The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from Arabidopsis leaves

Accepted Manuscript Title: The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from Arabidopsis leaves Author: Tat’yana Fedorchuk Natalia Rudenko Lyudmila Ignatova Boris Ivanov PII: DOI: Reference:

S0176-1617(14)00056-X http://dx.doi.org/doi:10.1016/j.jplph.2014.02.009 JPLPH 51904

To appear in: Received date: Revised date: Accepted date:

1-10-2013 5-2-2014 6-2-2014

Please cite this article as: Fedorchuk T, Rudenko N, Ignatova L, Ivanov B, The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from Arabidopsis leaves, Journal of Plant Physiology (2014), http://dx.doi.org/10.1016/j.jplph.2014.02.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from

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Arabidopsis leaves

3 Tat’yana Fedorchuk, Natalia Rudenko, Lyudmila Ignatova, Boris Ivanov

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Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, 142290, Russia

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6 Abstract

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Supernatant obtained after high-speed centrifugation of disrupted thylakoids that had been

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washed free from extrathylakoid carbonic anhydrases demonstrated carbonic anhydrase activity

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that was inhibited by the specific inhibitors acetazolamide and ethoxyzolamide. A distinctive

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feature of the effect of Triton X-100 on this activity also suggested that the source of the activity

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is a soluble protein. Native electrophoresis of a preparation obtained using chromatography with

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agarose/mafenide as an affinity sorbent revealed one protein band with carbonic anhydrase

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activity. The same protein was revealed in a mutant deficient in soluble stromal carbonic

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anhydrase β-CA1, and this indicated that the newly revealed carbonic anhydrase is not a product

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of the At3g01500 gene. These data imply the presence of soluble carbonic anhydrase in the

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thylakoid lumen of higher plants.

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Keywords: Carbonic anhydrase; Arabidopsis thaliana; Thylakoids; Thylakoid lumen

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Abbreviations: AZ, acetazolamide; BSA, bovine serum albumin; CA, carbonic anhydrase; Chl,

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chlorophyll; DTT, 1,4-dithio-DL-threitol; EZ, ethoxyzolamide; PAAG, polyacrylamide gel;

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PMSF, phenylmethylsulfonyl fluoride; PSI, photosystem I; PSII, photosystem II; Rubisco,

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ribulose-1,5-bisphosphate carboxylase/oxygenase; WT, wild type; β1-mut, the mutant deficient

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in soluble stromal carbonic anhydrase.

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Introduction

27 Carbonic anhydrases (CAs, EC.4.2.1.1) constitute a large group of enzymes that have been

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assigned to six families, which are commonly assumed to have originated in the process of

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convergent evolution (Hewett-Emmett and Tashian, 1996; Moroney et al., 2011). The

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Arabidopsis thaliana genome contains 19 genes encoding carbonic anhydrases (Fabre et al.,

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2007), but not all of their products have been identified. In chlorophyll-containing cells of plants

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and in algae, following the finding of soluble CAs in cytoplasm and in chloroplast stroma, CA

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activity of the thylakoids was revealed (Komarova et al., 1982; Vaklinova et al., 1982; Stemler,

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1986, 1997; Ignatova et al., 1998). At least two sources of activity have been found in thylakoid

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membrane fragments enriched with photosystem II (PSII), and an additional one in membrane

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fragments enriched with photosystem I (PSI) (Lu and Stemler, 2002; Pronina et al., 2002;

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Ignatova et al., 2006). In Chlamydomonas reinhardtii thylakoids, a CA was found (Karlsson et

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al., 1998) that was identified as α-CA (Cah3) and, according to the authors’ data, it is bound to

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the lumenal side of the thylakoid membrane near PSII. It was proposed that this CA catalyzes

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bicarbonate dehydration at the donor side of PSII, allowing immediate removal of the protons

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from the water oxidation complex, thus preventing its damage (Villarejo et al., 2002). This

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function was also proposed for the membrane CA bound to PSII in higher plants (Rudenko et al.,

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2007). Based on the other assumptions, namely, assuming the participation of CAs in the supply

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of CO2 to Rubisco, the presence of a lumenal CA was postulated in algae both from indirect

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experiments (Pronina and Borodin, 1993) and theoretical considerations (Raven, 1997). The first

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indication of the presence of soluble CA in the thylakoid lumen of higher plants appeared in

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work with pea thylakoids (Rudenko et al., 2007). The present study conducted with Arabidopsis

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confirms the presence of a soluble CA in the thylakoid lumen of higher plants, and shows that it

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differs from the stromal soluble CA.

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Materials and methods

53 A. thaliana (L.) ecotype Columbia (WT) and the β-CA1 knockout line (β1-mut) were grown in a

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chamber at 19ºC with an 8 h photoperiod under 100-120 µmol quanta m–2 s–1. The seeds of the

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mutant were obtained from the Arabidopsis Biological Resource Center as a T-DNA insertion

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line (SALK_106570). Total RNA was extracted from the leaves of WT plants and the

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homozygous mutant plants containing the T-DNA insert in At3g01500 (β1-mut) and was treated

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with DNase to eliminate any genomic DNA contamination. First-strand cDNA was synthesized

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using an Omniscript RT Kit (Qiagen) and oligo(dT) as a primer. cDNA specific for the

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At3g01500 gene primer pair was used, 5′- CCT CTC CGA AAC TAG CTC TGT TAA -3′ and

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5′- CTG TCC CCC AAG ATT TTA ATT CTG TAA A -3′, and polymerase chain reactions

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(PCR) were run in an IQ5 cycler (Bio-Rad). At3g01500 gene transcripts were absent in the β1-

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mut leaves (Supplement 1, Fig. 1).

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The thylakoids were isolated from the leaves of 1.5-2 month-old plants. The leaves were

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homogenized in medium containing 0.1 М Tris-H2SO4 (pH 8.0), 0.4 M sucrose, 2% Polyclar

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(w/v), 5 mM EDTA, 0.005% BSA, 1 mM benzamidine, 1 mM α-aminocaproic acid, and PMSF

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(Medium 1). The homogenate filtered through nylon cloth was centrifuged for 6 min at 3400×g,

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and the supernatant was centrifuged to remove the rest of the membranes for an additional1 h at

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175 000×g, yielding supernatant ‘a’. The pellet after the first centrifugation was suspended in

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Medium 1 diluted to one-tenth to break the chloroplast envelope, and the mixture was

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successively centrifuged twice as above, yielding finally supernatant ‘b’. The thylakoids were

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washed three times by suspending the pellets in Medium 1 followed by centrifugation for 1 h at

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175 000×g yielding supernatants ‘c’, ‘d’, and ‘e’. To obtain the soluble thylakoid proteins, the

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thylakoids, after the last washing were either incubated under stirring for 20 min at 0°C at the

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required Triton X-100 concentration, or passed through a French press (Thermo electron, USA)

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twice, each time at 10000 psi. Then the lysate in both the cases was centrifuged at 175 000×g for

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3 1 h to obtain supernatant ‘f’ and the pellet containing the thylakoid membranes. Supernatant ‘f’

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and the pellet did not contain Rubisco, which was verified with the Western-blot assay, using

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antibodies to large subunits of Rubisco and Alkaline Phosphatase Conjugate Substrate Kit (Bio-

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Rad). This indicated that the thylakoids were not contaminated before disrupting with stromal

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components.

Carbonic anhydrase activity was evaluated as the difference between the rates of pH

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decrease, measured with pH electrode, from 8.3 to 7.8 in the course of CO2 hydration at 2°С in

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13.6 mM Veronal buffer (pH 8.4) in the presence and in the absence of an aliquot of the

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preparation. The difference in the buffer capacities was taken into account to express CA activity

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as extent of proton release. The protein content in the supernatants and the chlorophyll content in

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the pellets were determined according to (Lowry et al., 1951) and (Winterman and De Mots,

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1965), respectively.

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Affinity chromatography was carried out by loading supernatant ‘f’ concentrated in a Millipore concentrator (50 kDa) and diluted with medium containing Тris-Н2SO4 (pH 8.0), 100

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mM NaCl, 5 mM EDTA, 1 mM benzamidine, and 1 mM α-aminocaproic acid (Medium 2) onto

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the column filled with agarose/mafenide (Sigma). After incubation for 40 min, the column was

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washed with Medium 2 with 0.05% Triton X-100 to remove nonspecifically bound substances.

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The CA was eluted from the column with Medium 2 containing 50 μM mafenide, which, being

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an inhibitor of the CA, was removed by centrifugation of the eluate in Millipore concentrators to

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restore the CA activity.

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Native electrophoresis was performed according to Davis (1964) using a Mini-Protean 3

Cell (Bio-Rad). It was carried out at current strength of 10 mA for 3-4 hours at 4°С in the dark in 10% PAAG containing 20% glycerol.

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The activity of CA in PAAG was visualized after incubation of the gel on ice for 20-

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30 min in 44 mM Veronal buffer (pH 8.1) with bromothymol blue followed by transfer into

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water saturated with CO2 at 0°C (Edwards and Patton, 1966). The blue gel became locally

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yellow at the place where a source of CA activity was situated. The effect of acetazolamide (AZ)

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was evaluated, placing the gels into the buffer with 10 mM AZ before the visualization of CA

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activity. The proteins in the gels were stained with Coomassie G-250 (Kashino, 2003).

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Figure 1 shows CA activities of supernatants ‘a’ – ‘f’ (see Methods) isolated from WT (Fig. 1A)

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and from β1-mut (Fig. 1B) plants. Perceptible activities in supernatants ‘a’ obtained after

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breaking the cells by homogenization and in supernatant ‘b’ obtained after also breaking the

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chloroplasts in hypoosmotic media obviously reflected the presence of extrathylakoid soluble

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CAs from cytoplasm and chloroplast stroma: β-CA1, β-CA2, β-CA3 (Fabre et al., 2007), and α-

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CA1 (Villarejo et al., 2005) in the mixtures containing thylakoids both from WT and β1-mut

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(without β-CA1). It should be noted that CA activity of supernatant ‘b’ from the mutant was

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lower than that of the supernatant from WT. CA activities were not detectible in supernatant ‘e’

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from WT (Fig. 1A) and already in supernatant ‘c’ from β1-mut (Fig. 1B). The absence of CA

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activity in supernatants ‘e’ obtained from leaves of both the WT and the mutant was confirmed

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by the failure to observe this activity in the gel following native electrophoresis of these

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supernatants, which were concentrated thirty-fold (Supplement 1, Fig. 2).

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Following the detergent treatment of the thylakoids to release the soluble lumen proteins,

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appreciable CA activity was observed in supernatants ‘f’ (see Methods) both from WT and β1-

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mut (Figs. 1A and 1B). The CA activity in the same supernatant was observed after breaking the

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thylakoids not only by the detergent, but also by the use of a French press (data not shown); the

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latter treatment does not lead to release into solution of the proteins that could adhere to the

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membrane surface according to Kieselbach and Schröder (1998). The CA activity of supernatant

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‘f’, i.e. of the soluble lumen proteins abandoning the thylakoids during the time of Triton

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treatment, gradually reached a maximal value at the concentration of the detergent corresponding

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to Triton/Chl ratio of 0.7, and further increase of the detergent concentration did not change the

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level of CA activity (Fig. 2A). However, the precipitate containing the thylakoid membranes

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demonstrated two peaks of CA activity at Triton/Chl ratio of 0.3 and 1.0 (Fig. 2B).

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The addition of BSA into all media in the course of thylakoid purification and breaking resulted in a moderate increase of the CA activity of the last precipitate containing the membrane

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fragments of the washed thylakoids, while the CA activity of supernatant ‘f’ increased 3.5-fold

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(Table). An increase of the CA activity of the thylakoid membranes is an expected effect of

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protective action of BSA preventing their disintegration (Wasserman and Fleischer, 1967).

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Because BSA can be attached to the membrane in a reversible manner, it is used to prevent

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nonspecific protein–membrane surface adsorption. This effect would decrease the CA activity of

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the last supernatant if the washed thylakoids, before breaking by detergent, contained the

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adsorbed soluble stromal and/or cytoplasmic CAs. The opposite effect of BSA addition

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observed, namely, a considerable increase of the CA activity of this supernatant, was obviously

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provided by preservation of the thylakoid integrity by BSA. That, in turn, blocks the soluble

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protein outflow from the thylakoids during isolation and washings. The additions of DTT to

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supernatant ‘f’ and to the corresponding membrane precipitate before measurements of the CA

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activities resulted in a decrease of the CA activity of the precipitate by approximately18% in the

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absence and in the presence of BSA. DTT increased the CA activity of the supernatant 2.2-fold

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in the absence and 1.3-fold in the presence of BSA (Table).

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Affinity chromatography was applied to purify and concentrate CA residing in

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supernatant ‘f’. Native electrophoresis revealed that only one protein band with an apparent

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molecular mass of 130 kDa was detected in that position in the gel, where CA activity appeared

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in the cases of thylakoid disruption using either Triton X-100 (Fig. 3) or French press treatment

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(not shown). No CA activity appeared after incubation of the gel in the presence of AZ, a

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specific CA inhibitor (Fig. 3, B in comparison with A).

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Discussion

157 Our data confirm the existence of a new member of the CA system of chloroplast

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thylakoids, namely, the soluble CA in the thylakoid lumen. The finding of such a protein in the

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mutant plants lacking the soluble stromal β-CA1 provides extremely strong evidence that the

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lumen CA was not the product of the At3g01500 gene. The soluble nature of the lumen CA, in

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addition to its presence in the supernatant after high speed centrifugation, is supported by that

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fact that this protein migrates in the gel in the course of electrophoresis in the absence of

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detergents, i.e. under conditions suitable for soluble proteins (Davis, 1964). The catalytic activity

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of soluble enzymes does not depend on the detergent concentration (Kabanov et al., 1991), and

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the absence of change of CA activity under increasing detergent concentration (Fig. 2) can be

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considered as additional evidence that the CA activity of supernatant ‘f’ originates from a

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soluble enzyme. The activity of a membrane-bound enzyme, in distinction from an activity of a

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soluble enzyme, depends on the detergent concentration, since the activity maximum exists as

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the active conformation for the given enzyme is maintained at a specific micelle size (Kabanov

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et al., 1991; Levashov et al., 1997). The presence of two peaks of the CA activity of the

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precipitate containing thylakoid membranes (Fig. 2B) agrees with the data indicating the

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presence of two membrane-bound CAs in thylakoid membranes of Arabidopsis (Ignatova et al.,

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2011).

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DTT had no effect on pea thylakoid membrane CAs (Rudenko et al., 2007), and even

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inhibited the activity of the membrane-bound Cah3 from Ch. reinhardtii (Mitra et al., 2005). An

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increase in the activity of the lumen CA in the presence of DTT (Table) implies that this CA is a

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β-CA, most of which are soluble proteins. β-CAs retain their catalytic activity when the

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sulfhydryl groups of cysteine residues are reduced. In the structure of β-CAs of higher plants,

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there are two conserved cysteines necessary for proper subunit interaction at the tetramer–

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tetramer interface (Björkbacka et al., 1997). Additional evidence to support the conclusion that

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7 the CA we have found is a member of the β-family is the characteristic of the effect of the

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specific CA inhibitor ethoxyzolamide on the CA activity of the lumen content, i.e. of supernatant

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‘f’ (Supplement 1, Fig. 3). It is known that CAs of the α-family are inhibited by sulfonamides in

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nanomolar concentrations, while β-CAs are inhibited in the micromolar concentration range

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(Karlsson et al., 1995). Moreover, CA, denoted by Villarejo et al. (2005) as CAH1, and

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belonging to the α-family, was found in the chloroplasts stroma.

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With respect to the possible physiological functions of the soluble lumen CA, in addition to the proposed role in providing Rubisco with CO2 (see Introduction), utilizing the protons in

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the dehydrase reaction with bicarbonate, it could be the very enzyme that stabilizes the

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intrathylakoid pH (Supplement 2). Experimental data have shown that this pH in vivo does not

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drop below 6.0 (Takizawa et al., 2007), permitting the functioning of lumen enzymes. If the

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proposition that the membrane CA situated like Cah3 near a water-oxidizing complex supports

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the operation of this complex by taking away protons ‘from’ water is correct, then the soluble

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lumen CA can work together with it and additionally fulfill such function for the protons ‘from’

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plastohydroquinone. It should be noted that as a rule several CAs work in cell compartments. For

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example, six CAs were found in mitochondria of A. thaliana (Sunderhaus et al., 2006; Fabre et

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al., 2007). Thus, the soluble lumen CA can be an important part of the carbonic anhydrase

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system of thylakoids.

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Acknowledgments

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The authors express their gratitude to Dr. G.T. Hanke of Osnabrueck University for

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providing with antibodies to Rubisco and to Dr. J.V. Moroney of Louisiana State University for

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production of the seeds of a homozygous line of knockout mutant in his laboratory, the

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possibility to conduct the primer design and PCR, and for valuable discussions.

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This work was supported by grants CRDF # RUB1-2911-PU-07, RFBR #14-04-32323,

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and the Ministry of Education and Science of the Russian Federation # 2012-1.2.2-12-000-1013-

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068.

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Figure legends

254 Fig. 1. Carbonic anhydrase activities of supernatants obtained in the course of isolation of the

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soluble proteins of thylakoid from leaves of WT (A) and β1-mut (B): ‘a’ – following

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centrifugations of homogenate; ‘b’ – following centrifugation of shocked chloroplasts; ‘c’, ‘d’

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and ‘e’ – following successive washings of thylakoids; ‘f’ – following centrifugation of

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thylakoids treated with Triton X-100 (for details, see Methods).

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Fig. 2. Effect of Triton to chlorophyll ratio (w/w) under Triton X-100 treatment of washed

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thylakoids (see Methods) on the carbonic anhydrase activities of both supernatant ‘f’ (A) and the

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pellet containing thylakoid membranes (B). 100% in the panel A corresponds to CA activity of

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377 μmol H+ (mg protein)-1 min-1.

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Fig. 3. Native electrophoresis of soluble thylakoid proteins obtained after affinity

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chromatography on agarose/mafenide. A and B – gels stained with bromothymol blue, gel B

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after incubation with acetazolamide; C – gel stained with Coomassie G-250. The numbers on the

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right are positions of the marker proteins with indicated molecular masses in kDa. The arrow

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shows the manifestation of CA activity.

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Table. Effects of presence of 0.005% BSA in all media in the course of isolation of the thylakoid

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soluble proteins, of 5 mM DTT added to the preparations before measurements of the CA

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activity, and superposition of these improvements on the CA activities of washed thylakoid

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membranes and the soluble proteins, pellet, and supernatant ‘f’, respectively (see Methods).

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μmol Н+/mg Chl×min

%

203 ± 12

100

Standard conditions

5884 ± 92

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160 ± 9

79

3540 ± 45

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229 ± 4

113

7392 ± 34

449

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measurements

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100

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+ DTT before

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1646 ± 153

%

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268 ± 2 media

+ BSA + DTT

μmol Н+/mg Protein×min

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+ BSA in all

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Thylakoid soluble proteins

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Thylakoid membranes Conditions

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Figure

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Fig. 1. Carbonic anhydrase activities of supernatants obtained in the course of isolation of the

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soluble proteins of thylakoid from leaves of WT (A) and β1-mut (B): ‘a’ – following centrifugations of homogenate; ‘b’ – following centrifugation of shocked chloroplasts; ‘c’, ‘d’

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and ‘e’ – following successive washings of thylakoids; ‘f’ – following centrifugation of

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thylakoids treated with Triton X-100 (for details, see Methods).

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Figure

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Fig. 2. Effect of Triton to chlorophyll ratio (w/w) under Triton X-100 treatment of washed

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thylakoids (see Methods) on the carbonic anhydrase activities of both supernatant ‘f’ (A) and the pellet containing thylakoid membranes (B). 100% in the panel A corresponds to CA activity of

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377 μmol H+ (mg protein)-1 min-1.

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Figure

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Fig. 3. Native electrophoresis of soluble thylakoid proteins obtained after affinity

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chromatography on agarose/mafenide. A and B – gels stained with bromothymol blue, gel B after incubation with acetazolamide; C – gel stained with Coomassie G-250. The numbers on the right are positions of the marker proteins with indicated molecular masses in kDa. The arrow

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shows the manifestation of CA activity.

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