Xunling Han's research works | Chinese Academy of Sciences and other places

This page lists works of an author who doesn't have a ResearchGate profile or hasn't added the works to their profile yet. It is automatically generated from public (personal) data to further our legitimate goal of comprehensive and accurate scientific recordkeeping. If you are this author and want this page removed, please let us know.

Carbonic anhydrase (CA) activity in Synechocystis and mutants as affected by dithiothreitol (DTT) and acetazolamide (AZA). (a) CA activity of recombinant purified EcaB was measured as proton production from CO2 hydration in the presence or absence of DTT (5 mM) or AZA (50 μM). CcmK (Sll1029) was used as an additional control. (b) CA activity of EcaB, measured as CO2 evolution from HCO3⁻ in the presence or absence of DTT (5 mM) or AZA (300 μM). CcmK was used as a control. Values are the averages of three independent measurements ± SE. (c) Comparison of CA CO2 hydration activity in wild‐type (WT) and various Synechocystis mutants in the presence (+) or absence (−) of DTT (5 mM), or AZA (50 μM). The barbital buffer solution alone was used as the control to measure the uncatalyzed rate of CO2 hydration. CA activity was calculated as the difference in the rate (20–60 s) of CO2 hydration between the control and the samples. CA activity was measured using 20 μg thylakoid membranes ml⁻¹ Chl from WT and mutants ::ecaB, ΔcupA, ΔcupB, ΔcupA/B and M55 (cultured at pH 8.0 under high‐CO2 conditions). Values are means ± SE (n = 3). Asterisk indicates statistically significant differences (t‐test; *, P < 0.05; **, P < 0.01).

Comparisons of growth in the Synechocystis wild‐type (WT) and ::ecaB mutant under growth light (GL; 40 μmol photons m⁻² s⁻¹) and high light (HL; 200 μmol photons m⁻² s⁻¹) conditions, and photosynthetic performance under HL. (a, b) Effect of pH on the growth of WT and ::ecaB under GL (a) and HL (b) conditions. Five microliters of cell suspension with OD730 values of 0.1, 0.01 and 0.001 were spotted on agar plates containing BG11 medium adjusted to various pHs and grown under high CO2 (HC, 2% CO2 v/v in air) and low CO2 (LC, air concentration of CO2) conditions for 5 d under GL and HL. (c) Maximal photosynthetic rate in the WT and mutant ::ecaB grown at different pHs under HL. The HC‐grown cells at the early logarithmic phase grown at pH 8.0 were shifted to the treatment condition (different pH values), to grow over the next 12–16 h until an OD730 of 0.5 was reached, before measurement. The illumination during O2 exchange measurements was 900 μmol photons m⁻² s⁻¹, 30°C, and the culture containing 0.2 mM inorganic carbon (Ci) was directly used for the measurement. (d) The effect of NaHCO3 concentration on the photosynthetic rate (O2 evolution) by cells at logarithmic phase grown under HC or LC conditions and under HL at pH 8.0. Experiments were performed as described earlier in this caption. Values are means ± SE of three independent measurements. Asterisk indicates significant differences (t‐test; *, P < 0.05; **, P < 0.01).

(a, b) Effect of light intensity on the quantum yield of photosystem II (yield(II)) (a) and the redox state of primary plastoquinone, QA (b), in the Synechocystis wild‐type (WT) and ::ecaB. The actinic light intensity was 100 μmol photons m⁻² s⁻¹. The culture containing 0.2 mM inorganic carbon (Ci) was directly used for the measurement. GL, cultured at growth light (40 μmol photons m⁻² s⁻¹); HL, cultured at high light (200 μmol photons m⁻² s⁻¹). The parameters were calculated from the fluorescence parameters of WT and ::ecaB grown at various pH values. Asterisks indicate significant differences in (b) (t‐test; **, P < 0.01). PAR, photosynthetically active radiation.

The location of EcaB in the wild‐type (WT) strain and the accumulation of EcaB, Ndh subunits and Cup proteins in treated Synechocystis WT and ::ecaB. (a) Immunodetection of NdhH and EcaB in total protein (T), supernatant (S) and membrane (M) of the WT grown at high CO2 (HC, 2% CO2 v/v in air), growth light (GL, 40 μmol photons m⁻² s⁻¹) and pH 8.0. (b) The localization of EcaB, BicA and NdhK to the thylakoid membrane (TM) and/or plasma membrane (PM) fractions. TM and PM were separated by two‐phase partitioning. (c) Immunodetection of Ndh subunits, Cup proteins and EcaB from the total protein fraction of WT cells grown under HC or low CO2 (LC, air concentration of CO2) at pH 8.0 and GL. (d) The accumulation of CupA, CupB and EcaB at different pH values in HC‐ and GL‐grown WT cells. The HC‐grown cells at the early logarithmic phase grown at pH 8.0 were shifted to the treatment condition (different pHs) to grow over the next 12–16 h until an OD730 of 0.5 was reached before the harvest. (e) The accumulation of CupA, NdhK and EcaB at different incubation times under high light (HL, 200 μmol photons m⁻² s⁻¹), at pH 8.0. Each lane was loaded with 15 μg total proteins. A replicate gel stained with Coomassie blue (Coom.) is shown as a loading control in the lower part of panels (c), (d) and (e).

The localization and interaction of EcaB with the Cup proteins and a proposed working model for EcaB function. (a) Comparison of EcaB levels in the thylakoid membranes and supernatants of wild‐type (WT), ΔcupA, ΔcupB and ΔcupA/B cultured at pH 8.0, under growth light (40 μmol photons m⁻² s⁻¹) and high CO2 (HC, 2% CO2 v/v in air). Immunoblotting was performed using antibody against EcaB. Each lane was loaded with 15 μg membrane protein or 25 μg supernatant protein. A replicate gel stained with Coomassie blue (Coom.) is shown in the lower panel as the loading control. (b) Thylakoid membrane proteins from WT and ::ecaB strains cultured at pH 8.0 and LC/GL (LC, low CO2, air concentration; GL, growth light, 40 μmol photons m⁻² s⁻¹) were separated by blue native polyacrylamide gel electrophoresis (BN‐PAGE) and subjected to two‐dimensional sodium dodecyl sulfate‐PAGE. The proteins were immunodetected with antibodies against CupA, CupB and EcaB. The positions of molecular mass markers in the BN gel are indicated. (c) Yeast two‐hybrid assay of EcaB interaction with CupA and CupB. AD, GAL4 activation domain; BD, GAL4 DNA‐binding domain. −WL and −WLHA indicate SD (synthetic dropout medium)/‐Trp‐Leu and SD/‐Trp‐Leu‐His‐Ade dropout plates, respectively. The ability to grow on −WLHA plates indicates an interaction between two proteins. Empty vectors were used as negative controls. (d) Coimmunoprecipitation analysis of EcaB with CupA and CupB. For CupA experiments, the Synechocystis cupA‐CFP strain was used as a source of membranes (cupA CFP‐M). CupA is linked to the CFP peptide for immunodetection using a GFP antibody. WT‐M, the thylakoid membrane of WT for immunodetection of CupB using CupB antibody. Control, preimmune rabbit serum. In the different lanes, (+) indicates the presence of, and (−) the absence of, antibodies, control serum and the membrane protein, respectively. (e) A scheme describing the proposed role of the thylakoid located EcaB in CO2 uptake in Synechocystis. CupA or CupB in CO2‐uptake systems NDH‐1MS′ or NDH‐1MS converts CO2 into HCO3⁻ under alkaline local conditions (Kaplan & Reinhold, 1999). Upon illumination, photosynthetic electron transfer couples the formation of trans‐thylakoid membrane proton gradient, subsequently forming a strong alkaline region inside the U‐type structure for CupA or CupB activity, which enables the release of HCO3⁻ after dehydration of CO2 that diffused into the cytosol. HCO3⁻ enters into carboxysome where it is converted to CO2 by carbonic anhydrase (CA) (CcaA/IcfA) confined to these bodies for CO2 assimilation by Rubisco. EcaB regulates and/or contributes to the CA activity of NDH‐1MS′ or NDH‐1MS by interacting with CupA and CupB, converting HCO3⁻ into CO2. The formed alkaline region through the CO2‐uptake systems is also in favor of ecaB expression (Fig. 4) and function (Figs 2). When the exciting light energy or inorganic carbon (HCO3⁻) is in excess over the carbon assimilation ability, such as under high illumination, the plastoquinone (PQ) pool would become over‐reduced and the induced expression of EcaB would convert HCO3⁻ to CO2, dissipating excess HCO3⁻ and minimizing the over‐reduction of the PQ pool, and resulting in optimization of photosynthesis.

A thylakoid‐located carbonic anhydrase regulates CO2 uptake in the cyanobacterium Synechocystis sp. PCC 6803

December 2018

507 Reads

32 Citations

[...]

Carbonic anhydrases (CAs) are involved in CO2 uptake and conversion, a fundamental process in photosynthetic organisms. Nevertheless, the mechanism underlying the regulation of CO2 uptake and intracellular conversion in cyanobacteria is largely unknown. We report the characterization of a previously unrecognized thylakoid‐located CA Slr0051 (EcaB) from the cyanobacterium Synechocystis sp. PCC 6803, which possesses CA activity to regulate CO2 uptake. Inactivation of ecaB stimulated CO2 hydration in the thylakoids, suppressed by the classical CA inhibitor acetazolamide. Absence of ecaB increased the reduced state of the photosynthetic electron transport system, lowered the rate of photosynthetic O2 evolution at high light (HL) and pH, and decreased the cellular affinity for extracellular inorganic carbon. Furthermore, EcaB was upregulated in cells grown at limiting CO2 concentration or HL in tandem with CupA. EcaB is mainly located in the thylakoid membranes where it interacts with CupA and CupB involved in CO2 uptake by converting it to bicarbonate. We propose that modulation of the EcaB level and activity in response to CO2 changes, illumination or pH reversibly regulates its conversion to HCO3 by the two CO2‐uptake systems (CupA, CupB), dissipating the excess HCO3⁻ and alleviating photoinhibition, and thereby optimizes photosynthesis, especially under HL and alkaline conditions.

Download

Supplementary_Table_S1_Figures_S1_S4

June 2017

6 Reads

Fig. 3. The localization of NDH-1MS and NDH-1MS′ complexes in wild type and different mutant backgrounds. The thylakoid membrane proteins from the wild type and indicated mutant strains were separated by BN-PAGE and further subjected to 2-D/SDS-PAGE. Then the proteins were immunodetected with the indicated antibodies against the Ndh subunits or Cup proteins. The co-localization of Ndh subunits and CupA in a larger molecular size band is defined as NDH-1MS while that with smaller molecular size is NDH-1S; the co-localization of Ndh subunits and CupB in the larger molecular band is NDH-1MS′ while that with smaller molecular size is NDH-1S′. (A) Comparison of accumulation of Ndh subunits, CupB and their assembly into NDH-1MS′ in wild type and ΔndhD1/D2D3. (B) Comparison of accumulation of CupA and CupB and their assembly into NDH-1MS and NDH-1MS′ in wild type and M55. (C) Comparison of accumulation of CupA and CupB and their assembly into NDH-1MS and NDH-1MS′ in wild type and ΔndhM. (D) Comparison of the accumulation of CupA and CupB and their assembly into NDH-1MS′ in wild type, ΔcupA, and ΔndhD3, and NDH-1MS in wild type, ΔcupB, and ΔndhD4. The red arrow which indicates the higher molecular site is NDH-1L and the arrow that indicates the lower one is NDH-1M. (This figure is available in color at JXB online.) Downloaded from https://academic.oup.com/jxb/article-abstract/68/14/3869/3811776 by guest on 03 April 2020

Fig. 5. A model of the proposed function of CO 2 uptake systems in Synechocystis sp. strain PCC6803. CupA or CupB converts CO 2 into HCO 3 -under alkaline conditions, while the conversion is reversed under acidic conditions. Under light conditions, photosynthetic electron transfer couples to the formation of a transthylakoid membrane proton gradient, subsequently forming a strong alkaline region inside the U-type structure for CupA or CupB activity, which leads to the accumulation of HCO 3 -after CO 2 has diffused into the cytosol. After HCO 3 -enters the carboxysome, it is converted into CO 2 by CA for carbon assimilation by Rubisco. (This figure is available in color at JXB online.) Downloaded from https://academic.oup.com/jxb/article-abstract/68/14/3869/3811776 by guest on 03 April 2020

Co-ordination of NDH and Cup proteins in CO2 uptake in cyanobacterium Synechocystis sp. PCC 6803

June 2017

149 Reads

33 Citations

Journal of Experimental Botany

High and low affinity CO2-uptake systems containing CupA (NDH-1MS) and CupB (NDH-1MS'), respectively, have been identified in Synechocystis sp. PCC 6803, but it is yet unknown how the complexes function in CO2 uptake. In this work, we found that deletion of cupB significantly lowered the growth of cells, and deletion of both cupA and cupB seriously suppressed the growth below pH 7.0 even under 3% CO2. The rate of photosynthetic oxygen evolution was decreased slightly by deletion of cupA but significantly by deletion of cupB and more severely by deletion of both cupA and cupB, especially in response to changed pH conditions under 3% CO2. Furthermore, we found that assembly of CupB into NDH-1MS' was dependent on NdhD4 and NdhF4. NDH-1MS' was not affected in the NDH-1MS-degradation mutant and NDH-1MS was not affected in the NDH-1MS'-degradation mutants, indicating the existence of independent CO2-uptake systems under high CO2 conditions. The light-induced proton gradient across thylakoid membranes was significantly inhibited in ndhD-deletion mutants, suggesting that NdhDs functions in proton pumping. The carbonic anhydrase activity was suppressed partly in the cupA- or cupB-deletion mutant but severely in the mutant with both cupA and cupB deletion, indicating that CupA and CupB function in conversion of CO2 to HCO3-. In turn, deletion of cup genes lowered the transthylakoid membrane proton gradient and deletion of ndhDs decreased the CO2 hydration. Our results suggest that NDH-1M provides an alkaline region to activate Cup proteins involved in CO2 uptake.

Download

... The CCM provides algae with an additional ecological advantage as it allows both CO 2 and HCO 3 − to be efficiently exploited by carbonic anhydrases (CAs), which significantly contribute to the transformation of CO 2 [57]. CAs are found in the archaea Methanosarcina [58,59], yeasts [60], microalgae and Cyanobacteria [61][62][63][64][65][66][67], diatom algae [68], and fungi [69][70][71]. Chloroplast stroma, mitochondria, periplasmic space, and chloroplast thylakoid lumens of eukaryotic algae have all been found to contain CAs [55]. ...
Reference:
The Hydration-Dependent Dynamics of Greenhouse Gas Fluxes of Epiphytic Lichens in the Permafrost-Affected Region

A thylakoid‐located carbonic anhydrase regulates CO2 uptake in the cyanobacterium Synechocystis sp. PCC 6803

Citing Article
Full-text available
December 2018

[...]

... No carbonic anhydrase has been detected in the cytoplasm of cyanobacteria, and contrary to the original expectations, expressing carbonic anhydrase in the cytoplasm leads to a high CO 2 requiring phenotype, further indicating that in the cytoplasm, bicarbonate concentration is high (Price and Badger 1989). Instead of carbonic anhydrase, two specialized NDH complexes, an inducible NDH-1 3 (NDH-1MS) and a constitutively expressed NDH-1 4 (NDH-1MS') complex have been suggested to convert CO 2 to HCO 3 À in the cytoplasm, thus increasing the net influx of CO 2 and minimizing the efflux of CO 2 Han et al. 2017;Schuller et al. 2020;Artier et al. 2022). Both specialized NDH complexes contain a carbonic anhydrase-like protein (Shibata et al. 2001): CupA is associated with the NDH-1 3 complex and CupB with the NDH-1 4 complex (Zhang et al. 2004;Xu et al. 2008b;Han et al. 2017). ...
Reference:
Inorganic carbon sensing and signalling in cyanobacteria

Co-ordination of NDH and Cup proteins in CO2 uptake in cyanobacterium Synechocystis sp. PCC 6803

Citing Article
Full-text available
June 2017

Journal of Experimental Botany

Xunling Han’s research while affiliated with Chinese Academy of Sciences and other places

What is this page?

Publications (3)

Citations (2)