Plant Mol Biol DOI 10.1007/s11103-014-0183-z
Regulation of CCM genes in Chlamydomonas reinhardtii during conditions of light–dark cycles in synchronous cultures Srikanth Tirumani · Mallikarjuna Kokkanti · Vishal Chaudhari · Manish Shukla · Basuthkar J. Rao
Received: 8 July 2013 / Accepted: 19 February 2014 © Springer Science+Business Media Dordrecht 2014
Abstract We have investigated transcript level changes of CO2-concentrating mechanism (CCM) genes during light– dark (12 h:12 h) cycles in synchronized Chlamydomonas reinhardtii at air-level CO2. CCM gene transcript levels vary at various times of light–dark cycles, even at same airlevel CO2. Transcripts of inorganic carbon transporter genes (HLA3, LCI1, CCP1, CCP2 and LCIA) and mitochondrial carbonic anhydrase genes (CAH4 and CAH5) are up regulated in light, following which their levels decline in dark. Contrastingly, transcripts of chloroplast carbonic anhydrases namely CAH6, CAH3 and LCIB are up regulated in dark. CAH3 and LCIB transcript levels reached maximum by the end of dark, followed by high expression into early light period. In contrast, CAH6 transcript level stayed high in dark, followed by high level even in light. Moreover, the up regulation of transcripts in dark was undone by high CO2, suggesting that the dark induced CCM transcripts were regulated by CO2 even in dark when CCM is absent. Thus while the CAH3 transcript level modulations appear Srikanth Tirumani and Mallikarjuna Kokkanti share co-firstauthorship in this work. Electronic supplementary material The online version of this article (doi:10.1007/s11103-014-0183-z) contains supplementary material, which is available to authorized users. S. Tirumani · V. Chaudhari · M. Shukla · B. J. Rao (*) B‑202, Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India e-mail: [email protected] URL: http://www.tifr.res.in/~dbs/faculty/bjr/Mission.html S. Tirumani · M. Kokkanti Department of Botany and Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur 522510, Andhra Pradesh, India
not to positively correlate with that of CCM, the protein regulation matched with CCM status: in spite of high transcript levels in dark, CAH3 protein reached peak level only in light and localized entirely to pyrenoid, a site functionally relevant for CCM. Moreover, in dark, CAH3 protein level not only reduced but also the protein localized as a diffused pattern in chloroplast. We propose that transcription of most CCM genes, followed by protein level changes including their intracellular localization of a subset is subject to light–dark cycles. Keywords Air-level CO2 · Bicarbonate transporter · Carbonic anhydrase · CO2-concentrating mechanism · Light–dark cycles · Transcriptional regulation Abbreviations CAs Carbonic anhydrases CO2 Carbon dioxide CCM CO2-concentrating mechanism Ci Inorganic carbon DIC Dissolved inorganic carbon L:D Light:dark ML 6 h of incubation in light (mid-light) CL 12 h of incubation in light (complete-light) MD 6 h of incubation in dark (mid-dark) CD 12 h of incubation in dark (complete-dark) qRT-PCR Quantitative real time PCR RT-PCR Reverse transcription PCR
Introduction CO2 undergoes reduction in photosynthetic reactions to form carbohydrates that supplies food and energy to the biosphere. Hence any change in the CO2 availability in local
13
environments would affect the growth and development of inhabitant plants profoundly. Algae and other aquatic plants experience significant constraints in acquiring CO2 from local environment due to very slow diffusion and low solubility of CO2 in water (104 times slower diffusion than that in the air). Moreover, low affinity and very low catalytic turnover of RuBisCO, the key enzyme involved in CO2 assimilation, imposes further constraints on carbon fixation efficiency (Spalding 2009). Algae and aquatic plants also experience limiting CO2 due to difference in CO2 supply dynamics between water and air. Under limiting CO2, RuBisCO shows oxygenase activity fixing O2 rather than CO2, thereby loosing photosynthetic carbon fixation efficiency. Chlamydomonas reinhardtii (C. reinhardtii) can use both CO2 and HCO3− as the inorganic sources of carbon, relative availability of which to cells is determined by solution pH. To overcome limitations in acquiring CO2, C. reinhardtii and a range of aquatic plants have adapted an elaborate CO2-concentrating mechanism (CCM) which actively uptake both forms of inorganic carbon (Ci) (CO2 and HCO3−) (Sultemeyer et al. 1989; Palmqvist et al. 1994) and facilitate maintenance of high levels of CO2 at the site of RuBisCO. Fundamental to CCM acclimation during low to very low-CO2 (air-level), is a mechanism to regulate the scavenging of Ci and concentrate it internally since CO2 supply gets limiting. It has been shown that the induction of CCM occurs at two limiting CO2 conditions, air level (0.03–0.04 %) and below air level CO2 (95 %) and properties, one each was chosen for northern analyses. As expected, all the five control gene transcripts showed specific up regulation in light (Fig. 2b), with hardly any detectable level of transcripts in dark, thereby corroborating our earlier results (Fig. 1a). It was interesting to note that in both the assays (qRT-PCR and northern), while the light regulated transcript levels showed specific rise in light incubations, those up regulated in dark were not entirely specific to dark, but also to light (see Discussion). Thus far, we have uncovered a set of CCM related genes that are up regulated in dark and light (CAH3, CAH6 and LCIB) and another set entirely in light (HLA3, LCI1, CCP1, CCP2, LCIA, CAH4 and CAH5). Most of these genes are implicated to be under the control of a “master regulator” CIA5/CCM1 transcription factor gene (Fang et al. 2012; Wang et al. 2005; Xiang et al. 2001). We therefore assessed CIA5 transcript levels in the same light– dark regime using RT-PCR by gel analyses. As expected of a master regulator gene that can control both light and dark-inducible transcripts, the transcript level of CIA5 was similar in all the four stages, namely MD, CD, ML and CL (Fig. 3a). CIA5 transcript level was low and appeared not to change significantly across MD, CD, ML and CL stages. Since CIA5 transcript level was too low and unchanging across various time-points, it was not analyzed further by either qRT-PCR or northern blot assays. In order to assess whether the observed increase in transcript levels is specific to low CO2 conditions of CCM, we repeated the analyses at high CO2 in dark. We compared CAH6, CAH3 and LCIB transcript levels in low (air- level)
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CAH3 is one of the moderately expressed CAs in C. reinhardtii that is induced in low CO2 conditions where CCM is active (Moroney et al. 2011). Since CCM is active only in light, we were intrigued by the observed increase in CAH3 transcript levels in dark (Fig. 1j, 2a), which prompted us to analyze protein level changes also. For comparison, we also chose CAH4/5 that are highly light induced and abundantly expressed in mitochondria during low CO2 conditions when CCM is active (Moroney et al. 2011). Since CAH4/5 transcript levels go up in light, their protein levels going up in light also serves as an active CCM mark. Western blot analyses showed that CAH3 protein level reached its maximum at 6–12 h in light, followed by a gradual drop in dark reaching the minimum into 9–12 h of dark (Fig. 4a). Therefore it appears that even though transcript levels increase in dark, peaking at 3 h of light, it is only in later half of light period (6–12 h) that CAH3 protein level reaches maximum where CCM is active, revealing a distinct difference in the time course of transcript versus protein levels during day/night cycles (12 h:12 h). In the same experiment, CAH3 immunofluorescence revealed another interesting facet: while the basal level of CAH3 protein present in dark showed a diffuse pattern of distribution, away from pyrenoid in dark, that of enhanced level of CAH3 in light was entirely concentrated to pyrenoid location, where it is most required for CCM (Fig. 4b, S2). This result suggested that CAH3 localization is perhaps well regulated in relation to CCM activity in the cell. This notion is highly consistent with the recent finding that CAH3 protein localization is critically dependent on CCM status of the cell where at high CO2 conditions the protein stays associated with stroma thylakoid, away from pyrenoid, but is recruited to pyrenoid specifically at low CO2 following CAH3 phosphorylation (BlancoRivero et al. 2012)..Western blot analyses using an antibody raised against CAH4/5 combine revealed an expected trend, namely their high expression at 9–12 h of light followed by a gradual drop through dark reaching a minimum towards the end of dark period (6–12 h into dark) (Fig. 5a), a pattern similar to transcript level changes observed earlier (Fig. 1f, g, 2b). These two abundant proteins associated with mitochondria revealed mitochondria-specific
Plant Mol Biol Fig. 4 CAH3 protein analyses: a Western blot analyses of CAH3 from 3 hourly samples (RbcL is the loading control). b Confocal immunofluorescence analyses of ML and MD sample fixed cells by anti-CAH3 Ab. Confocal image stacks arranged from left to right span most of the cell volume. (Scale bar: 3 μm)
Fig. 5 CAH4/5 protein analyses: a Western blot analyses of CAH4/5 from 3 hourly samples (RbcL is the loading control). b Confocal immunofluorescence analyses of ML and MD sample fixed cells by anti-CAH4/5 Ab. Confocal image stacks arranged from left to right span most of the cell volume. c Confocal analyses of mitochondrial staining in live-cells by mitotracker of ML and MD sample cells. Confocal image stacks arranged from left to right span most of the cell volume. (Scale bar: 3 μm)
changes in light versus dark incubation samples: CAH4/5 staining largely paralleled mitochondrial staining pattern as revealed by mitotracker dye (Fig. 5 compare b with c). Most of CAH4/5 was aligned as a continuous ring shaped morphology along the periphery of cell body in light treated cells whereas it was more interspersed towards the interior of the cell in dark (Fig. 5b, S3). We believe that
these changes were largely reflective of alterations in mitochondrial morphology in light versus dark incubations as revealed by mitotracker green staining (Fig. 5c, S3), rather than repositioning of CAH4/5, per se. All these results, put together, suggest that protein level changes in CAH3 and CAH4/5 are distinct and involve additional cellular regulation beyond the transcriptional control.
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Discussion Aquatic photosynthetic organisms acclimate to low CO2 stress by inducing CCM, which operate through the concerted functioning of a number of Ci transporters and CAs. It is well known that CCM is active in the light and inactive in the dark. Although physiological aspects of CO2 response in light and dark have been extensively studied, expression level regulation of CCM genes in light versus dark regime has not been well investigated. In this report, we describe transcriptional changes of CCM genes at different time points of day/night cycles (12 h:12 h) (3 or 6 hourly interval samples) using synchronized cells, grown photoautotrophically at air-level CO2. We show here that putative bicarbonate transporter gene transcripts (HLA3, LCI1 and LCIA) are up regulated in light at the air level CO2 conditions. The same trend is seen with chloroplast carrier protein genes (CCP1 and CCP2) (Fig. 1, 2b). Both type of Ci transporters, namely that of plasma membrane and chloroplast, are up regulated in light. The observed up regulation of Ci transporter gene transcripts in light is consistent with their projected role in CCM, which is specifically operational in light. Several reports indeed strongly implicate these transporters in CCM function (Duanmu et al. 2009a; Ohnishi et al. 2010). Interestingly, mitochondrial CA gene transcripts (CAH4 and CAH5) are also up regulated in light (Fig. 1f, g, 2b). It is likely that the light mediated up regulation of CAH4 and CAH5 transcripts via their protein products could facilitate an efficient augmentation of HCO3− production that can then be routed to chloroplast for efficient CCM. Our observation that CAH4 and CAH5 gene transcripts are up regulated in light is consistent with the results of Eriksson et al, where they had shown circadian up regulation of mitochondrial CA RNA’s specifically during the light periods in low CO2 conditions (Fig. 5 in Eriksson et al. 1998). A point of caution also needs to be emphasized here, namely, the conditions that favor mitochondrial CA expression usually also stimulate anaplerosis (Giordano et al. 2003). Protein level changes also showed that CAH4/5 reach maximum by 9–12 h into light, followed by a drop in dark, reaching minimum levels at 6–9 h into dark (Fig. 5a). As expected, immunofluorescence studies revealed that these proteins follow localization changes that are largely reflective of mitochondrial morphology changes in light–dark regimes (Fig. 5b–e, S3). Live staining pattern of cells by Mitotracker green is qualitatively similar to that of CAH4/5 specific immunofluorescence pattern in fixed cells, even though the sample processing conditions in these two experiments are distinctly different (compare Fig. 5b with c). Transcriptional regulation cascade that is driven by light-regulated promoters is poorly understood in C.
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Plant Mol Biol
reinhardtii. A recent bioinformatic study makes a compelling case for deciphering such regulatory network by experimental approaches (Riano-Pachon et al. 2008). In view of paucity of information, it is unclear whether the light up regulated transcript levels of HLA3, LCI1, LCIA, CCP1, CCP2, CAH4 and CAH5 reflects a direct consequence of light-regulated promoter activity, if any, or a consequence of an indirect regulation. We are currently trying to locate light response elements in some of these gene promoters. Counter-intuitively, unlike that of Ci transporter genes, transcript levels of CA’s (CAH6: chloroplast stromal CA; CAH3: thylakoid lumen CA) are up regulated in dark (Fig. 1, 2a). These CAs are strongly implicated in CCM. CAH1 was not studied, as it is not strongly implicated in CCM functions (Ohnishi et al. 2010; Van and Spalding 1999). CAH3 and CAH6 genes showed high accumulation of transcripts even in dark period. We also included chloroplast stromal soluble protein LCIB in our studies that functions downstream of CAH3 (Duanmu et al. 2009b). LCIB also showed up regulation by the end of dark period (Fig. 1). Northern blot analyses performed with closely spaced time-interval samples showed that CAH3 transcript level reached maximum by the end of dark, followed by high expression even into early light. CAH6 transcript level stayed high in dark for a longer duration, followed by high level even in light. Contrary to the pattern of CAH6, that of LCIB expression in dark was followed by its expression in early hours of light, very similar to that of CAH3 (Fig. 2a). Most observations reported in the literature so far on CA expression levels are related to light conditions where CCM is active. The observed up regulation of CAH3, CAH6 and LCIB transcripts in dark ensued in normal air level CO2 conditions. To test whether up regulation in dark of LCIB and its upstream component CAH3 transcript was dominant even in high CO2 conditions, we compared their transcript levels at high CO2 (~3 %). The up regulation seen at low CO2 (normal air-level CO2) in dark was undone in high CO2 (Fig. 3b). This result indicated that the up regulation of dark induced transcripts at air level of CO2 is not solely dependent on dark, but also on CO2 conditions, suggesting that CCM components are under regulation even in dark at air level of CO2 where CCM is inactive. This interesting, but not well appreciated, aspect had been pointed out by Rawat and Moroney several years ago (Rawat and Moroney 1995). We have schematized the transcript level changes observed so far in the study in the perspective of complete CCM regulation known to date (Supplementary Fig. S4). To understand transcriptional up regulation of some of the important CCM genes in dark when CCM is not operational, we performed further studies. Western analyses revealed that protein level changes follow a different trend: even though CAH3 transcript is up regulated in dark,
Plant Mol Biol
followed by that in light (Fig. 1, 2a), protein level reaches maximum only in light (6–12 h into light), followed by a drop in dark leading to a minimum at 12 h into dark (Fig. 4a). Protein level changes were consistent with its functional requirement in CCM during light. As observed by Yamano et al. for LCIB, immunofluorescence staining of CAH3 also revealed that the protein specifically localizes to pyrenoid during light. Intense staining of CAH3 protein was observed in pyrenoid at ML. In contrast, the protein diffuses away into a punctate distribution away from pyrenoid in dark (MD) (Fig. 4b, S2). This is very similar to Yamano et al. results that LCIB localizes to pyrenoid in light from where it diffuses away in dark (Yamano et al. 2010). Recent finding by (Blanco-Rivero et al. 2012) showing CAH3 localization to pyrenoid at low CO2 specifically following its phosphorylation strongly reinforces the view that some of these CCM proteins are under strict regulation with respect to their cellular localization. All these results strongly suggest parallels between CAH3 and LCIB, pointing towards a regulatory control that encompasses transcriptional up regulation of these genes in dark and protein translational up regulation in light. It is relevant to reiterate here, that CAH3 and LCIB transcript level changes in dark versus light time-points are nearly identical (Fig. 1, 2a). In addition, there is perhaps an added layer of regulation that promotes protein recruitment to pyrenoid in light, whose absence in dark leads to release of the protein from pyrenoid, followed by punctate distribution throughout the chloroplast (Fig. 4b, S2). Protein recruitment in and out of pyrenoid as a function of light–dark cues may in fact be an important regulation in C. reinhardtii cells even in the context of non-CCM functions as suggested by our recent study involving a UV-induced DNA-endonuclease (UVI31+) (Shukla et al. 2012). Our current study describes that CCM gene transcript level changes operative at air level CO2 conditions are perhaps critically shaped by light–dark rhythmic changes. The synchronized cells provided an opportunity to dissect the role of light–dark cycles on CCM regulation. Interestingly, the protein products of dark up regulated transcripts, namely, CAH3, CAH6 and LCIB are implicated in CO2 recapture mechanisms (Yamano et al. 2010) and we are tempted to speculate that such recapture steps might indeed be active and relevant in dark as well using the basal level of protein products formed in dark, so that CO2 does not leak out. Alternately, the transcripts formed in dark can serve as ready reservoir of mRNA for a rapid production of protein “on demand” for CCM in light. It is not clear why a small subset of CCM transcripts is up regulated in dark whereas the majority is specifically up regulated in light. It is interesting to mechanistically explore this dichotomy for which more detailed molecular and cellular analyses are required.
Acknowledgments This work would not been possible without the help and cooperation from BJ lab people whom we profusely thank. We want to thank Prof. J.V. Moroney for sharing CAH4/5 antibody preparation. A special acknowledgement is due to Dr Ullas Kolthur and Upasana Roy for their help in qPCR work. MK is thankful to M Vijayalakshmi (HOD), Botany and Microbiology Department, Acharya Nagarjuna University for sanctioning leave to carry out this work. MK is also thankful to Prof. G Dr PCO Reddy (YV University, Kadapa) for useful discussions and help. BJR acknowledges JC Bose fellowship grant (DST). Department of Atomic Energy (Government of India) grant to Tata Institute of Fundamental Research (TIFR), Mumbai.
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Regulation of CCM genes in Chlamydomonas reinhardtii during conditions of light–dark cycles in synchronous cultures Srikanth Tirumani · Mallikarjuna Kokkanti · Vishal Chaudhari · Manish Shukla · Basuthkar J. Rao
Received: 8 July 2013 / Accepted: 19 February 2014 © Springer Science+Business Media Dordrecht 2014
Abstract We have investigated transcript level changes of CO2-concentrating mechanism (CCM) genes during light– dark (12 h:12 h) cycles in synchronized Chlamydomonas reinhardtii at air-level CO2. CCM gene transcript levels vary at various times of light–dark cycles, even at same airlevel CO2. Transcripts of inorganic carbon transporter genes (HLA3, LCI1, CCP1, CCP2 and LCIA) and mitochondrial carbonic anhydrase genes (CAH4 and CAH5) are up regulated in light, following which their levels decline in dark. Contrastingly, transcripts of chloroplast carbonic anhydrases namely CAH6, CAH3 and LCIB are up regulated in dark. CAH3 and LCIB transcript levels reached maximum by the end of dark, followed by high expression into early light period. In contrast, CAH6 transcript level stayed high in dark, followed by high level even in light. Moreover, the up regulation of transcripts in dark was undone by high CO2, suggesting that the dark induced CCM transcripts were regulated by CO2 even in dark when CCM is absent. Thus while the CAH3 transcript level modulations appear Srikanth Tirumani and Mallikarjuna Kokkanti share co-firstauthorship in this work. Electronic supplementary material The online version of this article (doi:10.1007/s11103-014-0183-z) contains supplementary material, which is available to authorized users. S. Tirumani · V. Chaudhari · M. Shukla · B. J. Rao (*) B‑202, Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India e-mail: [email protected] URL: http://www.tifr.res.in/~dbs/faculty/bjr/Mission.html S. Tirumani · M. Kokkanti Department of Botany and Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur 522510, Andhra Pradesh, India
not to positively correlate with that of CCM, the protein regulation matched with CCM status: in spite of high transcript levels in dark, CAH3 protein reached peak level only in light and localized entirely to pyrenoid, a site functionally relevant for CCM. Moreover, in dark, CAH3 protein level not only reduced but also the protein localized as a diffused pattern in chloroplast. We propose that transcription of most CCM genes, followed by protein level changes including their intracellular localization of a subset is subject to light–dark cycles. Keywords Air-level CO2 · Bicarbonate transporter · Carbonic anhydrase · CO2-concentrating mechanism · Light–dark cycles · Transcriptional regulation Abbreviations CAs Carbonic anhydrases CO2 Carbon dioxide CCM CO2-concentrating mechanism Ci Inorganic carbon DIC Dissolved inorganic carbon L:D Light:dark ML 6 h of incubation in light (mid-light) CL 12 h of incubation in light (complete-light) MD 6 h of incubation in dark (mid-dark) CD 12 h of incubation in dark (complete-dark) qRT-PCR Quantitative real time PCR RT-PCR Reverse transcription PCR
Introduction CO2 undergoes reduction in photosynthetic reactions to form carbohydrates that supplies food and energy to the biosphere. Hence any change in the CO2 availability in local
13
environments would affect the growth and development of inhabitant plants profoundly. Algae and other aquatic plants experience significant constraints in acquiring CO2 from local environment due to very slow diffusion and low solubility of CO2 in water (104 times slower diffusion than that in the air). Moreover, low affinity and very low catalytic turnover of RuBisCO, the key enzyme involved in CO2 assimilation, imposes further constraints on carbon fixation efficiency (Spalding 2009). Algae and aquatic plants also experience limiting CO2 due to difference in CO2 supply dynamics between water and air. Under limiting CO2, RuBisCO shows oxygenase activity fixing O2 rather than CO2, thereby loosing photosynthetic carbon fixation efficiency. Chlamydomonas reinhardtii (C. reinhardtii) can use both CO2 and HCO3− as the inorganic sources of carbon, relative availability of which to cells is determined by solution pH. To overcome limitations in acquiring CO2, C. reinhardtii and a range of aquatic plants have adapted an elaborate CO2-concentrating mechanism (CCM) which actively uptake both forms of inorganic carbon (Ci) (CO2 and HCO3−) (Sultemeyer et al. 1989; Palmqvist et al. 1994) and facilitate maintenance of high levels of CO2 at the site of RuBisCO. Fundamental to CCM acclimation during low to very low-CO2 (air-level), is a mechanism to regulate the scavenging of Ci and concentrate it internally since CO2 supply gets limiting. It has been shown that the induction of CCM occurs at two limiting CO2 conditions, air level (0.03–0.04 %) and below air level CO2 (95 %) and properties, one each was chosen for northern analyses. As expected, all the five control gene transcripts showed specific up regulation in light (Fig. 2b), with hardly any detectable level of transcripts in dark, thereby corroborating our earlier results (Fig. 1a). It was interesting to note that in both the assays (qRT-PCR and northern), while the light regulated transcript levels showed specific rise in light incubations, those up regulated in dark were not entirely specific to dark, but also to light (see Discussion). Thus far, we have uncovered a set of CCM related genes that are up regulated in dark and light (CAH3, CAH6 and LCIB) and another set entirely in light (HLA3, LCI1, CCP1, CCP2, LCIA, CAH4 and CAH5). Most of these genes are implicated to be under the control of a “master regulator” CIA5/CCM1 transcription factor gene (Fang et al. 2012; Wang et al. 2005; Xiang et al. 2001). We therefore assessed CIA5 transcript levels in the same light– dark regime using RT-PCR by gel analyses. As expected of a master regulator gene that can control both light and dark-inducible transcripts, the transcript level of CIA5 was similar in all the four stages, namely MD, CD, ML and CL (Fig. 3a). CIA5 transcript level was low and appeared not to change significantly across MD, CD, ML and CL stages. Since CIA5 transcript level was too low and unchanging across various time-points, it was not analyzed further by either qRT-PCR or northern blot assays. In order to assess whether the observed increase in transcript levels is specific to low CO2 conditions of CCM, we repeated the analyses at high CO2 in dark. We compared CAH6, CAH3 and LCIB transcript levels in low (air- level)
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CAH3 is one of the moderately expressed CAs in C. reinhardtii that is induced in low CO2 conditions where CCM is active (Moroney et al. 2011). Since CCM is active only in light, we were intrigued by the observed increase in CAH3 transcript levels in dark (Fig. 1j, 2a), which prompted us to analyze protein level changes also. For comparison, we also chose CAH4/5 that are highly light induced and abundantly expressed in mitochondria during low CO2 conditions when CCM is active (Moroney et al. 2011). Since CAH4/5 transcript levels go up in light, their protein levels going up in light also serves as an active CCM mark. Western blot analyses showed that CAH3 protein level reached its maximum at 6–12 h in light, followed by a gradual drop in dark reaching the minimum into 9–12 h of dark (Fig. 4a). Therefore it appears that even though transcript levels increase in dark, peaking at 3 h of light, it is only in later half of light period (6–12 h) that CAH3 protein level reaches maximum where CCM is active, revealing a distinct difference in the time course of transcript versus protein levels during day/night cycles (12 h:12 h). In the same experiment, CAH3 immunofluorescence revealed another interesting facet: while the basal level of CAH3 protein present in dark showed a diffuse pattern of distribution, away from pyrenoid in dark, that of enhanced level of CAH3 in light was entirely concentrated to pyrenoid location, where it is most required for CCM (Fig. 4b, S2). This result suggested that CAH3 localization is perhaps well regulated in relation to CCM activity in the cell. This notion is highly consistent with the recent finding that CAH3 protein localization is critically dependent on CCM status of the cell where at high CO2 conditions the protein stays associated with stroma thylakoid, away from pyrenoid, but is recruited to pyrenoid specifically at low CO2 following CAH3 phosphorylation (BlancoRivero et al. 2012)..Western blot analyses using an antibody raised against CAH4/5 combine revealed an expected trend, namely their high expression at 9–12 h of light followed by a gradual drop through dark reaching a minimum towards the end of dark period (6–12 h into dark) (Fig. 5a), a pattern similar to transcript level changes observed earlier (Fig. 1f, g, 2b). These two abundant proteins associated with mitochondria revealed mitochondria-specific
Plant Mol Biol Fig. 4 CAH3 protein analyses: a Western blot analyses of CAH3 from 3 hourly samples (RbcL is the loading control). b Confocal immunofluorescence analyses of ML and MD sample fixed cells by anti-CAH3 Ab. Confocal image stacks arranged from left to right span most of the cell volume. (Scale bar: 3 μm)
Fig. 5 CAH4/5 protein analyses: a Western blot analyses of CAH4/5 from 3 hourly samples (RbcL is the loading control). b Confocal immunofluorescence analyses of ML and MD sample fixed cells by anti-CAH4/5 Ab. Confocal image stacks arranged from left to right span most of the cell volume. c Confocal analyses of mitochondrial staining in live-cells by mitotracker of ML and MD sample cells. Confocal image stacks arranged from left to right span most of the cell volume. (Scale bar: 3 μm)
changes in light versus dark incubation samples: CAH4/5 staining largely paralleled mitochondrial staining pattern as revealed by mitotracker dye (Fig. 5 compare b with c). Most of CAH4/5 was aligned as a continuous ring shaped morphology along the periphery of cell body in light treated cells whereas it was more interspersed towards the interior of the cell in dark (Fig. 5b, S3). We believe that
these changes were largely reflective of alterations in mitochondrial morphology in light versus dark incubations as revealed by mitotracker green staining (Fig. 5c, S3), rather than repositioning of CAH4/5, per se. All these results, put together, suggest that protein level changes in CAH3 and CAH4/5 are distinct and involve additional cellular regulation beyond the transcriptional control.
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Discussion Aquatic photosynthetic organisms acclimate to low CO2 stress by inducing CCM, which operate through the concerted functioning of a number of Ci transporters and CAs. It is well known that CCM is active in the light and inactive in the dark. Although physiological aspects of CO2 response in light and dark have been extensively studied, expression level regulation of CCM genes in light versus dark regime has not been well investigated. In this report, we describe transcriptional changes of CCM genes at different time points of day/night cycles (12 h:12 h) (3 or 6 hourly interval samples) using synchronized cells, grown photoautotrophically at air-level CO2. We show here that putative bicarbonate transporter gene transcripts (HLA3, LCI1 and LCIA) are up regulated in light at the air level CO2 conditions. The same trend is seen with chloroplast carrier protein genes (CCP1 and CCP2) (Fig. 1, 2b). Both type of Ci transporters, namely that of plasma membrane and chloroplast, are up regulated in light. The observed up regulation of Ci transporter gene transcripts in light is consistent with their projected role in CCM, which is specifically operational in light. Several reports indeed strongly implicate these transporters in CCM function (Duanmu et al. 2009a; Ohnishi et al. 2010). Interestingly, mitochondrial CA gene transcripts (CAH4 and CAH5) are also up regulated in light (Fig. 1f, g, 2b). It is likely that the light mediated up regulation of CAH4 and CAH5 transcripts via their protein products could facilitate an efficient augmentation of HCO3− production that can then be routed to chloroplast for efficient CCM. Our observation that CAH4 and CAH5 gene transcripts are up regulated in light is consistent with the results of Eriksson et al, where they had shown circadian up regulation of mitochondrial CA RNA’s specifically during the light periods in low CO2 conditions (Fig. 5 in Eriksson et al. 1998). A point of caution also needs to be emphasized here, namely, the conditions that favor mitochondrial CA expression usually also stimulate anaplerosis (Giordano et al. 2003). Protein level changes also showed that CAH4/5 reach maximum by 9–12 h into light, followed by a drop in dark, reaching minimum levels at 6–9 h into dark (Fig. 5a). As expected, immunofluorescence studies revealed that these proteins follow localization changes that are largely reflective of mitochondrial morphology changes in light–dark regimes (Fig. 5b–e, S3). Live staining pattern of cells by Mitotracker green is qualitatively similar to that of CAH4/5 specific immunofluorescence pattern in fixed cells, even though the sample processing conditions in these two experiments are distinctly different (compare Fig. 5b with c). Transcriptional regulation cascade that is driven by light-regulated promoters is poorly understood in C.
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Plant Mol Biol
reinhardtii. A recent bioinformatic study makes a compelling case for deciphering such regulatory network by experimental approaches (Riano-Pachon et al. 2008). In view of paucity of information, it is unclear whether the light up regulated transcript levels of HLA3, LCI1, LCIA, CCP1, CCP2, CAH4 and CAH5 reflects a direct consequence of light-regulated promoter activity, if any, or a consequence of an indirect regulation. We are currently trying to locate light response elements in some of these gene promoters. Counter-intuitively, unlike that of Ci transporter genes, transcript levels of CA’s (CAH6: chloroplast stromal CA; CAH3: thylakoid lumen CA) are up regulated in dark (Fig. 1, 2a). These CAs are strongly implicated in CCM. CAH1 was not studied, as it is not strongly implicated in CCM functions (Ohnishi et al. 2010; Van and Spalding 1999). CAH3 and CAH6 genes showed high accumulation of transcripts even in dark period. We also included chloroplast stromal soluble protein LCIB in our studies that functions downstream of CAH3 (Duanmu et al. 2009b). LCIB also showed up regulation by the end of dark period (Fig. 1). Northern blot analyses performed with closely spaced time-interval samples showed that CAH3 transcript level reached maximum by the end of dark, followed by high expression even into early light. CAH6 transcript level stayed high in dark for a longer duration, followed by high level even in light. Contrary to the pattern of CAH6, that of LCIB expression in dark was followed by its expression in early hours of light, very similar to that of CAH3 (Fig. 2a). Most observations reported in the literature so far on CA expression levels are related to light conditions where CCM is active. The observed up regulation of CAH3, CAH6 and LCIB transcripts in dark ensued in normal air level CO2 conditions. To test whether up regulation in dark of LCIB and its upstream component CAH3 transcript was dominant even in high CO2 conditions, we compared their transcript levels at high CO2 (~3 %). The up regulation seen at low CO2 (normal air-level CO2) in dark was undone in high CO2 (Fig. 3b). This result indicated that the up regulation of dark induced transcripts at air level of CO2 is not solely dependent on dark, but also on CO2 conditions, suggesting that CCM components are under regulation even in dark at air level of CO2 where CCM is inactive. This interesting, but not well appreciated, aspect had been pointed out by Rawat and Moroney several years ago (Rawat and Moroney 1995). We have schematized the transcript level changes observed so far in the study in the perspective of complete CCM regulation known to date (Supplementary Fig. S4). To understand transcriptional up regulation of some of the important CCM genes in dark when CCM is not operational, we performed further studies. Western analyses revealed that protein level changes follow a different trend: even though CAH3 transcript is up regulated in dark,
Plant Mol Biol
followed by that in light (Fig. 1, 2a), protein level reaches maximum only in light (6–12 h into light), followed by a drop in dark leading to a minimum at 12 h into dark (Fig. 4a). Protein level changes were consistent with its functional requirement in CCM during light. As observed by Yamano et al. for LCIB, immunofluorescence staining of CAH3 also revealed that the protein specifically localizes to pyrenoid during light. Intense staining of CAH3 protein was observed in pyrenoid at ML. In contrast, the protein diffuses away into a punctate distribution away from pyrenoid in dark (MD) (Fig. 4b, S2). This is very similar to Yamano et al. results that LCIB localizes to pyrenoid in light from where it diffuses away in dark (Yamano et al. 2010). Recent finding by (Blanco-Rivero et al. 2012) showing CAH3 localization to pyrenoid at low CO2 specifically following its phosphorylation strongly reinforces the view that some of these CCM proteins are under strict regulation with respect to their cellular localization. All these results strongly suggest parallels between CAH3 and LCIB, pointing towards a regulatory control that encompasses transcriptional up regulation of these genes in dark and protein translational up regulation in light. It is relevant to reiterate here, that CAH3 and LCIB transcript level changes in dark versus light time-points are nearly identical (Fig. 1, 2a). In addition, there is perhaps an added layer of regulation that promotes protein recruitment to pyrenoid in light, whose absence in dark leads to release of the protein from pyrenoid, followed by punctate distribution throughout the chloroplast (Fig. 4b, S2). Protein recruitment in and out of pyrenoid as a function of light–dark cues may in fact be an important regulation in C. reinhardtii cells even in the context of non-CCM functions as suggested by our recent study involving a UV-induced DNA-endonuclease (UVI31+) (Shukla et al. 2012). Our current study describes that CCM gene transcript level changes operative at air level CO2 conditions are perhaps critically shaped by light–dark rhythmic changes. The synchronized cells provided an opportunity to dissect the role of light–dark cycles on CCM regulation. Interestingly, the protein products of dark up regulated transcripts, namely, CAH3, CAH6 and LCIB are implicated in CO2 recapture mechanisms (Yamano et al. 2010) and we are tempted to speculate that such recapture steps might indeed be active and relevant in dark as well using the basal level of protein products formed in dark, so that CO2 does not leak out. Alternately, the transcripts formed in dark can serve as ready reservoir of mRNA for a rapid production of protein “on demand” for CCM in light. It is not clear why a small subset of CCM transcripts is up regulated in dark whereas the majority is specifically up regulated in light. It is interesting to mechanistically explore this dichotomy for which more detailed molecular and cellular analyses are required.
Acknowledgments This work would not been possible without the help and cooperation from BJ lab people whom we profusely thank. We want to thank Prof. J.V. Moroney for sharing CAH4/5 antibody preparation. A special acknowledgement is due to Dr Ullas Kolthur and Upasana Roy for their help in qPCR work. MK is thankful to M Vijayalakshmi (HOD), Botany and Microbiology Department, Acharya Nagarjuna University for sanctioning leave to carry out this work. MK is also thankful to Prof. G Dr PCO Reddy (YV University, Kadapa) for useful discussions and help. BJR acknowledges JC Bose fellowship grant (DST). Department of Atomic Energy (Government of India) grant to Tata Institute of Fundamental Research (TIFR), Mumbai.
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