A pyrenoid-localized protein SAGA1 is necessary for Ca 2+ -binding protein CAS-dependent expression of nuclear genes encoding inorganic carbon transporters in Chlamydomonas reinhardtii

doi:10.21203/rs.3.rs-2215327/v1

Download PDF

Research Article

A pyrenoid-localized protein SAGA1 is necessary for Ca 2+ -binding protein CAS-dependent expression of nuclear genes encoding inorganic carbon transporters in Chlamydomonas reinhardtii

https://doi.org/10.21203/rs.3.rs-2215327/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 19 Jan, 2023

Read the published version in Photosynthesis Research →

You are reading this latest preprint version

Microalgae induce a CO₂-concentrating mechanism (CCM) to maintain photosynthetic affinity for dissolved inorganic carbon (Ci) under CO₂-limiting conditions. In the model alga Chlamydomonas reinhardtii, the pyrenoid-localized Ca²⁺-binding protein CAS is required to express genes encoding the Ci transporters, high-light activated 3 (HLA3) and low-CO₂-inducible protein A (LCIA). To identify new factors related to the regulation or components of the CCM, we isolated CO₂-requiring mutants KO-60 and KO-62. These mutants had insertions of a hygromycin-resistant cartridge in the StArch Granules Abnormal 1 (SAGA1) gene, which is necessary to maintain the number of pyrenoids and the structure of pyrenoid tubules in the chloroplast. In both KO-60 and the previously identified saga1 mutant, expression levels of 532 genes were significantly reduced. Among them, 10 CAS-dependent genes, including HLA3 and LCIA, were not expressed in the saga1 mutants. While CAS was expressed normally at the protein levels, the localization of CAS was dispersed through the chloroplast rather than in the pyrenoid, even under CO₂-limiting conditions. These results suggest that SAGA1 is necessary not only for maintenance of the pyrenoid structure but also for regulation of the nuclear genes encoding Ci transporters through CAS-dependent retrograde signaling under CO₂-limiting stress.

CO2-concentrating mechanism

CCM1

CRISPR/Cas9

retrograde signaling

starch-binding protein

Photosynthetic organisms are capable of sensing and responding to various environmental stresses. Guard cells of terrestrial plants, for example, sense the concentrations of extracellular carbon dioxide (CO₂), the substrate for photosynthesis, and control the opening and closing of stomata to take it in (Hashimoto et al., 2006). In aquatic environments, CO₂ exists as inorganic carbon (Ci; CO₂ and HCO₃^–), and the diffusion rate of CO₂ is substantially slower than in the atmosphere. To maintain efficient photosynthetic activity under such CO₂-limiting conditions, most microalgae induce a CO₂-concentrating mechanism (CCM) by actively taking up extracellular Ci and increasing the CO₂ concentration around the CO₂-fixation enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)(Fukuzawa et al. 2012).

Chlamydomonas can adapt to at least two CO₂-limiting conditions (Vance and Spalding 2005): very low-CO₂ (VLC; < 7 µM CO₂) and low-CO₂ (LC; 7–70 µM CO₂). Under VLC conditions, active HCO₃^– uptake is driven by high-light activated 3 (HLA3), an ABC-type transporter located in the plasma membrane, low-CO₂-inducible protein A (LCIA), an anion channel located in the chloroplast envelope, and bestrophin-like proteins (BST1–3) located in the thylakoid membranes. Insertional mutants of HLA3 and LCIA show a reduced affinity for Ci, especially under alkaline conditions where HCO₃^– is the predominant form of Ci (Yamano et al., 2015), and RNAi mutants with reduced expression of BST1–3 are unable to grow in CO₂-limiting conditions, exhibit a reduced affinity for Ci, and show reduced Ci-uptake (Mukherjee et al., 2019). Moreover, a mutant of low-CO₂-inducible protein 1 (LCI1) shows reduced Ci-affinity in acidic conditions, where CO₂ is the predominant form of Ci, suggesting that LCI1 is involved in CO₂ uptake (Kono and Spalding, 2020). LCI1 has also been reported to localize to the plasma membrane (Ohnishi et al., 2010) and to interact with HLA3 (Mackinder et al., 2017), but the physiological significance of the interaction remains unclear.

In Chlamydomonas, a pyrenoid, a liquid-liquid phase-separated organelle in the chloroplast, plays an essential role in photosynthetic CO₂ fixation (Freeman Rosenzweig et al., 2017). The pyrenoid matrix aggregated with Rubisco is surrounded by a starch sheath and penetrated by thylakoid membranes called pyrenoid tubules (Engel et al., 2015). Recently, two Chlamydomonas proteins required for pyrenoid formation have been identified. One is Essential Pyrenoid Component 1 (EPYC1), the Rubisco-binding protein that localizes in the pyrenoid matrix (Mackinder et al., 2016). Considering that EPYC1 is required for a development of pyrenoid structure and that Rubisco and EPYC1 are both necessary and sufficient to phase separate and form liquid droplets in vitro, EPYC1 is shown to function as a linker protein of Rubisco to form a phase-separated pyrenoid (Wunder et al., 2018). The other is StArch Granules Abnormal 1 (SAGA1), the Rubisco- and starch-binding protein that localizes between the starch sheaths and the pyrenoid matrix (Itakura et al., 2019). The saga1 mutant forms multiple pyrenoids which lack pyrenoid tubules, which causes a decrease in photosynthetic Ci-affinity. Therefore, SAGA1 could be a linker between the starch sheath and Rubisco that functions to maintain pyrenoid morphology. Moreover, based on the analysis of the Rubisco-binding motif derived from SAGA1, a common motif that is necessary and sufficient for targeting the pyrenoid was revealed and novel pyrenoid proteins such as SAGA2, RBMP1, RBMP2, and CSP41A have been identified (Meyer et al., 2020).

Although the genes related to the pyrenoid formation tend to be constitutively expressed, the expression of genes that encode Ci-transporters and several carbonic anhydrases (CAs) are induced under CO₂-limiting conditions by CCM1/CIA5, a master regulator of the CCM (Fukuzawa et al., 2001; Miura et al., 2004; Fang et al., 2012). A recent study revealed that the basic leucine zipper transcription factor 8 (BLZ8) is involved in the expression of HLA3, CAH7, and CAH8 in response to oxidative stress conditions (Choi et al., 2022); however, the genetic relationship between CCM1/CIA5 and BLZ8 during CCM induction is not known. Additionally, the mutant strain H82 which lacks the Ca²⁺-binding protein CAS demonstrates reduced growth, Ci-affinity, and more than four-fold lower transcriptional levels for 13 genes (Wang et al., 2016), including HCO₃^– transporters (HLA3 and LCIA), mitochondrial-localized CAs (CAH4 and CAH5), mitochondrial membrane transporters (CCP1 and CCP2), chaperone proteins (DNJ31), proteins involved in non-photochemical quenching (LHCSR3.1 and LHCSR3.2), phosphoprotein phosphatase 2C-related (PPP30), and unknown proteins (LCID, Cre12.g541550, and Cre26.g756747). Considering that CAS is dispersed throughout the chloroplast under high-CO₂ (HC; > 70 µM CO₂) conditions but migrates into the pyrenoid through the pyrenoid tubules under CO₂-limiting conditions (Yamano et al., 2018), the CAS-mediated retrograde signal from pyrenoid to nucleus could play an important role in regulating the CCM (Wang et al., 2016).

Another essential CCM component is LC-inducible protein B (LCIB). LCIB has a structure like β-type CA (Jin et al., 2016) and is dispersed throughout the chloroplast under HC conditions but localizes around the starch sheath of the pyrenoid under VLC conditions (Yamano et al., 2010; Yamano et al., 2022). The starch sheath is required for LCIB localization around the pyrenoid and for maintaining the Ci-affinity (Toyokawa et al., 2020). These findings suggest that LCIB has a role in converting CO₂ leaking from the gaps in the starch sheath to HCO₃⁻ under VLC conditions.

Thus, it is becoming clear that the pyrenoid structure is not simply a site of CO₂ fixation but is necessary to the precise localization of CAS and LCIB and to efficiently transport Ci while maintaining relatively complex structures such as starch sheaths and pyrenoid tubules. However, the global impact of pyrenoid morphological abnormalities on gene expression has remained unclear. It is also important to know whether the reduced photosynthetic affinities for Ci of saga1 mutants were caused solely by structural disruption of the pyrenoid. In this study, we focused on the subcellular relocation of the CAS, which regulates nuclear genes encoding Ci-transporters involved in the CCM. It is interesting to know whether SAGA1-dependent pyrenoid structure contributes to the localization of CAS or not. We provide the physiological and gene expression data using comparative RNA-sequencing analysis of the SAGA1 allele mutants isolated from a random insertional mutant library and discuss the impact of pyrenoid morphological changes on nuclear gene expression.

Algal strains and cultural conditions

Chlamydomonas reinhardtii wild-type (WT) strain C9 was originally provided from IAM Culture Collection at the University of Tokyo and is now available from Microbial Culture Collection at the National Institute for Environmental Studies, Japan, as strain NIES-2235 (alternatively named CC-5098 in the Chlamydomonas Resource Center). Strains CC-5325, CC-5420 (saga1), and CC-5422 (SAGA1-Venus/saga1) were obtained from the Chlamydomonas resource center.

For physiological and biochemical experiments, cells were precultured in TAP medium as described previously (Wang et al., 2016). Then, cells were resuspended in 50 mL of modified HSM supplemented with MOPS and grown in air containing 5% (v/v) CO₂ with illumination at the intensity of 120 µmol photons·m^–2·s^–1 until cultures reached mid-log phase as described previously (Wang et al., 2016). For induction to VLC condition, HC-acclimated cells were centrifuged, resuspended in 50 mL of fresh HSM medium, and further cultured with bubbling of ordinary air containing 0.04% (v/v) CO₂ at 120 µmol photons·m^–2·s^–1 for 2 or 12 h.

Transformation Of Cells

A DNA cassette containing the hygromycin (hyg)-resistant gene aphVII driven by the β2-tubulin promoter was amplified by PCR from plasmid pHyg3 (Berthold et al., 2002) using PrimeSTAR GXL DNA Polymerase (Takara Bio). The PCR product was purified using a PCR purification kit (QIAGEN), and the concentration was adjusted to 100 ng µL^− 1. The gene cassette was used to transform C9 cells by electroporation using a NEPA-21 electroporator (NEPAGENE), as described previously (Yamano et al., 2013). The transformants were incubated at 25˚C for 24 h with gentle agitation under illumination of 1.5 µmol photons m^− 2·s^− 1 and then screened on TAP plates containing 30 µg mL^− 1 hyg at 25˚C under illumination of 80 µmol photons m^− 2·s^− 1. After 4 days, colonies of transformants appeared and were subjected to the screening process of HC-requiring mutants.

Screening Of Hc-requiring Mutants

Cells were replicated into 96-well microtiter plates containing liquid TAP medium. For the first screening, cells were spotted on agar plates at pH 6.2 and 8.4 and grown in 0.04% [v/v] CO₂ chambers for 4 days. Transformants that did not grow under 0.04% CO₂ conditions were selected as candidates for HC-requiring mutants. For the second screening, the candidate mutants grown in liquid TAP medium were diluted to an optical density at 700 nm (OD₇₀₀) of 0.15, 0.07, or 0.03, and 3 µL of each cell suspension was spotted onto agar plates at pH 6.2, 7.0, 7.8 and 9.0 and grown for 4 days in different chambers supplied with air containing 5% [v/v] CO₂, 0.04% CO₂ and 0.01%, respectively.

Measurement Of Photosynthetic O-evolution

Cells were harvested and suspended in Ci-depleted 20 mM MES-NaOH buffer (pH 6.2), MOPS-NaOH buffer (pH 7.0), HEPES-NaOH buffer (pH 7.8), or AMPSO-NaOH buffer (pH 9.0) to a density of 10 µg mL^− 1 chlorophyll. The photosynthetic O₂-evolving rate was measured using a Clark-type O₂ electrode (Hansatech Instruments), as described previously (Yamano et al., 2008).

Generation of ccm1 mutant by the CRISPR-Cas9 method

For CRISPR-Cas9-mediated genome editing, the guide RNAs was designed using CRISPOR (Concordet and Haeussler, 2018) and shown in the supplemental Figure S1a. Introduction of ribonucleoprotein complex and the aphVII cassette into cells were performed according to Tsuji et al. (2022). After transformation cells were incubated at 25˚C for 24 h in dim light and then screened on TAP plates containing 30 µg mL^− 1 hyg at 25˚C under 80 µmol photons m^− 2·s^− 1 of light for 7 days.

Immunoblotting Analysis

Extraction of total protein, SDS-polyacrylamide gel electrophoresis (SDS/PAGE), and immunoblotting analyses were performed as described previously (Wang et al., 2016). The primary antibodies were used at the indicated dilutions: anti-HLA3 (1:1,250), anti-LCIA (1:5,000), anti-LCI1 (1:5,000), anti-LCIB (1:5,000), anti-CAH1 (1:2,500), anti-CAH3 (1:2,000), anti-CCM1 (1:2,500), anti-CAS (1:5,000), and anti-Histone H3 (1:10,000). To recognize the primary antibody, a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Life Technologies) was used as a secondary antibody in a dilution of 1:10,000.

Rna-seq Analysis

Total RNA was extracted from cells cultured in LC conditions for 2 h using a RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s protocol. After RNA purification, total RNA was analyzed by GENEWIZ (https://www.genewiz.com/) using Illumina Novaseq. The resulting reads were aligned to version 5.5 of the Chlamydomonas reinhardtii genome annotation (downloaded from https://phytozome-next.jgi.doe.gov/). Alignments were performed using the HISAT2 program. Counts of the number of reads mapping to each gene were calculated by HTSeq. The resulting expression counts were normalized with the trimmed mean of the M-values method as implemented in the edgeR package. Differentially expressed genes with FDR < 0.01 between strains were plotted as volcano plots.

Indirect Immunofluorescence Assay

Cells cultured in VLC conditions were collected by centrifugation and rinsed with phosphate-buffered saline (PBS) buffer twice. Immunofluorescence staining by purified antibodies against CAS and LCIB was performed as described previously (Wang et al., 2016). The primary antibodies against CAS and LCIB were used at dilutions of 1:500 and 1:200, respectively. Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) was used at a 1:500 dilution.

Measurement Of Co Concentration In The Culture Medium

Cell suspensions were centrifuged to remove cells from the supernatant. Subsequently, aliquots of the supernatant were directly injected, and total Ci concentrations were measured using a gas chromatograph GC-8A (Shimadzu) with a methanizer MTN-1 (Shimadzu) as described previously (Toyokawa et al., 2020). CO₂ concentrations were also calculated as described previously (Toyokawa et al., 2020)

Isolation of CO₂-requiring mutants KO-60 and KO-62　

To obtain mutants defective in the induction of the CCM, we constructed a mutant library and isolated CO₂-requiring mutants, such as the cas mutant H82, which could not grow under CO₂-limiting conditions. First, we constructed a random insertional mutant library by transforming a wild-type (WT) strain, C9, with an aphVII cassette conferring hyg-resistance, yielding ~72,000 transformants. Next, because the mutant H82 showed a significantly reduced growth rate under LC conditions with air containing 0.04% (v/v) CO₂ (Wang et al., 2016), we evaluated the growth rate of the mutants on agar plates under LC conditions as a first screening and selected two strains, KO-60 and KO-62.

To further evaluate the growth under multiple CO₂-acclimated conditions, growth of C9, H82, KO-60 and KO-62 under 5% CO₂, 0.04% CO₂ or 0.01% CO₂ conditions was examined at pH 6.2 (HCO₃^–/CO₂ = 0.71), 7.0 (HCO₃^–/CO₂ = 4.46), 7.8 (HCO₃^–/CO₂ = 28.18) and 9.0 (HCO₃^–/CO₂ = 446.68) (Fig. 1a). Under 5% CO₂ conditions, there were no significant differences in growth rates among strains. In contrast, under LC conditions, growth rates of KO-60 and KO-62 were reduced at pH 6.2, 7.0, and 7.8 and severely reduced at pH 9.0. Furthermore, under VLC conditions, KO-60 and KO-62 showed severely reduced growth at all tested pH conditions, as was the case for H82.

To evaluate the induction of the CCM under LC conditions, the photosynthetic affinity for Ci was evaluated by measuring the rates of photosynthetic oxygen (O₂) evolution. The K_0.5(Ci) values, the Ci concentrations required for half of the maximal rate of O₂-evolving activity (V_max), in KO-60 (554 ± 130 µM) and KO-62 (471 ± 40 μM) cells were 4.4 and 3.7 times higher than that of C9 cells (126 ± 34 µM) at pH 9.0, respectively (Fig. 1b). Even at pH 6.2, 7.0 and 7.8, the K_0.5(Ci) values of KO-60 and KO-62 were over 1.5 times higher than that of C9 cells. The K_0.5(Ci) values of H82 cells were also higher than those of C9 cells at all tested pH conditions (Wang et al., 2016). At pH 6.2, 7.0, and 7.8, the V_max of KO-60 and KO-62 were 69–84% of that of C9 (Fig 1c). At pH 9.0, the V_max of KO-60 and KO-62 were 47–61% of that of C9. These results showed that both KO-60 and KO-62 had reduced Ci-affinity and V_max. On the other hand, the V_max of H82 cells is not significantly different from C9 at any pH condition (Wang et al., 2016). These results showed that KO-60 and KO-62 had reduced Ci-affinity regardless of pH conditions.

Protein levels of Ci transporters decreased in KO-60 and KO-62

To elucidate the cause of the impaired growth and reduced Ci-affinity of KO-60 and KO-62, we examined the accumulation levels of CCM-related proteins – including Ci-transporters (HLA3, LCIA, and LCI1), CAs (LCIB, CAH1, and CAH3), and regulatory factors (CCM1 and CAS) – in C9, ccm1-2 (a newly generated ccm1 mutant with a C9 background made using the CRISPR/Cas9 system; Fig. S1a-c), H82, KO-60, and KO-62. CCM1 and CAS were not detected in ccm1-2 and H82, respectively, and CAH3 was detected in all strains.

After shifting conditions from HC to LC, C9 induced the accumulation of HLA3, LCIA, LCI1, LCIB, and CAH1, while ccm1-2 showed little or markedly reduced accumulation. On the other hand, KO-60 and KO-62 showed little accumulation of HLA3, LCIA, and LCI1 as in the case of H82, even though CAS was accumulated at the same level as in C9 (Fig. 2a).

To determine the insertion site of the aphVII cassette in KO-60 and KO-62, thermal asymmetric interlaced (TAIL)-PCR was performed. In KO-60 and KO-62, the aphVII cassette was inserted in the 10^th and 15^th intron of the SAGA1 gene, respectively (Supplemental Fig. S1d and e). To confirm that SAGA1 is defective in KO-60 and KO-62, we examined the number of pyrenoids in C9, KO-60, and KO-62 cells by introducing a plasmid expressing Venus-tagged Rubisco small subunit 1 (RBCS1-Venus). Although a single fluorescent signal was observed in the C9 background, multiple fluorescent signals were observed in KO-60 and KO-62 background cells (Supplemental Fig. S1f), as in the case of the original saga1 mutant (Itakura et al., 2019), indicating that KO-60 and KO-62 were loss-of-function mutants of SAGA1.

Next, to further investigate whether the reduced accumulation of Ci transporters was due to the SAGA1 disruption, we also examined the accumulation of the above CCM-related proteins in the original saga1 mutant (Itakura et al., 2019). As in the case of KO-60 and KO-62, HLA3, LCIA, and LCI1 were not accumulated in saga1 compared to the parental strain, CC-5325. Furthermore, the complemented strain recovered the accumulation of these Ci transporters (Fig. 2b), suggesting that changes in pyrenoid morphology due to the disruption of SAGA1 are responsible for the reduced accumulation of Ci-transporters.

Decrease in the mRNA levels of CCM1-dependent genes in the saga1 mutants

To examine the effect of pyrenoid morphological changes caused by the SAGA1 mutation on nuclear gene expression, C9, KO-60, CC-5325, saga1, and complementary strain cells were cultured while bubbling with ordinary air for 2 h, and the respective transcriptome profiles were compared by RNA-seq analyses. Comparison of the transcriptomes of C9 and KO-60, and CC-5325 and saga1, showed that the expression levels of 2,288 and 3,633 genes, respectively, were significantly reduced (FDR [false discovery rate] < 0.01; Figs. 3 and S2a). Of the genes down-regulated as a result of SAGA1 mutations in KO-60 and saga1, 532 were recovered in the saga1 complemented strain (Supplemental Dataset S1).

To assess whether changes in pyrenoid morphology in saga1 mutants affect the expression of CCM-related genes, we compared the 532 genes down-regulated in KO-60 and saga1 but recovered in the complemented strain with those that are induced under CO₂-limiting conditions and regulated by CCM1/CIA5 (Fang et al., 2012). The expression levels of 41 CCM1-dependent genes were decreased in KO-60 and saga1 (Table 1). In particular, among the 13 genes regulated by CAS (Wang et al., 2016), the expression levels of 10 genes (HLA3, LCIA, LHCSR3.1, LHCSR3.2, CAH4, CAH5, CCP1, CCP2, LCID, and DNJ31) decreased in both KO-60 and saga1 and recovered in the saga1 complemented strain (Fig. 3). The expression of LCI1 was not significantly reduced in KO-60 but was reduced in saga1 and recovered in the complemented strain. These results indicate that the changes in pyrenoid morphology caused by the SAGA1 mutation affect the expression of most CAS-dependent genes.

In addition to CAS-dependent genes, CCM1-regulated LCIE and DNJ15 were also downregulated more than four-fold. LCIE is a homolog of LCIB (Yamano et al., 2010) and DNJ15 has a domain that acts as a chaperone that may interact with LCIB and LCIC (Mackinder et al., 2017).

Next, focusing on genes with upregulated expression in the saga1 mutants, we compared the transcriptomes of C9 and KO-60, and CC-5325 and saga1, and found that the expression levels of 2,352 and 3,725 genes were significantly induced in KO-60 and saga1, respectively (Fig. S2b). Of the genes upregulated in the saga1 mutants KO-60 and saga1, 570 were recovered in the saga1 complemented strain (Supplemental Dataset S2). When comparing those 570 genes with CCM1/CIA5-dependent genes, the expression levels of STA2, BST1, and BST2 were increased in KO-60 and saga1. STA2 is localized in the starch sheath (Mackinder et al., 2017), and pyrenoid morphology is altered in the sta2 mutant (Delrue et al., 1992). BST1 and BST2 are anion channels localized in the thylakoid membrane close to the pyrenoid (Mukherjee et al., 2019).

CAS and LCIB were dispersed throughout the chloroplast in the saga1 mutants

Given that CAS-dependent gene expression was impaired in the saga1 mutants and that CAS re-localizes into the pyrenoid upon CCM induction, abnormal pyrenoid formation may affect CAS localization. To test this hypothesis, we examined CAS localization in the saga1 mutants using an indirect immunofluorescence assay. Previous studies showed that CAS dispersed throughout the chloroplast stroma moves to the pyrenoid along the pyrenoid tubules under CO₂-limiting conditions (Yamano et al., 2018). In C9, CC-5325, and the complemented strain, CAS was localized inside the pyrenoid as a distinct wheel-like structure, whereas in KO-60 and saga1, CAS was dispersed in the chloroplast (Figs. 4a and S3).

To assess the effect of abnormal pyrenoid formation on another pyrenoid peripheral protein, we also examined the localization of LCIB. Because the localization changes of LCIB are strictly controlled by CO₂ concentration with ∼7 µM as the boundary (Yamano et al., 2022), the CO₂ concentration in the medium was calculated with the observation of LCIB localization. Although LCIB was localized around the pyrenoid in C9, CC-5325, and the complemented strain during aeration with air containing 0.04 % (v/v) CO₂ for 12 h, it was dispersed in the chloroplast in KO-60 and saga1 under VLC conditions (Figs. 4b and S4). These results suggest that the abnormal pyrenoid morphology caused by SAGA1 mutations impairs the localization of CAS and LCIB under CO₂-limiting conditions, resulting in defective CAS-dependent expression of CCM-related genes and a reduced Ci-affinity due to the lack of LCIB-based capture of CO₂ leaking from the pyrenoid.

In this study, we examined a broader range of phenotypes than previously thought to be caused by mutations in SAGA1, highlighting the importance of SAGA1 in the algal CCM. In WT cells, HLA3, LCI1, and LCIA take up Ci into the chloroplast stroma, and LCIB around the pyrenoid converts leaked CO₂ to HCO₃⁻ under VLC conditions. In addition, CAS migrates into the pyrenoid to induce the expression of CCM-related genes (Fig. 5a). However, in the saga1 mutants, not only is CO₂ recapture inhibited because LCIB is dispersed in the chloroplast, but the expression of CCM-related genes is impaired because CAS is also dispersed in the chloroplast (Fig. 5b). These results suggest that the reduced photosynthetic Ci-affinity exhibited by the saga1 mutants is not only due to abnormalities in pyrenoid formation but also to complex effects, including reduced expression of CCM-related genes and aberrant localization of LCIB and CAS.

Decreased accumulation of Ci transporter in saga1 mutants contributes to the reduced Ci-affinity

Although the saga1 mutants showed multiple pyrenoids, elongated starch sheaths, and loss of pyrenoid tubules (Itakura et al., 2019), it was not clear how these various phenotypes related to reduced photosynthetic activity. We showed that the accumulation levels of Ci-transporters, including HLA3, LCIA, and LCI1, were decreased in all saga1 allelic mutants (Fig. 5b). hla3 and lcia double mutants had reduced Ci-affinity, especially at pH 9.0 (Yamano et al., 2015), and the lci1 mutant had reduced Ci-affinity under pH 6.0 and 7.3 conditions (Kono and Spalding, 2020). These phenotypes are one of the pieces of evidence that strongly suggest that HLA3/LCIA and LCI1 are involved in HCO₃^– and CO₂ uptake, respectively.

The saga1 mutants showed two-times higher K_0.5 (Ci) values than the hla3 and lcia double mutants, suggesting that the pyrenoid abnormality in saga1 mutants may affect expression of CCM-related genes in addition to these two Ci-transporter genes. By RNA-seq analysis, we revealed that the expression levels of CAs CAH4 and CAH5, mitochondrial membrane proteins CCP1 and CCP2, and Ci-transporter genes were decreased in the saga1 mutants. Mutant strains of CAH4 and CAH5 have reduced Ci-affinity under aerated conditions (Rai et al., 2021), and it was suggested that the mitochondrial CAs might play a role in the recapture of CO₂ leaking from chloroplasts. CCP1 and CCP2 are induced under LC conditions (Fang et al., 2012; Wang et al., 2016) and localize to the mitochondria (Atkinson et al., 2016). Although the growth of CCP1/CCP2 knockdown strains was reduced under LC conditions, their Ci-affinity was not affected (Pollock et al., 2004). Therefore, the reduced growth and Ci-affinity observed in saga1 mutants may partly be due to the reduced accumulation of Ci-transporters and mitochondria-localized CCM factors.

The Presence Of Multiple Pyrenoids Inhibits Lcib Migration

Under VLC conditions, LCIB migrates to the basal region of the chloroplast and localizes around pyrenoids (Yamano et al., 2022). This suggests that there is a mechanism by which LCIB recognizes the position of the pyrenoid and moves to the basal region of the chloroplast. In the isa1 mutant, which has lost its starch sheath, LCIB does not localize with the ring-like structure under VLC conditions but moves to the basal region of the chloroplast (Toyokawa et al., 2020). In contrast, LCIB migration is inhibited in the saga1 mutants, which have multiple pyrenoids surrounded by a thin starch sheath. This suggests that multiple pyrenoids in the chloroplast may prevent LCIB from recognizing the chloroplast basal region. Another possibility is that morphological changes in pyrenoids, rather than just an increase in the number of pyrenoids, may affect LCIB localization. Considering that LCIB is localized as puncta around the pyrenoid periphery (Yamano et al., 2010) and BST3 localizes to the thylakoid membrane and interacts with LCIB and LCIC (Mukherjee et al., 2019; Mackinder et al., 2017), LCIB could localize at the site of thylakoid membrane penetration into the pyrenoid matrix and the presence of pyrenoid tubules is essential for the localization of LCIB around the pyrenoid. Therefore, the loss of the pyrenoid tubules in saga1 mutants may cause the inhibition of LCIB localization.

Morphological Changes In Pyrenoids Affect Ccm Regulation

Under CO₂-limiting conditions, Ca²⁺ concentration in the pyrenoid increases (Wang et al., 2016). Although the physiological significance of the increase is not yet clear, it is possible that the coexistence of CAS and high concentrations of Ca²⁺ in the pyrenoid is important for the activation of CAS function, as CAS exhibits low-affinity and high-capacity Ca²⁺ binding properties (Han et al., 2003). Thus, CAS dispersion and inhibition of CAS-Ca²⁺ binding in saga1 mutants may be responsible for the suppression of gene expression. However, several CAS-dependent genes (PPP30, Cre12.g541550, and Cre26.g756747) were expressed in saga1 mutants, suggesting that the binding of dispersed CAS to Ca²⁺ may induce the expression of these genes. Isolation and analysis novel mutants in which CAS does not localize to pyrenoid under LC conditions could clarify the relationship between CAS localization and gene regulation.

Accession Numbers

The accession number of the SAGA1 gene in the Phytozome database v6.1 for Chlamydomonas is Cre11.g467712.

Author contributions

H.F. and T.Y. conceived and designed the study; all authors contributed to the construction of a mutant library and mutant screening; D.S. contributed to characterization of the mutants; D.S. and T.Y. performed RNA-seq analysis; D.S., T.Y. and H.F. wrote the manuscript, and all authors edited and approved the final manuscript.

Acknowledgments

We thank Noriko Kozai, Tomoya Honjo, Toshiki Matsuoka, and Asuka Miyamoto for constructing the mutant library. We thank Lilly LaPlace for critical reading. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grants JP16H04805, JP16K07399, JP20H03073, and JP21K19145, Advanced Low Carbon Technology Research and Development Program (ALCA) of the Japan Science and Technology Agency, and the ImPACT program of the Council for Science, Technology, and Innovation of Japan.

Atkinson N, Feike D, Mackinder LCM, Meyer MT, Griffiths H, Jonikas MC, Smith AM, McCormick AJ (2016) Introducing an algal carbon-concentrating mechanism into higher plants: location and incorporation of key components. Plant Biotechnol J 14:1302–1315. https://doi.org/10.1111/pbi.12497
Berthold P, Schmitt R, Mages W (2002). An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist, 153:401–412. https://doi.org/10.1078/14344610260450136
Choi BY, Kim H, Shim D, Jang S, Yamaoka Y, Shin S, Yamano T, Kajikawa M, Jin E, Fukuzawa H, Lee Y. (2022) The Chlamydomonas bZIP transcription factor BLZ8 confers oxidative stress tolerance by inducing the carbon-concentrating mechanism. Plant Cell 34:910–926. https://doi.org/10.1093/plcell/koab293
Concordet JP, Haeussler M. (2018) CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res 46:242-245. https://doi.org/10.1093/nar/gky354
Delrue B, Fontaine T, Routier F, Decq A, Wieruszeski JM, Van Den Koornhuyse N, Maddelein ML, Fournet B, Ball S (1992) Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin. J Bacteriol 174:3612–3620. https://doi.org/10.1128/jb.174.11.3612-3620.1992
Engel BD, Schaffer M, Kuhn Cuellar L, Villa E, Plitzko JM, Baumeister W (2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. Elife 4: e04889. https://doi.org/10.7554/eLife.04889
Fang W, Si Y, Douglass S, Casero D, Merchant SS, Pellegrini M, Ladunga I, Liu P, Spalding MH (2012) Transcriptome-wide changes in Chlamydomonas reinhardtii gene expression regulated by carbon dioxide and the CO₂-concentrating mechanism regulator CIA5/CCM1. Plant Cell 24:1876–1893. https://doi.org/10.1105/tpc.112.097949
Freeman Rosenzweig ES, Xu B, Kuhn Cuellar L, Martinez-Sanchez A, Schaffer M, Strauss M, Cartwright HN, Ronceray P, Plitzko JM, Förster F, Wingreen NS, Engel BD, Mackinder LCM, Jonikas MC (2017) The Eukaryotic CO₂-concentrating organelle is liquid-like and exhibits dynamic reorganization. Cell 171:148-162. https://doi.org/10.1016/j.cell.2017.08.008
Fukuzawa H, Miura K, Ishizaki K, Kucho K, Saito T, Kohinata T, Ohyama K (2001) Ccm1, a regulatory gene controlling the induction of a carbon-concentrating mechanism in Chlamydomonas reinhardtii by sensing CO₂ availability. Proc Natl Acad Sci USA 98:5347–5352. https://doi.org/10.1073/pnas.081593498
Fukuzawa H, Ogawa T, Kaplan A (2012) The uptake of CO₂ by cyanobacteria and microalgae. In: Eaton-Rye J, Tripathy B, Sharkey T (eds) Photosynthesis. Advances in Photosynthesis and Respiration, vol 34. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-1579-0_25
Han S, Tang R, Anderson LK, Woerner TE, Pei ZM (2003) A cell surface receptor mediates extracellular Ca²⁺ sensing in guard cells. Nature, 425:196–200. https://doi.org/10.1038/nature01932
Hashimoto M, Negi J, Young J, Israelsson M, Schroeder JI, Iba K (2006) Arabidopsis HT1 kinase controls stomatal movements in response to CO₂. Nat Cell Biol 8:391–397. https://doi.org/10.1038/ncb1387
Jin S, Sun J, Wunder T, Tang D, Cousins AB, Sze SK, Mueller-Cajar O, Gao Y-G (2016) Structural insights into the LCIB protein family reveals a new group of β-carbonic anhydrases. Proc Natl Acad Sci USA 113:14716–14721. https://doi.org/10.1073/pnas.1616294113
Itakura AK, Chan KX, Atkinson N, Pallesen L, Wang L, Reeves G, Patena W, Caspari O, Roth R, Goodenough U, McCormick AJ, Griffiths H, Jonikas MC. (2019) A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 116:18445–18454. https://doi.org/10.1073/pnas.1904587116
Kono A, Spalding MH (2020) LCI1, a Chlamydomonas reinhardtii plasma membrane protein, functions in active CO₂ uptake under low CO₂. Plant J 102: 1127–1141. https://doi.org/10.1111/tpj.14761
Mackinder LCM, Meyer MT, Mettler-Altmann T, Chen VK, Mitchell MC, Caspari O, Freeman Rosenzweig ES, Pallesen L, Reeves G, Itakura A, Roth R, Sommer F, Geimer S, Mühlhaus T, Schroda M, Goodenough U, Stitt M, Griffiths H, Jonikas MC. (2016) A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle. Proc Natl Acad Sci USA 113:5958–5963. https://doi.org/10.1073/pnas.1522866113
Mackinder LCM, Chen C, Leib RD, Patena W, Blum SR, Rodman M, Ramundo S, Adams CM, Jonikas MC (2017) A spatial interactome reveals the protein organization of the algal CO₂-concentrating mechanism. Cell 171:133-147. https://doi.org/10.1016/j.cell.2017.08.044
Meyer MT, Itakura AK, Patena W, Wang L, He S, Emrich-Mills T, Lau CS, Yates G, Mackinder LCM, Jonikas MC (2020) Assembly of the algal CO₂-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif. Sci Adv 6: eabd2408. https://doi.org/10.1126/sciadv.abd2408
Miura K, Yamano T, Yoshioka S, Kohinata T, Inoue Y, Taniguchi F, Asamizu E, Nakamura Y, Tabata S, Yamato KT, Ohyama K, Fukuzawa H (2004) Expression profiling-based identification of CO₂-responsive genes regulated by CCM1 controlling a carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol 135:1595–1607. https://doi.org/10.1104/pp.104.041400
Mukherjee A, Lau CS, Walker CE, Rai AK, Prejean CI, Yates G, Emrich-Mills T, Lemoine SG, Vinyard DJ, Mackinder LCM, Moroney JV (2019) Thylakoid localized bestrophin-like proteins are essential for the CO₂ concentrating mechanism of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 116:16915–16920. https://doi.org/10.1073/pnas.1909706116
Ohnishi N, Mukherjee B, Tsujikawa T, Yanase M, Nakano H, Moroney JV, Fukuzawa H (2010) Expression of a low CO₂-inducible protein, LCI1, increases inorganic carbon uptake in the green alga Chlamydomonas reinhardtii. Plant Cell 22:3105–3117. https://doi.org/10.1105/tpc.109.071811
Pollock SV, Prout DL, Godfrey AC, Lemaire SD, Moroney JV (2004) The Chlamydomonas reinhardtii proteins Ccp1 and Ccp2 are required for long-term growth, but are not necessary for efficient photosynthesis, in a low-CO₂ environment. Plant Mol Biol 56:125–132. https://doi.org/10.1007/s11103-004-2650-4
Rai AK, Chen T, Moroney JV (2021) Mitochondrial carbonic anhydrases are needed for optimal photosynthesis at low CO₂ levels in Chlamydomonas. Plant Physiol 187:1387–1398. https://doi.org/10.1093/plphys/kiab351
Toyokawa C, Yamano T, Fukuzawa H (2020) Pyrenoid Starch Sheath Is Required for LCIB Localization and the CO₂-Concentrating Mechanism in Green Algae. Plant Physiol 182:1883–1893. https://doi.org/10.1104/pp.19.01587
Tsuji Y, Kinoshita A, Tsukahara M, Ishikawa T, Shinkawa H, Yamano T, Fukuzawa H (2022) A YAK1-type protein kinase, triacylglycerol accumulation regulator 1, in the green alga Chlamydomonas reinhardtii is a potential regulator of cell division and differentiation into gametes during photoautotrophic nitrogen deficiency. J Gen Appl Microbiol in press. https://doi.org/10.2323/jgam.2022.08.001
Vance P, Spalding M (2005) Growth, photosynthesis, and gene expression in Chlamydomonas over a range of CO₂ concentrations and CO₂/O₂ ratios: CO₂ regulates multiple acclimation states. Can J Bot 83:796–809. https://doi.org/10.1139/b05-064
Wang L, Yamano T, Takane S, Niikawa Y, Toyokawa C, Ozawa SI, Tokutsu R, Takahashi Y, Minagawa J, Kanesaki Y, Yoshikawa H, Fukuzawa H (2016) Chloroplast-mediated regulation of CO₂-concentrating mechanism by Ca²⁺-binding protein CAS in the green alga Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 113:12586–12591. https://doi.org/10.1073/pnas.1606519113
Wunder T, Cheng SLH, Lai S-K, Li H-Y, Mueller-Cajar O (2018) The phase separation underlying the pyrenoid-based microalgal Rubisco supercharger. Nat Commun 9:5076. https://doi.org/10.1038/s41467-018-07624-w
Yamano T, Miura K, Fukuzawa H (2008) Expression analysis of genes associated with the induction of the carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol 147:340–354. https://doi.org/10.1104/pp.107.114652
Yamano T, Tsujikawa T, Hatano K, Ozawa S, Takahashi Y, Fukuzawa H (2010). Light and low-CO₂-dependent LCIB-LCIC complex localization in the chloroplast supports the carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Cell Physiol, 51:1453–1468. https://doi.org/10.1093/pcp/pcq105
Yamano T, Iguchi H, Fukuzawa H (2013) Rapid transformation of Chlamydomonas reinhardtii without cell-wall removal. J Biosci Bioeng 115:691–694. https://doi.org/10.1016/j.jbiosc.2012.12.020
Yamano T, Sato E, Iguchi H, Fukuda Y, Fukuzawa H (2015) Characterization of cooperative bicarbonate uptake into chloroplast stroma in the green alga Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 112:7315–7320. https://doi.org/10.1073/pnas.1501659112
Yamano T, Toyokawa C, Fukuzawa H (2018) High-resolution suborganellar localization of Ca²⁺-binding protein CAS, a novel regulator of CO₂-concentrating mechanism. Protoplasma 255:1015–1022. https://doi.org/10.1007/s00709-018-1208-2
Yamano T, Toyokawa C, Shimamura D, Matsuoka T, Fukuzawa H (2022). CO₂-dependent migration and relocation of LCIB, a pyrenoid-peripheral protein in Chlamydomonas reinhardtii. Plant Physiol 188:1081–1094. https://doi.org/10.1093/plphys/kiab528

Table 1 CCM1-dependent and LC-inducible genes downregulated in KO-60 and saga1 in LC conditions

gene ID	gene name	description	C9 / KO-60		saga1 / CC-5325		saga1 / SAGA1-Venus/saga1
gene ID	gene name	description	logFC	FDR	logFC	FDR	logFC	FDR
*Cre03.g204577*	*DNJ31*	DnaJ-like protein	-4.0	4.4E-41	-2.9	2.3E-21	-1.4	1.2E-09
Cre07.g321800	—	—	-3.8	5.7E-54	-2.7	2.3E-26	-1.4	2.0E-06
Cre04.g223250	LCIE	Low-CO₂ inducible protein E	-3.8	8.5E-77	-2.3	7.1E-15	-2.5	2.7E-22
*Cre02.g097800*	*HLA3*	Bicarbonate ABC transporter	-3.0	3.8E-63	-2.4	2.7E-39	-1.0	7.7E-19
Cre12.g555700	DNJ15	DnaJ-like protein	-2.7	1.3E-11	-5.6	1.5E-44	-3.1	2.4E-31
*Cre08.g367500*	*LHCSR3.1*	Stress-related chlorophyll a/b binding protein 2	-2.6	1.0E-54	-1.0	2.2E-13	-0.9	9.8E-25
*Cre08.g367400*	*LHCSR3.2*	Stress-related chlorophyll a/b binding protein 3	-2.2	1.3E-33	-0.8	6.4E-19	-0.4	2.2E-04
Cre09.g399252	—	—	-2.1	8.3E-38	-0.3	2.4E-04	-0.4	1.1E-06
Cre06.g281600	LCI23	Low-CO₂-inducible protein 23	-1.7	2.0E-28	-1.7	1.3E-34	-1.2	2.7E-25
Cre05.g234652	—	—	-1.7	2.6E-22	-1.2	1.8E-41	-0.8	1.1E-25
Cre02.g080800	—	—	-1.7	5.4E-20	-0.9	1.6E-08	-0.5	9.6E-04
Cre12.g547100	CGL2	putative methyltransferase	-1.5	2.9E-05	-1.4	9.4E-21	-0.6	1.0E-04
Cre13.g591550	SRR20	Scavenger receptor cysteine-rich protein	-1.5	6.6E-07	-1.5	1.6E-10	-1.3	5.0E-08
Cre05.g237800	CYG64	Adenylate/guanylate cyclase	-1.4	2.3E-15	-1.6	5.3E-33	-0.9	1.1E-10
Cre02.g095294	—	—	-1.4	3.1E-11	-1.2	6.5E-07	-0.7	9.2E-03
*Cre05.g248450*	*CAH5*	Mitochondrial carbonic anhydrase	-1.4	2.0E-09	-3.6	2.9E-108	-2.4	2.0E-84
*Cre04.g223300*	*CCP1*	Low-CO₂-inducible membrane protein	-1.3	5.9E-14	-2.8	1.5E-197	-1.6	3.0E-117
*Cre04.g222750*	*CCP2*	Low-CO₂-inducible membrane protein	-1.3	1.7E-17	-0.9	4.5E-12	-0.4	1.7E-03
Cre02.g096700	—	—	-1.3	1.2E-15	-0.8	2.1E-18	-0.5	2.6E-09
*Cre05.g248400*	*CAH4*	Mitochondrial carbonic anhydrase	-1.2	4.8E-08	-3.5	1.9E-139	-2.4	2.9E-125
Cre11.g467617	LCI19	Gamma hydroxybutyrate dehydrogenase	-1.2	8.7E-12	-1.6	2.7E-53	-1.0	5.1E-26
*Cre06.g309000*	*LCIA*	Inorganic carbon channel localized at chloroplast membrane	-1.2	1.1E-15	-1.7	1.1E-37	-1.4	2.7E-42
*Cre04.g222800*	*LCID*	Low-CO₂ inducible protein D	-1.1	7.9E-13	-2.0	6.7E-39	-0.4	3.3E-03
Cre17.g696850	—	—	-1.0	3.9E-10	-0.9	1.5E-20	-0.8	1.3E-35
Cre02.g105800	—	—	-0.9	3.5E-07	-0.4	1.7E-04	-0.8	9.9E-19
Cre04.g214000	—	—	-0.9	6.1E-07	-1.3	7.4E-42	-1.1	4.6E-29
Cre03.g189550	ZIP3	Zinc/iron transporter	-0.7	2.0E-04	-1.0	1.6E-21	-0.7	1.9E-12
Cre02.g145800	MDN3	NAD-dependent malate dehydrogenase	-0.7	1.5E-05	-0.7	3.2E-17	-0.6	2.4E-15
Cre12.g485600	—	—	-0.6	1.6E-04	-1.2	4.4E-37	-0.8	5.4E-40
Cre16.g682100	TDR1	Tropine dehydrogenase/reductase	-0.6	5.0E-04	-0.8	2.8E-15	-0.5	1.9E-09
Cre05.g241950	VDAC2	Voltage-dependent anion-selective channel protein	-0.5	2.6E-03	-1.0	1.3E-30	-0.6	8.5E-32

CCM1-dependent and LC-inducible genes downregulated in KO-60 and saga1 compared with those in C9, CC-5325, and SAGA1-Venus/saga1 with FDR < 0.01 in LC conditions for 2 h are listed. Genes with upregulation >4-fold in C9 compared to H82 (cas) are indicated in bold letters (Wang et al., 2016).

Table 2 Genes encoding pyrenoid-localized proteins upregulated in KO-60 and saga1 in LC condition

gene ID	gene name	description	C9 / KO60		saga1 / CC-5325		saga1/ SAGA1-Venus/saga1
gene ID	gene name	description	logFC	FDR	logFC	FDR	logFC	FDR
Cre06.g271850	—	ATP-binding transport protein-related	1.46	3.12E-08	1.17	2.54E-32	1.01	3.06E-23
Cre16.g663400	BST2	Low-CO₂-inducible membrane protein	0.89	3.80E-08	0.55	1.73E-09	0.26	5.96E-06
Cre17.g721500	STA2	Granule-bound starch synthase I	0.76	3.12E-06	0.61	1.46E-13	0.37	6.12E-11
Cre16.g662600	BST1	Low-CO₂-inducible membrane protein	0.55	7.43E-03	0.87	3.88E-08	1.58	4.41E-18

Genes encoding pyrenoid-localized proteins upregulated in KO-60 and saga1 compared with those in C9, CC-5325, and SAGA1-Venus/saga1 with FDR < 0.01 in LC conditions for 2 h are shown.

No competing interests reported.

Download PDF

Journal Publication

published 19 Jan, 2023

Read the published version in Photosynthesis Research →

Editorial decision: Major revision
15 Nov, 2022
Reviews received at journal
04 Nov, 2022
Reviewers agreed at journal
03 Nov, 2022
Reviewers invited by journal
03 Nov, 2022
Editor assigned by journal
31 Oct, 2022
Submission checks completed at journal
29 Oct, 2022
First submitted to journal
29 Oct, 2022

You are reading this latest preprint version

A pyrenoid-localized protein SAGA1 is necessary for Ca 2+ -binding protein CAS-dependent expression of nuclear genes encoding inorganic carbon transporters in Chlamydomonas reinhardtii

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Algal strains and cultural conditions

Transformation Of Cells

Screening Of Hc-requiring Mutants

Measurement Of Photosynthetic O-evolution

Immunoblotting Analysis

Rna-seq Analysis

Indirect Immunofluorescence Assay

Measurement Of Co Concentration In The Culture Medium

Results

Discussion

The Presence Of Multiple Pyrenoids Inhibits Lcib Migration

Morphological Changes In Pyrenoids Affect Ccm Regulation

Accession Numbers

Declarations

Author contributions

Acknowledgments

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1