA type, but as multiple isoforms. By contrast, higher plants, algae, and cyanobacteria may contain members of all three CA families. In algae, CAs has been found in mitochondria and chloroplasts and in the cytoplasm and apoplasm.Many fresh-water and soil-living microalgae face limiting concentrations of inorganic carbon (Ci) in their environments. To overcome this, the green microalga Chlamydomonas reinhardtii, as well as most other unicellular algae and cyanobacteria, actively accumulate Ci inside the cells. This mechanism is known as the carbon-concentrating mechanism (CCM; Raven, 1997; Wang et al., 2011; Meyer and Griffiths, 2013). CCM allows the algae to maintain a high concentration of CO₂ around the carboxylating enzyme, Rubisco, even under limiting external Ci. The increased concentration of CO₂ in the chloroplast increases the CO₂/O₂ specificity for Rubisco that leads to a decreased oxygenation reaction, and hence carboxylation becomes more efficient.CCM can be induced in C. reinhardtii cultures by bubbling air containing CO₂ at ambient or concentrations (≤0.04%; Vance and Spalding, 2005). Full metabolic adaptation is usually reached within 10 to 12 h after transfer to air CO₂ conditions (Renberg et al., 2010). Already within the first few hours after induction, several genes are either up- or down-regulated (Miura et al., 2004; Yamano et al., 2008; Fang et al., 2012). Surprisingly, the global changes in protein expression do not correspond to those in the gene expression; only few proteins are either up- or down-regulated during CCM induction (Manuel and Moroney, 1988; Spalding and Jeffrey, 1989). CAs are important components of the CCM. In C. reinhardtii, 12 genes are expressed that encode for CA isoforms (Moroney et al., 2011). Among the many genes that are significantly up-regulated during CCM induction, there is one encoding for an apoplastic CA (CrCAH1) and two encoding for mitochondrial CAs (CrCAH4 and CrCAH5; Fujiwara et al., 1990; Eriksson et al., 1996).An α-type CA (CrCAH3) located in the thylakoid lumen in C. reinhardtii has also been identified as important at low CO₂ levels (Karlsson et al., 1998). The sequence indicates that it is transported through the thylakoid membrane via the Twin Arg Translocation pathway (Albiniak et al., 2012). A mutant not expressing CrCAH3 (knockout of the cah3 gene) shows no or poor growth under air CO₂ levels (Spalding et al., 1983; Moroney et al., 1986) and has a severely impaired photosynthetic capacity under low Ci conditions. This mutant, called CrCIA3, has been a valuable tool for resolving the CrCAH3 function.It is also established that CrCAH3 is associated with PSII (Stemler, 1997; Villarejo et al., 2002; Blanco-Rivero et al., 2012). Using isolated PSII membranes from C. reinhardtii, Shutova et al. (2008) presented data suggesting that CrCAH3 is important for efficient water oxidation by facilitating the removal of protons that are produced when water is oxidized by PSII. This is in line with recent studies (Zaharieva et al., 2011; Klauss et al., 2012) showing that it is crucial to have alternating electron and proton removals from the oxygen-evolving complex (OEC) during the five-state catalytic cycle, i.e. the Kok cycle (Kok et al., 1970), of photosynthetic water oxidation. If proton removal is slow, this leads to less efficient O₂ production and consequently may lead to donor side photoinhibition (Minagawa et al., 1996). That HCO₃^– acts as a mobile proton carrier has been recently demonstrated for spinach (Spinacia oleracea) PSII membrane fragments using membrane inlet mass spectrometry (MIMS; Koroidov et al., 2014). These results show that PSII possesses a light- and HCO₃^–-dependent CO₂ production for up to 50% of the O₂ produced.Taken together, these data suggest that CrCAH3 plays an important role in regulating PSII reactions. In this work, we present further evidence for its function in PSII primary reactions, in particular at low Ci concentrations. We determined crystal structures of CrCAH3 at 2.6 to 2.7 Å resolution in complex with acetazolamide (AZM) or phosphate ions. Our results support a zinc-hydroxide catalytic mechanism of CrCAH3 similar to that of other α-CAs. CrCAH3 has, however, an activity optimum at lower pH values than CAs of the same type, which normally operate at pH 7.0 and higher (Demir et al., 2000). The activity optimum of CrCAH3 makes it more suitable for CO₂/HCO₃^– interconversion at the pH levels present in the thylakoid lumen under light exposure.     

Subunit Q Is Required to Stabilize the Large Complex of NADPH Dehydrogenase in Synechocystis sp. Strain PCC 6803

Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1, NdhF1, and NdhP, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, NDH-1-dependent cyclic electron transport around photosystem I, and CO₂ uptake. Two mutants sensitive to high light for growth and impaired in cyclic electron transport around photosystem I were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in an open reading frame encoding a product highly homologous to NdhQ, a single-transmembrane small subunit of the NDH-1L complex, identified in Thermosynechococcus elongatus by proteomics strategy. Deletion of ndhQ disassembled about one-half of the NDH-1L to NDH-1M and consequently impaired respiration, but not CO₂ uptake. During prolonged incubation of the thylakoid membrane with n-dodecyl-β-d-maltoside at room temperature, the rest of the NDH-1L in ΔndhQ was disassembled completely to NDH-1M and was much faster than in the wild type. In the ndhP-deletion mutant (ΔndhP) background, absence of NdhQ almost completely disassembled the NDH-1L to NDH-1M, similar to the results observed in the ΔndhD1/ΔndhD2 mutant. We therefore conclude that both NdhQ and NdhP are essential to stabilize the NDH-1L complex.Cyanobacterial NADPH dehydrogenase (NDH-1) complexes are localized in the thylakoid membrane (Ohkawa et al., 2001, 2002; Zhang et al., 2004; Xu et al., 2008; Battchikova et al., 2011a) and participate in a variety of bioenergetic reactions, such as respiration, cyclic electron transport around PSI, and CO₂ uptake (Ogawa, 1991; Mi et al., 1992; Ohkawa et al., 2000). Structurally, the cyanobacterial NDH-1 complexes closely resemble energy-converting complex I in eubacteria and the mitochondrial respiratory chain, regardless of the absence of homologs of three subunits in cyanobacterial genomes that constitute the catalytically active core of complex I (Friedrich et al., 1995; Friedrich and Scheide, 2000; Arteni et al., 2006). Over the past few years, significant achievements have been made in resolving the subunit compositions and functions of the multiple NDH-1 complexes in several cyanobacterial strains (for review, see Battchikova and Aro, 2007; Ogawa and Mi, 2007; Ma, 2009; Battchikova et al., 2011b; Ma and Ogawa, 2015). Four types of NDH-1 have been identified in the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter, Synechocystis 6803), and all four types of NDH-1 are involved in NDH-1-dependent cyclic electron transport (CET) around PSI (NDH-CET; Bernát et al., 2011). The NDH-CET plays an important role in coping with various environmental stresses, regardless of its elusive mechanism. For example, this function can greatly alleviate high light-sensitive growth phenotypes (Endo et al., 1999; Battchikova et al., 2011a; Dai et al., 2013; Zhang et al., 2014; Zhao et al., 2014). Therefore, high light strategy can help in identifying the proteins essential to NDH-CET.Proteomics studies revealed the presence of three major NDH-1 complexes in cyanobacteria: a large complex (NDH-1L), a medium size complex (NDH-1M), and a small complex (NDH-1S) with molecular masses of about 460, 350, and 200 kD, respectively (Herranen et al., 2004). NDH-1M consists of 14 subunits (i.e. NdhA–NdhC, NdhE, NdhG–NdhO, and NdhS). In addition to these subunits, the NDH-1L complex contains NdhD1, NdhF1, NdhP, and NdhQ (Prommeenate et al., 2004; Battchikova et al., 2005, 2011b; Zhang et al., 2005, 2014; Nowaczyk et al., 2011; Wulfhorst et al., 2014; Ma and Ogawa, 2015) and is involved in respiration (Zhang et al., 2004). NDH-1S is composed of NdhD3, NdhF3, CO₂ uptake A (CupA), and CupS (Ogawa and Mi, 2007) and is considered to be associated with NDH-1M in the cells as a functional complex NDH-1MS (Zhang et al., 2004, 2005) participating in CO₂ uptake. Among the several copies of ndhD and ndhF genes found in cyanobacterial genomes, ndhD1 and ndhF1 show the highest homology to chloroplast ndhD and ndhF genes, respectively, and CupA and CupS subunits of the cyanobacteria have no counterparts in higher plants. These facts suggest that the structure and composition of NDH-1L, but not the NDH-1MS complex, are similar to those of the chloroplast NDH-1 complex (Battchikova and Aro, 2007; Ogawa and Mi, 2007; Shikanai, 2007; Ma, 2009; Suorsa et al., 2009; Battchikova et al., 2011b; Ifuku et al., 2011; Peng et al., 2011a; Ma and Ogawa, 2015). Despite their similarity, a large number of subunits that constitute the chloroplast NDH-1 complex, including ferredoxin-binding subcomplex subunits NdhT and NdhU and all the subunits of subcomplex B and lumen subcomplex, are absent in the cyanobacterial NDH-1L complex (Battchikova et al., 2011b; Ifuku et al., 2011; Peng et al., 2011a). This implies that the stabilization strategies for the cyanobacterial NDH-1L complex and chloroplastic NDH-1 complex might be significantly different.Recently, a new oxygenic photosynthesis-specific small subunit NdhQ was identified in the NDH-1L complex purified by Ni²⁺ affinity chromatography from Thermosynechococcus elongatus (Nowaczyk et al., 2011). NdhQ is extensively present in cyanobacteria, but its homolog is absent in higher plants (Nowaczyk et al., 2011). In this study, we demonstrate that deletion of NdhQ disassembled the NDH-1L into NDH-1M, but not NDH-1MS, in Synechocystis 6803 and consequently impaired respiration, but not CO₂ uptake. NdhQ and NdhP stabilize the NDH-1L complex. Thus, the stabilization strategy of cyanobacterial NDH-1L is distinctly different from that of the chloroplastic NDH-1 complex.     

Does Low Stomatal Conductance or Photosynthetic Capacity Enhance Growth at Elevated CO2 in Arabidopsis?

The objective of this study was to determine if low stomatal conductance (g) increases growth, nitrate (NO₃⁻) assimilation, and nitrogen (N) utilization at elevated CO₂ concentration. Four Arabidopsis (Arabidopsis thaliana) near isogenic lines (NILs) differing in g were grown at ambient and elevated CO₂ concentration under low and high NO₃⁻ supply as the sole source of N. Although g varied by 32% among NILs at elevated CO₂, leaf intercellular CO₂ concentration varied by only 4% and genotype had no effect on shoot NO₃^– concentration in any treatment. Low-g NILs showed the greatest CO₂ growth increase under N limitation but had the lowest CO₂ growth enhancement under N-sufficient conditions. NILs with the highest and lowest g had similar rates of shoot NO₃^– assimilation following N deprivation at elevated CO₂ concentration. After 5 d of N deprivation, the lowest g NIL had 27% lower maximum carboxylation rate and 23% lower photosynthetic electron transport compared with the highest g NIL. These results suggest that increased growth of low-g NILs under N limitation most likely resulted from more conservative N investment in photosynthetic biochemistry rather than from low g.The availability of water varies in time and space, and plants in a given environment are expected to evolve a stomatal behavior that optimizes the tradeoff of CO₂ uptake for photosynthesis at the cost of transpirational water loss. The resource of CO₂ also varies over time, and plant fossils indicate that stomatal characteristics have changed in response to periods of high and low atmospheric CO₂ over the past 65 million years (Beerling and Chaloner, 1993; Van Der Burgh et al., 1993; Beerling, 1998; Kürschner, 2001; Royer et al., 2001). Relatively low atmospheric CO₂ concentrations (less than 320 µmol mol⁻¹) over the last 23 million years (Pearson and Palmer, 2000) are associated with increased stomatal conductance (g) to avoid CO₂ starvation (Beerling and Chaloner, 1993). Atmospheric CO₂ concentration has risen rapidly from 280 to 400 µmol mol⁻¹ since 1800 and has resulted in lower stomatal density (Woodward, 1987; Woodward and Bazzaz, 1988; Lammertsma et al., 2011). At the current atmospheric CO₂ concentration (400 µmol mol⁻¹), further decreases in g reduce water loss but also restrict CO₂ assimilation and, thus, limit the effectiveness of low g in water-stressed environments (Comstock and Ehleringer, 1993; Virgona and Farquhar, 1996). Elevated CO₂ concentration enhances the diffusion gradient for CO₂ into leaves, which allows g to decrease without severely restricting photosynthetic carbon gain (Herrick et al., 2004). Most consider such an improvement in water use efficiency in C₃ plants to be the main driving force for decreased g at elevated CO₂ concentration, especially in dry environments (Woodward, 1987; Beerling and Chaloner, 1993; Brodribb et al., 2009; Franks and Beerling, 2009; Katul et al., 2010).Water is the most common factor limiting terrestrial plant productivity, but declining stomatal density has also occurred in wetland environments where water stress is uncommon (Wagner et al., 2005). Improved water use efficiency at elevated CO₂ concentration may be shifting the most common factor limiting plant productivity from water to nitrogen (N). In herbarium specimens of 14 species of trees, shrubs, and herbs, leaf N decreased 31% as atmospheric CO₂ increased from about 270 to 400 μmol mol⁻¹ since 1750 (Penuelas and Matamala, 1990). Indeed, many studies have shown that N availability limits the stimulation of plant growth at elevated CO₂ concentration (Luo et al., 2004; Dukes et al., 2005; Reich et al., 2006). That most plants at elevated CO₂ concentration exhibit both lower g and greater N limitation suggests a relationship between these factors.Plants primarily absorb N as nitrate (NO₃^–) in most temperate soils and assimilate a major portion of this NO₃^– in shoots (Epstein and Bloom, 2005). Elevated CO₂ increases the ratio of CO₂ to oxygen in the chloroplast, decreasing photorespiration and improving photosynthetic efficiency (Sharkey, 1988) but inhibiting photorespiration-dependent NO₃^– assimilation (Rachmilevitch et al., 2004; Bloom et al., 2010, 2012; Bloom, 2014). Greater rhizosphere NO₃^– availability tends to enhance root NO₃^– assimilation and decrease the influence of elevated CO₂ concentration on plant organic N accumulation (Kruse et al., 2002, 2003; Bloom et al., 2010).The most important factor regulating chloroplast CO₂ concentration among natural accessions of Arabidopsis (Arabidopsis thaliana) is g and to a lesser extent mesophyll conductance (Easlon et al., 2014). Low g may decrease the ratio of CO₂ to oxygen in the chloroplast at elevated CO₂ concentration, enhancing photorespiration-dependent NO₃^– assimilation. Alternatively, increasing atmospheric CO₂ may down-regulate the need to synthesize enzymes such as Rubisco to support photosynthesis, which conserves organic N, and g may decline as a by-product of lower photosynthetic capacity (Sage et al., 1989; Moore et al., 1998).Here, we examined the influence of atmospheric CO₂ concentration and NO₃^– supply on photosynthesis, leaf N, and growth in near isogenic lines (NILs) of Arabidopsis differing in g. Arabidopsis accessions differ in many traits (including g) and likewise differ in DNA sequence at a large percentage of genes across the genome (Cao et al., 2011). Use of these NILs greatly reduces the proportion of the genome that varies and minimizes the influence of variation in other traits that are frequently associated with low g and could limit growth (Arp et al., 1998). We tested the extent to which (1) low g was associated with greater CO₂ growth enhancement at low and high NO₃⁻ supply; (2) low leaf intercellular CO₂ concentration (C_i) increased shoot NO₃^– assimilation; and (3) low g at elevated CO₂ concentration was associated with altered N utilization in photosynthetic biochemistry.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins

Spatial segregation of metabolism, such as cellular-localized CO₂ fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concentrating them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are self-assembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biology and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be especially useful for developing synthetic carboxysomes for plant engineering.A key enzyme in photosynthesis is the CO₂ fixation enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco not only fixes CO₂, resulting in carbon assimilation, but it can also fix O₂, leading to photorespiration. Suppressing the unwanted oxygenase activity of Rubisco by sequestering Rubisco with a source of CO₂ is Nature’s solution to this substrate discrimination problem. While C4 plants compartmentalize CO₂ fixation in specific cells (Hibberd et al., 2008; Parry et al., 2011), cyanobacteria have evolved a specialized organelle composed entirely of protein to encapsulate Rubisco—the carboxysome.The carboxysome is just one type of bacterial microcompartment (BMC), widespread, functionally diverse bacterial organelles (Axen et al., 2014). All BMCs consist of an enzymatic core surrounded by a selectively permeable protein shell (Kerfeld et al., 2005; Tanaka et al., 2008; Chowdhury et al., 2014; Kerfeld and Erbilgin, 2015). While the encapsulated enzymes differ among functionally distinct BMCs, they share an architecturally similar shell composed of three types of proteins: BMC-H, BMC-T, and BMC-P forming hexamers, pseudohexamers, and pentamers, respectively (Kerfeld and Erbilgin, 2015). These constitute the building blocks of a self-assembling, apparently icosahedral shell with a diameter ranging from 40 to 400 nm (Shively et al., 1973a,b, 1998; Price and Badger, 1991; Bobik et al., 1999; Iancu et al., 2007, 2010; Petit et al., 2013; Erbilgin et al., 2014). Recent studies have also shown that in the biogenesis of BMCs an encapsulation peptide (EP) (Fan and Bobik, 2011; Kinney et al., 2012; Aussignargues et al., 2015; Jakobson et al., 2015), a short (approximately 18 residues) amphipathic α-helix mediates interactions between a subset of core protein and the shell (Fan and Bobik, 2011; Choudhary et al., 2012; Kinney et al., 2012; Lawrence et al., 2014; Lin et al., 2014; Aussignargues et al., 2015). Indeed, because they are self-assembling organelles composed entirely of protein, BMCs hold great promise for diverse applications in bioengineering and development of bionanomaterials (Frank et al., 2013; Chowdhury et al., 2014; Chessher et al., 2015; Kerfeld and Erbilgin, 2015); the key features of BMCs include selective permeability, spatial colocalization of enzymes, the establishment of private cofactor pools, and the potentially beneficial effects of confinement on protein stability. For example, introducing carboxysomes into plants could provide a saltational enhancement of crop photosynthesis (Price et al., 2013; Zarzycki et al., 2013; Lin et al., 2014; McGrath and Long, 2014).The β-carboxysome, which sequesters form 1B Rubisco, has been an important model system for the study of the structural basis of carboxysome function, assembly, and engineering (Kerfeld et al., 2005; Tanaka et al., 2008; Cameron et al., 2013; Aussignargues et al., 2015; Cai et al., 2015). Beta-carboxysomes assemble from the inside out (Cameron et al., 2013; Gonzalez-Esquer et al., 2015). Two proteins that are absolutely conserved and unique to β-carboxysomes, CcmM and CcmN, play essential roles in this process: CcmM crosslinks Rubisco through its C-terminal Rubisco small subunit-like domains (SSLDs; pfam00101); CcmM and CcmN interact through their N-terminal domains; and C-terminal EP of CcmN interacts with the carboxysome shell.Here we describe a system for producing synthetic β-carboxysome shells and encapsulating nonnative cargo. We constructed a synthetic operon composed of ccmK1, ccmK2, ccmL, and ccmO, genes encoding, respectively, two BMC-H proteins, a BMC-P protein, and a BMC-T protein of the carboxysome shell of the halotolerant cyanobacterium, Halothece sp. PCC 7418 (Halo hereafter). Recombinant shells composed of all four proteins were produced and purified. We also demonstrated that the terminal α-helices of CcmK1 and CcmK2 are not, as had been proposed (Samborska and Kimber, 2012), required for the shell formation, and that the synthetic shell is a single-layered protein membrane. Cargo could be targeted to the interior of the synthetic shells using either the EP of CcmN or the N-terminal domain of CcmM; the latter observation provides new insight into the organization of the β-carboxysome. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly but also provide a platform to study carboxysome shell permeability. These results will be useful in guiding the design and optimization of carboxysomes and other BMCs for introduction into plants.     

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

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