Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 894-910 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 7, pp. 974-992.

894

Features of Photosynthesis in Arabidopsis thaliana

Plants with Knocked out Genes Encoding

Chloroplast Carbonic Anhydrases αCA1 and βCA1

Natalia N. Rudenko

1,a

*, Maria Yu. Ruppert

, Lyudmila K. Ignatova

Elena M. Nadeeva

, Daria V. Vetoshkina

, and Boris N. Ivanov

Institute of Basic Biological Problems, Pushchino Scientific Center for Biological Research

of the Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia

e-mail: nataliacherry413@gmail.com

Received March 2, 2025

Revised May 26, 2025

Accepted June 9, 2025

Abstract— The knockout of either At3g01500 or At3g52720 gene encoding Arabidopsis thaliana βCA1 and

αCA1 carbonic anhydrase (CA), respectively, led to a lower CA activity of the chloroplast stroma prepara-

tions from the knockout mutant plants (αCA1-KO and βCA1-KO) compared to such preparations from the

wild-type (WT) plants. To identify the differences in the photosynthetic characteristics of mutant and WT

plants, they were grown in low light (LL; 50-70 µmol quanta∙m

−2

∙s

−1

, natural conditions) and high light

(HL; 400 µmol quanta∙m

−2

∙s

−1

, stressful conditions). The rate of CO

assimilation measured at 400 µmol

quanta∙m

−2

∙s

−1

in plants grown under LL was lower in αCA1-KO and βCA1-KO mutants compared to WT

plants. The rate of photosynthetic electron transport was lower in αCA1-KO plants and higher in βCA1-KO

plants than in WT plants; the content of CO

in chloroplasts was lower in βCA1-KO plants than in both WT

and αCA1-KO plants, where it differed little. The value of the proton-motive force was higher in βCA1-KO

plants and lower in αCA1-KO plants than in WT plants due to changes in ΔpH value. The obtained results

suggest that βCA1 facilitates the intake of inorganic carbon into chloroplasts, while αCA1 ensures the con-

version of bicarbonate into CO

in the chloroplast stroma for its use in the reaction catalyzed by Ribulose

bisphosphate carboxylase/oxygenase (RuBisCO). In both αCA1-KO and βCA1-KO mutants, the expression lev-

els of genes encoding other chloroplast CAs differed markedly from those in WT plants; the pattern of the

changes in the genes expression depended on the light intensity during cultivation. The content of hydrogen

peroxide in the leaves of both αCA1-KO and βCA1-KO mutants was higher in LL and lower in HL than in

WT plants. The expression levels of stress marker genes changed similarly in both types of mutant plants.

A possible involvement of the chloroplast stroma CAs in the transmission of stress signals in higher plants

is discussed.

DOI: 10.1134/S0006297925600954

Keywords: photosynthesis, Arabidopsis, chloroplasts, carbonic anhydrase, light intensity

* To whom correspondence should be addressed.

INTRODUCTION

Carbon dioxide(CO

) molecules from the air enter

cells of terrestrial plants, where photosynthesis takes

place, and are converted into bicarbonate(HCO

−

) ion

in the aqueous medium of the cells. In the light, the

stroma of chloroplasts in C3 plants is weakly alkaline

(pH  7.7-8.0) [1]. Under these conditions, bicarbonate

makes 96-98% of the total inorganic carbon (C

inorg

i.e., its level is almost 30-50 times higher than the level

of CO

. Ribulose bisphosphate carboxylase/oxygen-

ase (RuBisCO), a key enzyme of the Calvin–Benson–

Bassham(CBB) cycle, which is localized in the stroma

of chloroplasts, incorporates C

inorg

into organic com-

pounds in a form of CO

. Therefore, it is important

that C

inorg

in converted to CO

in close proximity to

the carboxylation centers. According to the existing

concepts, a relatively slow spontaneous conversion of

FEATURES OF PHOTOSYNTHESIS IN A.thaliana PLANTS 895

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

bicarbonate into CO

against the background of fast

inorg

consumption in its reaction with ribulose phos-

phate can limit photosynthesis [2]. Carbonic anhy-

drase (CA; carbonate hydrolase EC 4.2.1.1) accelerates

interconversion of C

inorg

forms. The role of CAs located

in the chloroplast stroma has been traditionally dis-

cussed in regard to their potential involvement in the

transport of C

inorg

to carboxylation centers (RuBisCO)

or in the conversion of bicarbonate, the predominant

form of C

inorg

in the alkaline stroma, into CO

that

serves as a substrate for RuBisCO [3-5].

Higher C3 plants contain about twenty genes en-

coding CAs that belong to the α, β, and γ families.

According to the TAIR database, the genome of Ara-

bidopsis thaliana contains 8 genes of CAs from the

α-family (αCAs), 6 genes of βCAs, 3 genes of γCAs,

and 2 genes for γCA-like proteins[6]. Often, the same

cell compartment contains several CA isoforms, both

membrane-bound and soluble, that belong to differ-

ent families. βCA1 is a long-known and well-studied

protein [7]; its content in the cells is exceeded only

by the content of RuBisCO[8], the most abundant pro-

tein in plant leaves. Similar to RuBisCO, this enzyme

is located in the stroma of chloroplasts[9]. A. thaliana

contains 4βCA1 isoforms formed by alternative splic-

ing; one of them was found in the chloroplast enve-

lope[10]. These isoforms differ in both their structure

and cellular localization [10].

αCA1 was found in 2005 [11], and the data on

its localization are still contradictory. The presence of

this enzyme in the stroma of Arabidopsis chloroplasts

had been shown by using GFP fusion proteins and

labeling with anti-αCA1 antibodies with attached gold

particles [11]. Later, Hines et al. [12] found αCA1 in

the plasma membrane of Nicotiana tabacum leaves.

However, the study conducted in 2023 showed that

in rice plants, this protein localized to the stroma of

chloroplasts [13]. The authors demonstrated that the

CA mutants lagged behind the wild type (WT) plants

in growth, contained less starch, and exhibited a low-

er rate of CO

assimilation (A

CO2

) and reduced water

use efficiency.

Arabidopsis chloroplasts also contain βCA5[6] lo-

cated in the chloroplast stroma (similar to βCA1 and

αCA1) [14]. The thylakoid membrane of A.  thaliana

chloroplasts was found to include αCA4 [15] locat-

ed apparently in the grana [16]. αCA5 was identified

in the preparations of A.  thaliana stromal thylakoid

membranes [17]. The lumen of thylakoids from Ara-

bidopsis and pea plants contained CA belonging to the

β-family(according to its properties) [18,19]; however,

the amino acid sequence of this protein is yet to be

determined. The shutdown of αCA2 synthesis in Ara-

bidopsis produced multiple effects on the functioning

of its photosynthetic apparatus [20, 21], which also

suggests the presence of αCA2 in chloroplasts. A large

number of CAs catalyzing the same reaction consid-

erably complicates the studies of individual functions

of each enzyme.

The studies on the role of CAs in the chloro-

plast stroma showed that reducing the level of βCA1

to 1% in tobacco plants by using the corresponding

antisense RNA (which bound to the βCA1 mRNA and

induced its degradation, resulting in the reduced en-

zyme synthesis) caused no changes in the phenotype

or photosynthetic parameters [22]. Studying the ef-

fects of single mutations in βCA1 and simultaneous

shutdown of two chloroplast CAs (ΔβCA1/βCA5) [12]

led to the assumption that the function of stromal

CAs is not associated with photosynthesis. It has

been shown that βCA1 is important at the stage of

appearance and growth of cotyledon leaves in Arabi-

dopsis [23], as well as involved, together with βCA4,

in the regulation of stomatal movements [24]. In rice

leaves, this mechanism seems to involve only one βCA,

which is structurally similar to βCA4 but localizes

to chloroplasts [25]. In contrast, βCA4.1 and βCA4.2

from Arabidopsis have been identified in the plasma

membrane [6, 26] and cytoplasm [26], respectively.

Tobacco ΔβCA1/βCA5 mutants demonstrated a lower

rate of seed germination, reduced mass, and growth

retardation compared to WT plants [12] because of

the reduced rate of fatty acid synthesis in chloroplasts.

The involvement of stromal CA in fatty acid synthe-

sis was hypothesized as early as in 2002 [27]. Beside

playing an important structural role, fatty acids serve

as precursors in the synthesis of jasmonates, includ-

ing jasmonic acid (JA) [28]. Jasmonates are a group

of plant hormones involved in the triggering of plant

response to various stress factors. It was also found

that CAs bind salicylic acid (SA), another stress hor-

mone in plants [29]. This ability has been shown for

a number of CAs, especially βCA1 [30]. The authors

demonstrated that the binding of βCA1 with SA and

major cognitive receptor proteins, including NPR1

(nonexpressor of pathogenesis-related genes), promote

transduction of stress signals to the nucleus.

Exposure of plants to stress conditions promote

the manifestation of effects caused by the absence

of particular CA proteins in mutant plants[31-33]. Ex-

pression of CA genes varies depending on the growth

conditions, thus demonstrating that the needs of plants

for CAs depends on external conditions [34, 35].

So far, the direct involvement of stromal CAs (βCA1

and αCA1) in the photosynthesis has not been prov-

en experimentally. Here, we studied the properties

of βCA1- or αCA1-deficient Arabidopsis plants were

grown under natural and stress (excess light) condi-

tions. The goal of this study was to further elucidate

the physiological role of these CAs and investigate

their involvement in the photosynthesis and stress

signaling in higher plants.

RUDENKO et al.896

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

MATERIALS AND METHODS

Plants and growth conditions. The plants used

in the study were A.  thaliana WT plants (Columbia

ecotype), plants with the knocked out At3g52720

gene encoding αCA1 (αCA1-KO; homozygous lines 082

and 029) that were derived from the SALK_082033C

and SALK_029393C lines, respectively, and plants with

the knocked out At3g01500 gene encoding βCA1 (βCA1-

KO; homozygous line) obtained from the SALK_106570

line. The plants were grown to the age of 36 days in

a climate chamber at a low light (LL) intensity corre-

sponding to the natural conditions (50-70µmol quan-

ta∙m

−2

∙s

−1

) with an 8 h/16 h day/night photoperiod at

the atmospheric CO

concentration (450ppm) at 19°C.

Next, some plants were left under the control condi-

tions described above(LL), while the other plantswere

adapted to high light (HL, 400 µmol quanta∙m

−2

∙s

−1

)

for 14-28 days. The seeds of WT Arabidopsis plants

were kindly provided by Professor R.  Scheibe from

the Collection of the Plant Physiology Department at

the University of Osnabrueck, Germany. The seeds of

homozygous mutant plants were kindly provided by

Professor J.  V.  Moroney (Louisiana State University,

United States).

Isolation of stromal preparations from the

leaves of WT and mutant Arabidopsis plants was per-

formed as described by Rudenkoetal.[35]. The leaves

were homogenized in the isolation medium contain-

ing 0.4 M sucrose, 35 mM K

HPO

, 15 mM NaH

3mM MgSO

, 10mM KCl, 20mM Na ascorbate, 1mM

KHCO

, 0.5mM Na ethylenediaminetetraacetate, 1mM

dithiothreitol, 1  mM benzamidine, 1  mM α-amino-

caproic acid, and 1mM phenylmethylsulfonyl fluoride.

The homogenate was filtered through a nylon tissue

and centrifuged at 150g for 1.5 min at 2°C to remove

large leaf fragments. The supernatant was centri-

fuged at 2500g for 5 min at 2°C; the resulting pellet

(chloroplasts) was resuspended in the shock medi-

um (isolation medium diluted 10-fold with distilled

water) to destroy chloroplast envelopes. The chloro-

plast suspension was centrifuged at 2500g for 5 min

at 2°C to obtain a pellet of thylakoids and a superna-

tant enriched with proteins of the chloroplast stroma.

The supernatant was additionally centrifuged at

175,000g for 1 h at 4°C to remove membrane debris,

resulting in the stromal protein preparation for fur-

ther analysis.

Protein assay was performed according to Brad-

ford [36].

CA activity assay. The activity of CAs was assessed

as a difference in the rates of pH decrease measured

with a pH electrode (from8.4 to7.9) during CO

hydra-

tion at 2°C in 13.6 mM Veronal buffer (pH 8.4) in the

presence and absence of stromal protein preparation

and expressed in µmolH

permg protein per min[37].

Measurement of gas exchange in plants. The

rate of A

CO2

and stomatal conductance were mea-

sured in unseparated leaves using an LI-6800 porta-

ble system for the analysis of photosynthetic processes

(Li-Cor, USA) at 400ppm CO

in a leaf chamber at a light

intensity changing discretely from 50 to 1600 µmol

quanta∙m

−2

∙s

−1

(90% red light, 10% blue light) at 23°C

and 50% relative humidity. The leaf area was mea-

sured with the Petiole mobile app (Petiole LTD).

Electrochromic shift (ECS) of carotenoid ab-

sorption for determining the values of the proton mo-

tive force (pmf) components was measured at 515nm

in unseparated leaves using a Dual-PAM-100 Fluores-

cence Measuring System equipped with a P515/535

emitter-detector module (Heinz Walz, Germany) [38].

The measurements were made using 2.5-µs flashes al-

ternating with 5-s dark periods for the excitation of

electron transfer in the reaction centers of photosys-

tems I and II (PSI and PSII). The averaged signal of

50 flashes was accumulated in the fast kinetics mode;

the amplitude of the averaged signal was calculated in

OriginPro and used as the ECT standard (ECSst) value.

The values of pmf and its components (ΔpH and ΔΨ)

were determined in leaves after illumination for 5-min

with the actinic light of increasing intensity (84, 155,

279, 364, 577, and 904 µmol quanta∙m

−2

∙s

−1

). The pmf

value was determined by the difference between the

ECS values at the moment immediately before the light

was turned off and the minimum of the inverted signal

in the dark after the light was turned off. The differ-

ence was normalized to the ECSst determined for a giv-

en leaf[39]. The ΔpH value was found as the difference

of ECS values corresponding to the minimum inverted

dark signal and the maximum signal after relaxation

of the inverted signal. The ΔΨ value was determined

by the difference between the pmf and ΔpH values.

Photosynthetic parameters were determined by

simultaneously measuring the gas exchange parame-

ters in plants and chlorophylla(Chla) fluorescence in

leaves using an LI-6800 portable system for the analy-

sis of photosynthetic processes. Before the measure-

ment, the plants were adapted in the dark for 1.5  h.

After a saturating flash in the dark, the actinic light

(400µmol quanta∙m

−2

∙s

−1

) was turned on, followed by

light flashes every 30  s. The measurements were made

for 10min at the CO

concentration of 400ppm in the

chamber; then the concentration of CO

was reduced

to 150  ppm and the measurements were continued

for another 5 min. Next, the CO

concentration was

increased to 1200 ppm for 5 min and then returned

again to the initial value of 400ppmfor 2.5min. The

electron transport rate (ETR), A

CO2

, and number of

closed PSII reaction centers (1-qL) were determined

by the formulas (1-3):

ETR = Y(II) × 0.50 × PPFDa, (1)

FEATURES OF PHOTOSYNTHESIS IN A.thaliana PLANTS 897

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

where PPFDa is the rate of light absorption by a sam-

ple, µmol m

−2

∙s

−1

;

YII = (Fm′ − Fs)/Fm′, (2)

where Fm′ is the maximum yield of fluorescence in

response to the saturated impulse upon illumina-

tion with the actinic light; Fs is the stationary level

of fluorescence upon illumination with the actinic

light [40, 41];

1 − qL = 1 − [(Fm′ − Fs)/(Fm′ − F

′)] × F

′/Fs, (3)

where F

′ is the minimum level of fluorescence in the

light-adapted state.

The content of CO

in the chloroplasts (Cc,

µmol CO

∙mol

−1

of air) was calculated by the formu-

la (4):

Cc = G* × [ETR + 8(A + Rd)]/[ETR − 4(A + Rd)][13], (4)

where G* is the CO

compensation point in the ab-

sence of respiration (µmolCO

mol

−1

of air); Rdis the

rate of CO

release from the mitochondria during res-

piration (µmol CO

−2

∙s

−1

) and does not depend on

illumination. For G* and Rd, the following empirical

values were used: 49µmol CO

mol

−1

[42] and 1µmol

−2

∙s

−1

 [43], respectively.

Starch content measurement. The leaves were

cut off in the morning after 3-h illumination under

the growing conditions. The content of starch was

determined by measuring light absorption at 620 nm

in the aqueous extracts of leaves after incubation

with 0.12% KI [44].

Measurement of Chl and carotenoid levels.

The content of carotenoids and Chls was determined

after extraction from plant leaves with 96% etha-

nol and expressed in mg pigment per wet weight of

leaves [45].

Hydrogen peroxide content in leaves was de-

termined by the luminol-peroxidase reaction [46].

Leaves (50-100 mg) were frozen in liquid nitrogen,

transferred into 0.4 mL 2 M trichloroacetic acid, and

homogenized. Hydrogen peroxide was extracted by

adding 3 mL of 0.05  M K-phosphate buffer (pH 8.5).

To remove Chls and carotenoids, the extract was in-

cubated with 5% polyvinylpyrrolidone (PVP) and then

centrifuged at 10,000g for 10 min. The supernatant

was collected and its pH was adjusted to 8.5 with

2  M  KOH. Luminol (1  mL; 2.26  ×  10

−4

  M) and peroxi-

dase (1  ×  10

−6

  M) mixture (1  mL) was added to 50  µL

of the extract directly in the measurement cuvette to

determine the H

level. Chemiluminescence was re-

corded with a Lum-100 luminometer (DISoft, Russia)

using solutions with the known H

concentration

for calibration.

Reverse transcription-quantitative polymerase

chain reaction (RT-qPCR). Total RNA was extract-

ed from the frozen Arabidopsis leaves using an Au-

rum Total RNA Mini kit (Bio-Rad, USA) and treated

with DNase I to eliminate any contamination of ge-

nomic DNA. cDNA was synthesized using an RT-1 re-

verse transcription kit (Syntol, Russia) using oligo(dT)

as a primer in a LightCycler 96 Instrument (Roche

Diagnostics, Switzerland). RT-qPCR was performed

with a ready-to-use qPCRmix-HS SYBR reagent mixture

(Evrogen, Russia), using the primers for the CA-en-

coding genes, as well as COR414-TM1(At1g29395),

ANAC019(At1g52890), NPR1(At1g64280), and JAZ1

(At1g19180) genes. Gene expression was calculated

with the formula 2

−(Ct sample − Ct control)

, where C

is

the threshold number of PCR cycles. The At5g09810

(actin 7) gene was used as the C

 control [34]. The

content of βCA1 transcripts was determined jointly in

pairs (βCA1.1+βCA1.2 and βCA1.3+βCA1.4), as these

alternative splicing forms possess identical sequences

at the 3′end. The sequences of primer (Table S1 in the

Online Resource 1) were from Rudenko et al. [34, 35]

and Borisova-Mubarakshina et al. [47].

Statistical analysis was performed with

OriginPro. The data are presented as mean values

with standard errors of the mean (SEM). Statistical

significance was evaluated by ANOVA with the paired

comparison plot using the Holm–Bonferroni method.

RESULTS

The effect of light intensity on the pigment

content in the leaves of WT plants and plants de-

ficient by stromal CAs. No phenotypic differences

were observed between the WT, αCA1-KO, and βCA1-

KO plants grown under HL and LL (natural for Ara-

bidopsis) conditions. The levels of Chl a, Chl b, and

carotenoids in the leaves of αCA1-KO grown under

LL were 10% lower compared to WT plants, while the

content of these pigments in βCA1-KO mutants was

close to that in WT plants (Table1). At the same time,

the Chl a/Chl b ratio in the leaves of WT, αCA1-KO,

and βCA1-KO plants was very similar. These findings

demonstrate that under LL conditions, the absence

of αCA1 or βCA1 had no significant effect on the

pigment biosynthesis. After the three-week adapta-

tion to HL, the amount of both Chl forms decreased

similarly in βCA1-KO and WT plants, while αCA1-KO

plants demonstrated a minor (7%) increase in the

Chl  a content (Table  1). At the same time, the Chl  a/

Chl  b ratio increased by 20-25% in all studied plants,

although in WT and βCA1-KO plants, it was due to a

decrease in the Chl  b levels, while in αCA1-KOplants,

the increase resulted from the elevation of the Chl  a

content. Although the level of carotenoids decreased

RUDENKO et al.898

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Table 1. Effects of light intensity on the pigment content and Chl a/Chl b ratio in the leaves of Arabidopsis

WT, αCA1-KO, and βCA1-KO plants grown at 50-70µmol quanta∙m

−2

∙s

−1

(LL) and 400µmol quanta∙m

−2

∙s

−1

(HL)

Light intensity,

µmol quanta,

−2

∙s

−1

Plant

Pigment content, mg/g wet weight

Chl a/Chl b

Chl a Chl b Chl a + Chl b Carotenoids

50-70 (LL)

WT 0.80 ± 0.05 0.38 ± 0.04 1.17 ± 0.08 0.17 ± 0.01 2.04 ± 0.02

αCA1-KO 0.71 ± 0.06 0.34 ± 0.01 1.06 ± 0.10 0.15 ± 0.01 2.08 ± 0.12

βCA1-KO 0.82 ± 0.12 0.40 ± 0.07 1.22 ± 0.19 0.17 ± 0.03 2.05 ± 0.01

400 (HL)

0.62 ± 0.05

(78%)

0.24 ± 0.02

(63%)

0.86 ± 0.08

(74%)

0.12 ± 0.02

(71%)

2.55 ± 0.06

(125%)

αCA1-KO

0.76 ± 0.02

(107%)

0.31 ± 0.01

(91%)

1.07 ± 0.03

(101%)

0.14 ± 0.02

(93%)

2.50 ± 0.02

(120%)

βCA1-KO

0.56 ± 0.01

(68%)

0.22 ± 0.01

(55%)

0.78 ± 0.02

(64%)

0.10 ± 0.01

(58%)

2.49 ± 0.02

(121%)

Note. The table shows the data of a typical experiment (n≥4). Similar results were obtained in six independent experiments.

The values in parentheses represent a percentage of the respective value under LL.

in WT plants and both mutant lines, it remained the

highest in αCA1-KO plants.

The CA activity in the stroma of WT and mu-

tant plants. The soluble fractions enriched in stro-

mal proteins were obtained from the leaves of WT,

αCA1-KO, and βCA1-KO plants grown under LL. The

CA activity in the preparations from WT plants was

the highest, since it was determined by the presence

of at least three CAs (αCA1, βCA1, and βCA5) (Fig. 1),

while knocking out either αCA1 or βCA1 led to a 5-fold

decrease in the CA activity, indicating that both βCA1

and αCA1 are soluble stromal enzymes.

The effects of the knockout of stromal CAs on

the expression levels of genes coding for chloro-

plast CAs in the plants grown at LL and after ac-

climation to HL. No αCA1 expression was observed

in αCA1-KO mutants, while βCA1-KO plants lacked

βCA1.1  +  βCA1.2 and βCA1.3  +  βCA1.4 transcripts

(data not shown). The relative expression of βCA1

was high in both WT and αCA1-KO plants under

both LL and HL (Fig. 2, a and b), with the content of

βCA1.3  +  βCA1.4 transcripts being the highest. Under

the LL conditions, expression of the βCA1.1 + βCA1.2

and βCA1.3  +  βCA1.4 transcripts in αCA1-KO mutants

was approximately 2 times higher than in WT plants

(Fig. 2, a and b). Under the same conditions, expres-

sion of αCA2 in these mutants was 3 times higher and

expression of βCA5 was 4.5 times lower compared to

WT plants (Fig. 2, d and e). Illumination of βCA1-KO

plants with LL led to the 3- and 9-fold downregula-

tion of the αCA1 and βCA5 genes, respectively (Fig.2,

c and e).

After adaptation to HL, WT plants showed an in-

crease in the expression levels of all studied CA genes

(Fig. 2) except for βCA5 (similar effect was described

previously by us in[34]). At the same time, in βCA1-KO

plants, adaptation to HL caused upregulation of all

studied CA genes, including βCA5 (Fig.2e). On the con-

trary, inαCA1-KO plants, expression of most investigat-

ed CA genes was downregulated under HL (Fig. 2, a,

b, d, and f), this decrease being especially noticeable

(30-fold) for the αCA2 gene transcripts (Fig. 2d).

Fig. 1. The CA activity in the preparations of soluble chlo-

roplast stromal proteins from the leaves of WT, αCA1-KO

and βCA1-KO plants grown under LL. The activity of stro-

mal proteins isolated from WT plants (1060 and 3560 µmol

∙mg protein

−1

∙s

−1

in two experimental replicates, n ≥ 6)

was taken as 100%.

FEATURES OF PHOTOSYNTHESIS IN A.thaliana PLANTS 899

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Fig. 2. Expression levels of genes coding for the chloroplast enzymes βCA1(a,b), αCA1(c), αCA2(d), βCA5(e), and αCA4(f)

in WT, αCA1-KO (line082), and βCA1-KO plants grown at LL(50-70µmol quanta∙m

−2

∙s

−1

; white columns) and HL (400µmol

quanta∙m

−2

∙s

−1

; gray columns). Expression of four alternative splicing transcripts of the βCA1 gene were determined pair-

wise: βCA1.1  +  βCA1.2 (a) and βCA1.3  +  βCA1.4 (b). Significance of differences was determined by the Holm–Bonferroni

method (n  ≥  9). Similar results were obtained in three experiments (3 biological and 3 analytical replicates).

RUDENKO et al.900

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 3. Dependence of stomatal conductance (a) and CO

assimilation rate (A

CO2

) (b) on the light intensity in the measure-

ment chamber in WT, αCA1-KO, and βCA1-KO plants grown at 50-70 µmol quanta∙m

−2

∙s

−1

(closed symbols, solid curves)

and 400 µmol quanta∙m

−2

∙s

−1

(semi-closed symbols, dashed curves). The measurements were performed at 400 ppm CO

The differences were considered significant at p ≤0.05 as determined by the Holm–Bonferroni method (n ≥ 14). The figure

presents the results of 6-7 biological replicates from four independent experiments.

The effect of the lack of stromal CAs on the

gas exchange parameters and starch metabolism.

The assessment of gas exchange parameters in WT

and mutant plants at a discretely increasing light

intensity (from 50 to 1600 µmol quanta∙m

−2

∙s

−1

) has

shown that stomatal conductance in the leaves of both

αCA1-KO and βCA1-KO mutants grown under LL was

higher than in WT plants grown at any light inten-

sity (Fig. 3a). No such differences were observed for

the HL-adapted plants. The A

CO2

values in the plants

grown under LL were the same in WT and mutant

specimens (Fig.3b). In the HL-adapted WT plants, A

CO2

was found to be significantly higher than in WT plants

grown under LL already at 400 µmol quanta∙m

−2

∙s

−1

whereas in HL-adapted αCA1-KO and βCA1-KO mu-

tants, the increases in A

CO2

and stomatal conduc-

tance were much less pronounced than in WT plants

(Fig. 3, a and b). Therefore, both parameters in the

HL-exposed αCA1-KO and βCA1-KO plants were lower

compared to WT plants.

In αCA1-KO and βCA1-KO mutants exposed to LL,

the content of starch (reserve polysaccharide and one

of the main products of photosynthesis) determined at

57 days of age was 25-30% lower than in WT plants

of the same age (Table 2). At the age of 64 days, the

content of starch was ~60% lower in αCA1-KO plants

and 85% lower in βCA1-KO compared to WT plants,

i.e., the lack of any of these stromal CAs significantly

reduced the formation of starch under LL. After three

weeks of adaptation to HL, the content of starch in

the leaves of WT plants increased. A much more sig-

nificant elevation of the starch content was found in

αCA1-KO and βCA1-KO plants compared to the plants

grown under LL (Table 2). At the same time, the con-

tent of starch in the leaves of αCA1-KO and βCA1-KO

mutants was 1.5-2.5 times higher than in WT plants,

despite a lower A

CO2

value (Fig. 3b). However, after

another week of exposure to HL, the starch content

in the mutant plants was only insignificantly higher

than in WT plants (Table 2).

FEATURES OF PHOTOSYNTHESIS IN A.thaliana PLANTS 901

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Table 2. The effects of light intensity on the starch content in the leaves of WT, αCA1-KO, and βCA1-KO plants

grown at 50-70 µmol quanta∙m

−2

∙s

−1

 (LL) and 400 µmol quanta∙m

−2

∙s

−1

 (HL)

Light intensity, µmol

quanta∙m

−2

∙s

−1

Plant

Starch content in leaves, mg/g wet weight

Age

57 days 64 days

50-70 (LL)

WT 1.54 ± 0.05 (100%) 1.28 ± 0.81 (100%)

αCA1-KO 1.31 ± 0.21 (85%) 0.47 ± 0.30 (37%)

βCA1-KO 1.10 ± 0.25 (71%) 0.18 ± 0.04 (15%)

400 (HL)

WT 2.70 ± 0.21 (100%) 24.23 ± 1.98 (100%)

αCA1-KO 6.21 ± 0.10 (230%) 31.06 ± 1.20 (128%)

βCA1-KO 4.39 ± 0.43 (163%) 26.24 ± 2.41 (108%)

Note. The table shows representative results of one experiment (n ≥ 4). The content of starch in HL-adapted plants was

determined after three (57-day old plants) and four (64-day old plants) weeks of adaptation. The value for WT plants under

the corresponding growing conditions was taken as 100%. Similar results were obtained in six growing of plants.

Assessment of pmf components in unseparated

leaves of WT, αCA1-KO, and βCA1-KO plants grown

under LL was carried out at the increasing light

intensity. Already at 364 µmol quanta∙m

−2

∙s

−1

, the

pmf value was higher in βCA1-KO plants and lower in

αCA1-KO plants compared to WT plants (Fig. 4a) due

to the corresponding changes in ΔpH (Fig. 4b). At the

light intensities below 904 µmol quanta∙m

−2

∙s

−1

, ΔΨ

did not differ significantly between the mutant and

WT plants. At 904µmol quanta∙m

−2

∙s

−1

, ΔΨ was high-

er in αCA1-KO mutants than in WT plants (Fig. 4c),

which allowed to partially compensate for the differ-

ences in the pmf value under these conditions.

Photosynthetic parameters of WT plants and

mutant plants deficient by stromal CAs. Despite a

pronounced effect caused by the knockout of genes

coding for stromal CAs on the CO

assimilation

(Fig.  3b) and starch synthesis (Table  2), there were

no differences between the ETR values in the leaves

of WT and mutant plants grown under LL and HL

(data not shown). However, when the illumination of

plants grown under the LL conditions was increased

to 400 µmol quanta∙m

−2

∙s

−1

and the CO

level in

the measurement chamber was decreased from 400

to 150 ppm, the ETR in αCA1-KO plants was lower

and the ETR in βCA1-KO plants was higher compared

to WT plants (Fig. 5a). It was also shown that under

these conditions, the value of 1-qL parameter charac-

terizing the proportion of closed PSII reaction centers

and relative reduction in the plastoquinone pool was

higher in αCA1-KO mutants and lower in βCA1-KO

mutants compared to WT plants (Fig. 5b). The differ-

ences between WT and βCA1-KO plants persisted even

when the CO

concentration in the measurement

chamber was increased to 1200 ppm (saturating con-

centration), while the differences between the WT and

αCA1-KO plants disappeared.

The rate of CO

assimilation measured at 400ppm

under the same conditions (after the dark adap-

tation of plants) was lower in αCA1-KO and βCA1-KO

Table 3. The effect of light intensity on the H

content

in the leaves of WT, αCA1-KO (line082), and βCA1-KO

plants grown at 50-70 µmol quanta∙m

−2

∙s

−1

 (LL) and

400  µmol quanta∙m

−2

∙s

−1

 (HL) after 3 weeks of accli-

mation to HL

Light

intensity, µmol

quanta∙m

−2

∙s

−1

Plant

content,

µmol H

wet weight

50-70 (LL)

WT 0.94 ± 0.06

αCA1-KO 1.29 ± 0.09**

βCA1-KO 1.60 ± 0.10***

400 (HL)

WT 1.0 ± 0.10

αCA1-KO 0.32 ± 0.01***

βCA1-KO 0.26 ± 0.01***

Note. The table shows the data of a representative experi-

ment; *** p ≤ 0.001 and ** p ≤ 0.01, significant difference

according to the Holm–Bonferroni test. Ineach case, the mu-

tants were compared to WT plants grown under the same

experimental conditions. Similar results were obtained in 3-4

biological replicates from three growing of plants.

RUDENKO et al.902

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 4. The effects of the lack of αCA1 and βCA1 on the pmf (a), ∆pH (b), and ΔΨ (c) on the thylakoid membrane in the

leaves of A. thaliana plants grown under LL. The differences were considered significant at p ≤ 0.05 as determined by

the Holm–Bonferroni method (n ≥ 14). The results were obtained in 7-8 biological replicates in four independent growing.

mutants than in WT plants (Fig. 5c); this difference

was even more pronounced at 1200 ppm CO

in the

measurement chamber. The Cc value determined at

400ppm CO

(atmospheric concentration of carbon di-

oxide) as two times lower in βCA1-KO mutants than

in WT and αCA1-KO plants (Fig. 5d).

The effects of illumination on the hydrogen

peroxide concentration and expression of stress

markers in the leaves of WT and mutant plants.

High light intensity, salinization, drought, temperature

changes, and other stress factors promote generation

of reactive oxygen species (ROS) [48, 49]. In response

to stress, plants trigger adaptation mechanisms in-

cluding the so-called ROS wave, i.e., the propagation

of stress signal activating multiple physiological, mo-

lecular, and metabolic responses necessary for the

plant acclimation to stress [49]. The intensity of the

ROS wave can be increased by various stress hor-

mones, such as SA, JA, abscisic acid (ABA), and others,

that can affect expression of specific stress response

genes [50]. In our experiments, the levels of H

in αCA1-KO and βCA1-KO mutants under LL were 40-

60% higher than in WT plants (Table 3). The acclima-

tion to HL for 21 days caused no changes in the H

content in WT plants, while the H

concentration

in αCA1-KO and βCA1-KO mutants decreased 4 and

6 times, respectively, compared to the LL conditions.

The expression levels of the ABA-induced genes

COR414-TM1(At1g29395) and ANAC019(At1g52890),

the SA-induced gene At1g64280(NPR1), and the

JA-induced gene At1g19180(JAZ1; JA pathway re-

pressor) were determined after 21 days of expo-

sure to HL [50, 51]. Under LL, expression of all four

stress genes in αCA1-KO and βCA1-KO mutant plants

FEATURES OF PHOTOSYNTHESIS IN A.thaliana PLANTS 903

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 5. The effects of the lack of αCA1 and βCA1 on the ETR(a), proportion of closed PSII reaction centers (1-qL)(b), A

CO2

(c),

and CO

level in chloroplasts(Cc)(d) in WT, αCA1-KO, and βCA1-KO plants grown at 50-70µmol quanta∙m

−2

∙s

−1

. The Ccval-

ues are shown for the stationary conditions (400ppm CO

). The differences were determined the Holm–Bonferroni method

separately for each region of the curve corresponding to particular CO

concentrations and were considered significant

p < 0.05 (n ≥ 14). The results were obtained in 6-7 biological replicates in four independent cultivations are presented.

The measurements were performed at 400 µmol quanta∙m

−2

∙s

−1

was 3-5times higher than in WT plants (Fig.6), except

for NPR1, whose expression was 12 times higher in

αCA1-KO mutants than in WT plants (Fig. 6b). After

acclimation to HL, the expression of At1g52890 and

JAZ1 increased 3-fold (Fig. 6, a and c) and expression

of NPR1 increased 17-fold in WT plants (Fig. 6b).

On the contrary, in αCA1-KO and in βCA1-KO mutants

exposed to HL, the expression levels of all studied

stress marker genes decreased 3.5-10 times and, ac-

cordingly, were found to be lower than in WT plants.

Hence, the changes in the expression levels of genes

induced by stress hormones in WT plants and mutants

deficient by stromal CAs corresponded to the relative

changes in the hydrogen peroxide level in these plants

both under LL and HL.

DISCUSSION

The assumptions on the physiological role of CAs

in the cells of higher C3 plants mostly consider their

potential involvement in C

inorg

transport to chloroplasts

RUDENKO et al.904

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 6. Expression levels of genes induced by phytohormones in the leaves of Arabidopsis plants grown at LL (50-70 µmol

quanta∙m

−2

∙s

−1

; white columns) and plants of the same age after 14-21 days of acclimation to HL (400 µmol quan-

ta∙m

−2

∙s

−1

; gray columns). a)At1g29395 (COR414-TM1) and b) At1g52890(ANAC019) induced by ABA; c) At1g64280(NPR1)

induced by SA; d) At1g19180(JAZ1) induced by JA. The data were normalized to the actin gene expression. Significant

differences were determined by the Holm–Bonferroni method, n ≥ 9. Similar results were obtained in three growing of

plants in 3 biological and 3 analytical replicates.

or the necessity of HCO

−

conversion into CO

(direct

substrate for RuBisCO) [3-5]. At the same time, it was

found that RuBisCO and stromal CAs are functionally

related and colocalize in the chloroplasts of different

plant species [52, 53]. However, the attempts to study

the effects of single mutations in CAs [22, 24] or si-

multaneous shutdown of two and more CAs [12, 26]

have not led to the development of generally accepted

concepts on the functions of stromal CAs.

The knockout of both αCA1 and βCA1 resulted in

a 5-fold decrease in the CA activity of stromal prepa-

rations (Fig.1), which confirmed the presence of both

enzymes in the stroma of chloroplasts. The effects of

mutations in αCA1 and βCA1 were more pronounced

when the plants were exposed to a higher light in-

tensity compared to the conditions under which they

had been grown. When exposed to HL, αCA1-KO,

and βCA1-KO plants grown under LL demonstrated

a lower A

CO2

value at 400 ppm CO

(compared to

WT plants) and even a more pronounced difference

at 1200 ppm CO

(Fig. 5c) after exposure in the dark,

followed by turning on the light, i.e., upon activation

of the CBB cycle. At the same time, the Cc value in

βCA1-KO mutants was two times lower than in WT

and αCA1-KO plants (Fig. 5d), suggesting that the de-

crease in the A

CO2

in βCA1-KO mutants (Fig.3b and5c)

was due to the insufficient rate of C

inorg

inflow into

chloroplasts, while in αCA1-KO plants that had the

same Cc value as WT plants (Fig. 5d), the observed

reduction in A

CO2

(compared to WT plants) was due

FEATURES OF PHOTOSYNTHESIS IN A.thaliana PLANTS 905

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

to the insufficient intensity of stromal HCO

−

con-

version to CO

for providing the CBB cycle. This was

also evidenced by the differences between the ΔpH

values in these mutants: in βCA1-KO plants, ΔpH was

higher than in WT plants (Fig. 4), which was appar-

ently determined by a higher (compared to WT) pH

value of the stroma in the case of insufficient CO

inflow. Under normal conditions, CO

entering a

chloroplast is converted to HCO

−

with the release of

, which shifts stromal pH to a more acidic region.

In the βCA1-deficient mutants, the inflow of CO

can

be impaired and, accordingly, the generation of H

in the stroma is reduced, leading to the pH increase.

At the same time, the absence of CA supplying CO

for RuBisCO through its conversion from bicarbonate

should lead to a slight decrease in stromal pH and,

therefore, to a decrease in ΔpH on the thylakoid mem-

brane. This was observed in αCA1-KO plants, in which

ΔpH was lower than in WT plants and, accordingly,

even lower than in βCA1-KO plants (Fig. 4).

The convergence of A

CO2

values in βCA1-KO and

WT plants at 1200  ppm CO

(Fig. 5c), compared to

the respective values at 400  ppm, can be explained

with regard to the presumptive function of βCA1 (see

above): if βCA1 is responsible for the CO

inflow into

chloroplasts, then the increased CO

concentration in

the air can partially compensate for its absence.

In αCA1-KO plants, the ETR was lower than in

WT and βCA1-KO plants at all CO

concentrations

(Fig. 5a), while the A

CO2

value at 400 ppm CO

was

lower than in WT plants but higher than in βCA1-KO

mutants (Fig. 5c). The increase in the CO

concentra-

tion to 1200 ppm caused no convergence of A

CO2

 val-

ues for αCA1-KO and WT plants, but rather “distanced”

them due to the reduction of this parameter in αCA1-

KO plants. The latter fact can also be explained by

the hypothetical function of αCA1 (see above): if

αCA1 catalyzes bicarbonate dehydration coupled with

supplying CO

directly to RuBisCO, then its absence

at a high CO

concentration in the air, which increas-

es the level of bicarbonate in the stroma, is capable

even to limit even to a greater extent (compared to

its absence at lower levels of CO

and, accordingly,

bicarbonate) this supply compared to WT plants.

No differences between A

CO2

values in the leaves

of mutant and WT plants grown under LL were ob-

served under the stationary photosynthesis conditions

(Fig. 3b), probably because the CA mutants lacking

stromal CAs compensated for the insufficient sup-

ply of chloroplasts with C

inorg

via enhanced stomatal

conductance (Fig. 3a). An increase (compared to WT

plants) in the stomatal conductance of mutants defi-

cient by both αCA1 and βCA1 has been shown pre-

viously in rice and tobacco plants [13, 25]. For some

reason, plants grown under HL were unable to main-

tain such compensatory mechanism (Fig. 3a), which

might be due to the fact that in αCA1-KO and βCA1-KO

mutants, expression of stress-related genes induced by

ABA (Fig. 6, a and b), the known regulator of stoma-

tal closure, was reduced, possible because of the de-

creased ABA content in mutant plants.

A convincing evidence of the involvement of

stromal CAs in the mechanisms of C

inorg

supply for

photosynthetic processes has been obtained in the ex-

periments on the knockout of both the αCA1 gene[13]

(see “Introduction”) and gene encoding stromal βCA in

rice plants [25]. The abolishment of βCA synthesis led

to a decrease in the plant biomass and net photosyn-

thesis rate, which was compensated by the increased

stomatal conductance, stomatal pore opening ratio,

water loss rate, and RuBisCO activity [25]. The lack

of pronounced effects of single mutations in CAs in

dicotyledonous plants might be due to the cooperative

function of CAs in the cells of these plants.

Physiological manifestations of CA knockouts in

Arabidopsis and tobacco plants were observed only

when the synthesis of two or more CAs was simulta-

neously shut down. Thus, the knockout of βCA1 and

plasma membrane βCA4.1 has shown that these en-

zymes are jointly involved in the regulation of stoma-

tal conductance for carbon dioxide [24]. Suppression

of plant growth at low CO

levels was observed upon

the simultaneous shutdown of at least two out of five

genes coding for mitochondrial CAs [54], as well as

two genes for cytoplasmic CAs in Δβ-CA4.2/β-CA2 mu-

tants [26]. The absence of pronounced effect of the

abolishment of stromal CAs under typical growth con-

ditions (such effect was observed only upon dramat-

ic changes in illumination and CO

level in the air;

Figs. 3-5) can also be explained by the cooperative

function of CAs in plant cells.

Both expression of CA genes and CA activity

varied depending on the growth conditions, thus

demonstrating different needs of plants for CAs un-

der different external conditions. Growth of plants at

a low carbon dioxide level (150 ppm) led to a signif-

icant increase in the CA activity in the stromal and

thylakoid preparations isolated from the leaves of

WT plants compared to the plants grown at the nor-

mal (450ppm) or elevated (1200ppm) CO

concentra-

tions[35]. In rice seedlings, expression of gene encod-

ing CAs of the β-family, as well as the total CA activity

in leaf extracts, increased after exposure to the osmot-

ic stress[55]. Previously, we have shown that exposure

of plants to HL upregulated expression of most genes

coding for chloroplast CAs, with the exception of the

βCA5 gene[34]. Here, we confirmed this effect in WT

plants (Fig. 2) by demonstrating that in the HL-ex-

posed βCA1-KO mutants, the changes in the content

of transcripts were the same as in WT plants, while

in αCA1-KO mutants, these changes were the opposite,

i.e., the amount of βCA1.1  +  βCA1.2, βCA1.3  +  βCA1.4,

RUDENKO et al.906

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

and αCA2 transcripts decreased, the content of the

βCA5 transcript increased, while the content of the

αCA4 transcript remained the same (Fig. 2). The lev-

els of βCA1.1  +  βCA1.2, βCA1.3+  βCA1 and αCA2 tran-

scripts in the mutants grown under LL was initial-

ly higher than in WT plants. The only gene with a

higher expression level was αCA4 in βCA1-KO plants.

Hence, the knockout of αCA1 and βCA1 influenced the

synthesis of other CAs in chloroplasts and this effect

depended on the light intensity at which the plants

were grown. The changes in the expression of some

CA genes with the knockout of genes coding for oth-

er CAs have also been shown by Sharma et al. [14],

Nadeeva et al. [21], and Rudenko et al. [56].

The effects of the αCA1 and βCA1 abolishment

are either directly caused by consequences of the ab-

sence of these enzymes in chloroplasts (e.g., changes

in ΔpH, decrease in A

CO2

, reduction of Cc in chloro-

plasts) or are associated with the compensation for

their absence (changes in the expression of other CA

genes and increase in the stomatal conductance of

leaves). Apparently, these compensatory mechanisms

enable plants to successfully compensate for the lack

of stromal αCA1 and βCA1. In αCA1-KO plants, the ETR

was only 10% lower and the proportion of closed re-

action centers of PSII was approximately 10% high-

er than in WT plants (Fig. 5, a and b). In βCA1-KO

plants, the ETR was higher and, accordingly, the value

of 1-qL was lower than in WT plants.

Despite the presence of mechanisms compensat-

ing for the absence of αCA1 and βCA1, the content

of starch, the main structural carbohydrate and prod-

uct of photosynthesis, in the mutants deficient by

these enzymes was lower than in WT plants when

the plants grown under LL conditions (Table 2): by

20-30% at the age of less than 2 months it and by 60

and 85% in αCA1-KO and βCA1-KO plants, respectively,

that were older than 2months. After 3 weeks of adap-

tation to HL, the starch content, on the contrary, was

1.6-2.3 times higher in αCA1-KO and βCA1-KO plants

than in WT plants grown under the same conditions.

These differences could be caused by the reduced

starch degradation resulting from the increased ABS

content, which was evidenced by the downregulated

expression of ABA-inducible genes under HL condi-

tions (Fig. 6, a and b). ABA promotes starch degra-

dation [57, 58], which enhances stress resistance of

plants. After one more week under HL, the starch

content increased approximately 9-fold in WT plants

vs. 5-6-fold in the mutants (Table  2), so it became

almost the same in αCA1-KO, βCA1-KO, and WT plants.

The number of chloroplasts in a cell increases

under HL [59] while the size of the PSII light-harvest-

ing antenna PSII decreases, which is accompanied by

the reduction in the Chl content and changes in the

composition and amount of carotenoids. After adap-

tation to HL, the content of both Chl forms decreased

in WT and βCA1-KO plants originally grown under LL,

i.e., these plants showed similar changes in the con-

tent of pigments during adaptation to HL (Table1). In

αCA1-KO plants, the level of Chl a slightly increased

(by 7%). As a result, there was a similar 20-25% in-

crease in the Chl a/Chl b ratio; however, in αCA1-KO

plants, it was due to the elevation in the Chl a con-

tent, while in WT and βCA1-KO plants, it was caused

by the decrease in the Chl b amount. The content of

carotenoids decreased in WT plants and both mutant

lines, but still remained the highest in αCA1-KO plants.

Thus, in the absence of αCA1 and with initially lower

levels of pigments under LL, αCA1-KO plants demon-

strated the ability to maintain a higher content of pig-

ments under HL compared to WT and βCA1-KO plants.

There is a growing body of data on the involve-

ment of βCA1 in stress signaling [30, 33] due to its

ability bind both SA [29], as well as NPR1 and NRB4,

the key proteins of the SA-mediated stress defense

pathway [30]. In addition, a convincing evidence has

been obtained that βCA1 participates in plant protec-

tion against stress as an enzyme of JA biosynthesis

(see “Introduction”). The knockout of the βCA1 and

αCA1 genes resulted in similar changes in the H

content and expression levels of ABA-, SA-, and JA-in-

ducible stress marker genes. In the mutant plants, the

above parameters were higher under LL and lower

under HL compared to WT plants (Table  3; Fig.  6),

suggesting the involvement of not only βCA, but αCA1

as well. One of the indications of insufficient synthe-

sis of JA under LL in the absence of stromal CAs is

higher (compared to WT) expression levels of the JAZ1

gene (Fig. 6d) coding for the JA pathway inhibitor,

NPR1 gene (Fig. 6c) encoding a major inducer of the

SA pathway, and marker genes involved in the induc-

tion of the ABA pathway (Fig. 6, a and b). The action

of these genesis antagonistic to the JA pathway; they

are activated when this pathway is suppressed [60].

In addition, SA promotes the ROS wave, whereas JA

suppresses it [49], which can explain a higher level

of H

in the mutants deficient by the stromal Cas

under LL compared to WT plants (Table 3). After ad-

aptation of plants to HL, as well as upon exposure

to other stress factors, plants activate generation of

ROS [49, 50], including H

, a molecule that induc-

es changes in the expression of many genes. The in-

crease in the H

levels was observed only at the

very beginning of adaptation to HL; even a few days

later, the H

concentration decreased to the level

observed under LL conditions [61]. This explains the

same content of H

in WT plants exposed to LL

and HL (Table 3). At the same time, the expression

levels of stress marker genes in WT plants under HL

were several times higher than under LL (Fig. 6). In

αCA1-KO and βCA1-KO mutants, the expression of

FEATURES OF PHOTOSYNTHESIS IN A.thaliana PLANTS 907

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

these genes under HL proved to be lower than in WT

plants, probably due to the higher activation of anti-

oxidant systems under normal conditions and greater

“readiness” to stress, which might explain the absence

of need for further enhancement of the antioxidant

response to HL in these plants.

Abbreviations. αCA1-KO, homozygous plants

with knocked-out At3g52720 gene encoding αCA1;

βCA1-KO, homozygous plants with knocked-out

At3g01500 gene encoding βCA1; ABA, abscisic acid;

CO2

, CO

 assimilation rate; CA, carbonic anhydrase;

Cc, CO

level in chloroplasts; Chl, chlorophyll; C

inorg

inorganic carbon; ECS, electrochromic shift of the ab-

sorption band of carotenoids in the thylakoid mem-

brane; ETR, electron transport rate; HL, high light;

JA,jasmonic acid; LL,low light; RuBisCO,ribulose bis-

phosphate carboxylase/oxygenase; pmf,proton motive

force; PSI and PSII, photosystem I and II; ROS, reac-

tive oxygen species; SA,salicylic acid; WT,wild type.

Supplementary information. The online version

contains supplementary material available at https://

doi.org/10.1134/S0006297925600954.

Acknowledgments. The authors are grateful to

Professor J. V. Moroney for kindly providing the seeds

of homozygous mutant plants and to Dr. M. A. Kozule-

va for assistance in developing the method for eval-

uating the gas exchange parameters in plants and

measuring Chl a fluorescence in plant leaves at dra-

matically changing carbon dioxide concentrations in

the measurement chamber. The study was carried out

with the equipment of the Center for Collective Use of

the Pushchino Scientific Center for Biological Research

(https://www.ckp-rf.ru/ckp/670266/).

Contributions. N.N.R. developed the concept and

supervised the study; N.N.R., M.Yu.R., L.K.I., E.M.N.,

and D.V.V. performed the experiments; N.N.R., M.Yu.R.,

L.K.I., E.M.N., D.V.V., and B.N.I. discussed the research

results; N.N.R. wrote the text of the article; B.N.I. edit-

ed the manuscript.

Funding. The work was supported by the State

Scientific Program (project no.125051305922-5).

Ethics approval and consent to participate.

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest. The authors of this work de-

clare that they have no conflicts of interest.

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