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The Functions of Chloroplastic Ascorbate in Vascular Plants and Algae

Authors:
  • Biological Research Centre Szeged

Abstract and Figures

Ascorbate (Asc) is a multifunctional metabolite essential for various cellular processes in plants and animals. The best-known property of Asc is to scavenge reactive oxygen species (ROS), in a highly regulated manner. Besides being an effective antioxidant, Asc also acts as a chaperone for 2-oxoglutarate-dependent dioxygenases that are involved in the hormone metabolism of plants and the synthesis of various secondary metabolites. Asc also essential for the epigenetic regulation of gene expression, signaling and iron transport. Thus, Asc affects plant growth, development, and stress resistance via various mechanisms. In this review, the intricate relationship between Asc and photosynthesis in plants and algae is summarized in the following major points: (i) regulation of Asc biosynthesis by light, (ii) interaction between photosynthetic and mitochondrial electron transport in relation to Asc biosynthesis, (iii) Asc acting as an alternative electron donor of photosystem II, (iv) Asc inactivating the oxygen-evolving complex, (v) the role of Asc in non-photochemical quenching, and (vi) the role of Asc in ROS management in the chloroplast. The review also discusses differences in the regulation of Asc biosynthesis and the effects of Asc on photosynthesis in algae and vascular plants.
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Citation: Tóth, S.Z. The Functions of
Chloroplastic Ascorbate in Vascular
Plants and Algae. Int. J. Mol. Sci.
2023,24, 2537. https://doi.org/
10.3390/ijms24032537
Academic Editor: Sean M. Bulley
Received: 21 December 2022
Revised: 17 January 2023
Accepted: 24 January 2023
Published: 28 January 2023
Copyright: © 2023 by the author.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Review
The Functions of Chloroplastic Ascorbate in Vascular Plants
and Algae
Szilvia Z. Tóth
Laboratory for Molecular Photobioenergetics, Institute of Plant Biology, Biological Research Centre,
Temesvári krt 62, H-6726 Szeged, Hungary; toth.szilviazita@brc.hu
Abstract:
Ascorbate (Asc) is a multifunctional metabolite essential for various cellular processes in
plants and animals. The best-known property of Asc is to scavenge reactive oxygen species (ROS),
in a highly regulated manner. Besides being an effective antioxidant, Asc also acts as a chaperone
for 2-oxoglutarate-dependent dioxygenases that are involved in the hormone metabolism of plants
and the synthesis of various secondary metabolites. Asc also essential for the epigenetic regulation
of gene expression, signaling and iron transport. Thus, Asc affects plant growth, development, and
stress resistance via various mechanisms. In this review, the intricate relationship between Asc and
photosynthesis in plants and algae is summarized in the following major points: (i) regulation of Asc
biosynthesis by light, (ii) interaction between photosynthetic and mitochondrial electron transport in
relation to Asc biosynthesis, (iii) Asc acting as an alternative electron donor of photosystem II, (iv) Asc
inactivating the oxygen-evolving complex, (v) the role of Asc in non-photochemical quenching, and
(vi) the role of Asc in ROS management in the chloroplast. The review also discusses differences in the
regulation of Asc biosynthesis and the effects of Asc on photosynthesis in algae and vascular plants.
Keywords:
ascorbate; non-photochemical quenching; oxygen-evolving complex; photosynthesis;
photosystem II; reactive oxygen species; vitamin C
1. Introduction
Ascorbate (Asc) is the most abundant water-soluble metabolite in plants and is essen-
tial for plant growth and development. Asc is also required in the human diet; therefore,
serious efforts are underway to increase the Asc contents of fruits and vegetables (reviewed,
e.g., by [
1
,
2
]). In vascular plants and green algae, Asc is synthesized via the Smirnoff-
Wheeler pathway, in which GDP-D-mannose is converted to L-galactono-1,4-lactone in the
cytoplasm by GDP-D-mannose 3
0
,5
0
epimerase (GME), GDP-L-galactose phosphorylase
(GGP), L-galactose-1-P phosphatase (GPP), and L-galactose dehydrogenase (GDH) [
3
,
4
].
The final step, the conversion of L-galactono-1,4-lactone to Asc, occurs in the mitochondria
and is catalyzed by L-galactono-1,4-lactone dehydrogenase (GLDH) at Complex I [
4
,
5
]
(Figure 1). VTC2, encoding GGP, plays a vital role in the regulation of Asc biosynthesis,
both in vascular plants and green algae [
6
,
7
]. In vascular plants, not only its expression but
also a feedback mechanism on GGP translation by Asc and a small ORF largely determines
the rate of Asc biosynthesis, thereby its cellular concentration [811].
Alternative Asc biosynthesis routes, namely, the galacturonate, the L-gulose pathway,
and the myoinositol pathway, have been suggested to contribute to Asc biosynthesis
(reviewed by [
12
]). However, various lines of evidence show that they do not contribute
significantly to Asc biosynthesis in plant leaves [13].
The best-known role of Asc is to scavenge reactive oxygen species (ROS), in a highly
regulated manner in each cellular compartment (reviewed by [
14
]). It is linked to its capacity
to act as a weak reductant and to the non-toxicity of the generated monodehydroascor-
bate (MDA) and the bicyclic dehydroascorbate (DHA) [
15
,
16
]. Asc effectively eliminates
superoxide and tocopherol radicals, though it reacts slowly with H
2
O
2
. Plants use Asc
Int. J. Mol. Sci. 2023,24, 2537. https://doi.org/10.3390/ijms24032537 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023,24, 2537 2 of 17
peroxidases (APX) to eliminate H
2
O
2
more effectively (reviewed by [
14
,
15
]). The reaction
oxidizes Asc into MDA radicals, which can spontaneously disproportionate into DHA and
Asc. The MDA radicals are then recycled back to Asc through MDA reductases (MDARs)
or reduced ferredoxin produced at the acceptor side of photosystem I (PSI) [
14
,
17
], while
DHA is reduced by DHA reductase (DHAR) in glutathione-dependent non-enzymatic or
enzymatic reactions [18,19] (Figure 1).
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 2 of 17
Figure 1. Ascorbate (Asc) biosynthesis and the roles of chloroplastic Asc in vascular plants. Asc is
synthesized via the Smirnoff-Wheeler pathway with the majority of the steps taking place in the
cytosol and the last step in the mitochondria at Complex I. Asc biosynthesis is regulated by light,
circadian clock, photosynthetic electron transport, and a feedback inhibition by Asc. Asc is found in
all cell compartments; PHT4;4 transports it into the chloroplast. Chloroplastic Asc plays multiple
roles: (i) it is an alternative electron donor of photosystem II (PSII) when the oxygen-evolving com-
plex (OEC) is inactive, (ii) it may inactivate the OEC under specific circumstances, (iii) it is a co-
substrate of violaxanthin de-epoxidase (VDE) thereby plays a role in non-photochemical quenching,
(iv) Asc participates in reactive oxygen species management. Abbreviations: APX, Asc peroxidase;
cp, chloroplast; cs, cytosol; Cytb6f, cytochrome b6f; Cytc, cytochrome c; DHA, dehydroascorbate;
DHAR, dehydroascorbate reductase; DTX25, vacuolar ascorbate transporter; Fd, ferredoxin; FNR,
ferredoxin-NADP oxidoreductase; GDH, L-galactose dehydrogenase; GGP, GDP-L-galactose phos-
phorylase (encoded by VTC2); GLDH, L-galactono-1,4 lactone dehydrogenase; GME, GDP-D-man-
nose 3,5-epimerase; GMP, GDP-D-mannose phosphorylase; GPP, L-galactose-1-P phosphatase;
GR, glutathione reductase; L-Gal-L, L-galactono-lactone; LHC, Light-harvesting complex; m, mito-
chondrion; MDA, monodehydroascorbate; MDAR Monodehydroascorbate reductase; n, nucleus;
PHT4;4, chloroplastic Asc transporter; pm, plasma membrane; PSI, photosystem I; SOD, superoxide
dismutase; t, thylakoid; TCA, tricarboxylic acid; UQ, ubiquitine; v, vacuole.
LHC LHC
Photosys tem II Photosystem I
PQ-pool
Calvin-Benson cycle
FNR
CO2
NADP+
NADPH
Fd
Chloro plast stroma
QA
e-
cytb6f
e-
PC
P700
P680
OEC
e-
2 H2O
O2 +4H+
Thylakoid lumen
O2O2-
H+
SOD
H2O2
H2O
APX
Asc
MDA
DHA
DHAR
MDAR
GR
GSH
GSSR
Asc
TyrZ+VDE
Asc
MDA
DHA
I
IV
UQ
Intermembrane space
III
e-
NADH
NAD+
Matrix
H+H+
H2O2
H2O
APX
Asc
DHA
Asc
Succinate
Fumarate
II
e-
H+
H+
H+H+
Cyt c
GLDH
L-Gal-L
H2OO2
TCA cycle
AOX
H2O
O2
cs
nD-mannos e-1P
GDP -D- mannose
GDP -L-galactose
L-galactose-1P
L-galactose
L-galactono-lactone
GMP
GME
GGP (VTC2)
GPP
GDH
v
cp
PHT4;4
Asc
GLDH
pm
t
m
Asc
Feedback inhib ition
Retrograd e signaling
VTC2
Asc
Asc
Asc
DTX25
Asc
DHA
Figure 1.
Ascorbate (Asc) biosynthesis and the roles of chloroplastic Asc in vascular plants. Asc is
synthesized via the Smirnoff-Wheeler pathway with the majority of the steps taking place in the
cytosol and the last step in the mitochondria at Complex I. Asc biosynthesis is regulated by light,
circadian clock, photosynthetic electron transport, and a feedback inhibition by Asc. Asc is found in
all cell compartments; PHT4;4 transports it into the chloroplast. Chloroplastic Asc plays multiple
roles: (i) it is an alternative electron donor of photosystem II (PSII) when the oxygen-evolving complex
(OEC) is inactive, (ii) it may inactivate the OEC under specific circumstances, (iii) it is a co-substrate
of violaxanthin de-epoxidase (VDE) thereby plays a role in non-photochemical quenching, (iv) Asc
Int. J. Mol. Sci. 2023,24, 2537 3 of 17
participates in reactive oxygen species management. Abbreviations: APX, Asc peroxidase; cp,
chloroplast; cs, cytosol; Cytb
6
f, cytochrome b
6
f; Cytc, cytochrome c; DHA, dehydroascorbate; DHAR,
dehydroascorbate reductase; DTX25, vacuolar ascorbate transporter; Fd, ferredoxin; FNR, ferredoxin-
NADP oxidoreductase; GDH, L-galactose dehydrogenase; GGP, GDP-L-galactose phosphorylase
(encoded by VTC2); GLDH, L-galactono-1,4 lactone dehydrogenase; GME, GDP-D-mannose 3
0
,5
0
-
epimerase; GMP, GDP-D-mannose phosphorylase; GPP, L-galactose-1-P phosphatase; GR, glutathione
reductase; L-Gal-L, L-galactono-lactone; LHC, Light-harvesting complex; m, mitochondrion; MDA,
monodehydroascorbate; MDAR Monodehydroascorbate reductase; n, nucleus; PHT4;4, chloroplastic
Asc transporter; pm, plasma membrane; PSI, photosystem I; SOD, superoxide dismutase; t, thylakoid;
TCA, tricarboxylic acid; UQ, ubiquitine; v, vacuole.
DHA is unstable; therefore, if it is not recycled back to Asc, it is rapidly and irreversibly
degraded into oxalate, L-threonate, or tartaric acid, depending on the plant species [
20
,
21
].
Nevertheless, the regeneration of DHA and MDA occurs very effectively, as most of the Asc
pool is in the reduced state under both non-stress and mild stress conditions. An exception
in this respect is the apoplast in which the oxidation of Asc occurs at a relatively high rate
due to the activity of Asc oxidase; this peculiarity is most probably related to cell wall
loosening and growth [22,23].
Besides being an effective antioxidant, Asc also acts as a chaperone for 2-oxoglutarate-
dependent dioxygenases (2-ODDs). Plant 2-ODDs are involved, for instance, in hormone
metabolism (ethylene, abscisic acid, gibberellins, indole-3-acetic acid) and the synthesis of
various secondary metabolites, such as anthocyanins and glucosinolates [
24
26
]. Asc is also
involved in the epigenetic regulation of gene expression [
27
] via modifying the activities
of ten-eleven translocation (TET) and Jumonji C-domain-containing histone demethylases
that are both 2-ODDs [
27
30
]. Asc has also been proposed to be involved in iron transport
and Ca2+ signaling [31,32].
Thus Asc affects plant growth, development, and stress resistance via various mech-
anisms that are challenging to dissect. Growth defects have been reported for the Asc-
deficient vtc2-1 Arabidopsis mutant containing about 20% Asc relative to the wild type,
but later it turned out to be due at least partially to cryptic mutations [
33
]. The more re-
cently identified T-DNA mutant vtc2-4 did not show any alteration in the phenotype under
optimal growth conditions in [
33
], but a mild phenotype was observed in [
34
]. Moreover,
the approx. 80% reduction in Asc level observed in the vtc2 mutants only moderately
enhances high light sensitivity [
35
]; thus, this low level of Asc seems to be sufficient for the
housekeeping functions under laboratory conditions. On the other hand, the situation can
be different in the field, and it may also be species-dependent because in rice, for instance,
Asc content reduction did lead to diminished growth and photosynthesis rate [36].
Several excellent reviews have already been published on the biosynthesis and the
various functions of Asc (e.g., [
11
,
14
,
15
,
37
40
]). This review focuses on less extensively
discussed issues, such as the relationship between photosynthesis and Asc biosynthesis
and the effects of Asc on photosynthetic electron transport in vascular plants and algae.
2. Regulation of Ascorbate Biosynthesis by Light
Under regular growth and moderate light conditions (at approx. 100
µ
mol m
2
s
1
),
the Asc level of Arabidopsis leaves is in the range of 2 to 5
µ
mol/g fresh weight (FW). The
Asc content varies by a factor of two depending on the time of day, with a minimum at
night and a maximum in the afternoon [
41
43
]. Under stress conditions, including high
light [
9
,
42
,
44
,
45
], ozone [
46
,
47
], salt [
48
,
49
], drought stress [
50
], and nitrogen starvation [
51
]
a two- to three-fold increase in Asc level occurs on a timescale of days, with an apparent
upper limit of about 8–10
µ
mol Asc/g FW. Using stable and inducible expression systems
in Arabidopsis, the maximum Asc concentration that can be reached is in the same range of
maximum 10
µ
mol Asc/g FW (e.g., [
7
]; reviewed by [
11
,
38
]). Asc content can be somewhat
more effectively increased by L-galactono-1,4-lactone treatment to the range of 15
µ
mol
Asc/g FW [52,53].
Int. J. Mol. Sci. 2023,24, 2537 4 of 17
These results demonstrate that Asc biosynthesis is highly regulated, to keep Asc in
the optimum concentration range during the normal life cycle of plants and under stress
conditions. Therefore, understanding the signaling pathways and the control mechanism
of Asc biosynthesis are of primary importance to achieve a substantial increase in the Asc
content of crops.
One major factor controlling Asc biosynthesis is light. Several Smirnoff-Wheeler
pathway genes are induced by illumination. These include GDP-D-mannose pyrophospho-
rylase (GMP), GGP, GPP, and GLDH [
54
56
]. Transcript levels of these biosynthesis genes
also follow a circadian rhythm [
42
]. It has been shown that 3-(3,4-dichlorophenyl)-1,1-
dimethylurea (DCMU), an inhibitor of photosystem II (PSII), prevents the increase in Asc
pool size in Arabidopsis upon a shift from light-dark cycle to continuous light conditions,
along with a decrease in the transcript levels of GMP, GGP, GPP, and GLDH [55].
When Arabidopsis plants were transferred to a medium containing sucrose, the leaf
Asc levels decreased along with a decrease in the rate of CO
2
fixation. Asc content diminish-
ment upon sucrose feeding did not occur in a sugar-insensitive Arabidopsis abi4/sun6 mu-
tant [
55
]. Exogenous application of glucose in pea seedlings did not affect Asc content [
57
],
whereas sucrose feeding in detached broccoli inflorescences delayed Asc depletion [
58
],
and in tomato fruit, it increased the Asc content [59].
The Asc content decreases markedly in darkness (at a rate of approx. 2% per hour),
while the levels of its degradation products increase [
21
], and dark-grown plants produce
no Asc at all [
60
]. Interestingly, the downregulation of VTC2 expression by DCMU was
slightly reversed in Arabidopsis mutants lacking GENOMES UNCOUPLED 1 (GUN1); [
61
],
the master regulator of chloroplast-to-nucleus retrograde signaling.
Thus, the results suggest that photosynthesis-derived signal(s) participate in the light
activation of Asc biosynthesis, and the role of photosynthesis is not solely to provide a
carbon source for Asc biosynthesis. On the other hand, soluble carbohydrates may affect
the expression of Asc biosynthesis genes and the regeneration of Asc. The molecular
mechanisms underlying the regulation of Asc biosynthesis by photosynthesis remain
to be explored. It is conceivable that the redox state of the plastoquinone pool plays a
regulatory role in Asc biosynthesis. In principle, H
2
O
2
and other ROS produced during
photosynthesis could also influence the expression of Asc biosynthesis genes; however,
experimental evidence is still lacking on such roles.
Light may also directly affect Asc biosynthesis, degradation, and regeneration. Indeed,
light can regulate GMP activity, namely, via CSN5B, a photomorphogenic factor that is part
of a CSN complex, negatively regulating photomorphogenesis in Arabidopsis via protea-
somal degradation [
62
64
]. Wang et al. [
64
] demonstrated that GMP is polyubiquitinated
and degraded in darkness via interaction with CSN5B. They also found that loss of CSN5B
function impaired the effect of light on Asc synthesis in response to continuous light or
darkness, showing that CSN5B is a posttranslational regulator in Asc biosynthesis.
A F-box type repressor, known as Asc acid mannose pathway regulator 1 (AMR1),
also regulates Asc biosynthesis in a light-dependent manner [
65
]. DNA knockout lines for
AMR1 accumulated two-fold greater foliar Asc than the wild type. AMR1 also negatively
affected the expression levels of most Asc biosynthesis genes, including GMP, GME, GGP,
GPP, L-GalDH, and GLDH. In addition, AMR1 expression was higher in aging leaves, and
lower at medium light than at low light intensity.
A HD-Zip I family transcription factor in tomato, SlHZ24, positively regulates the
accumulation of Asc by binding to the promoter of SlGMP3, and it also regulates the expres-
sion of GME and GGP. Accordingly, the Asc content fluctuated following the expression of
SlHZ24 in a light-dependent manner [66].
GGP also shows a significant response to different light levels due to the regulation
by light-responsive cis-elements in its promoter, namely, a G-box motif [67]. Furthermore,
light-responsive cis-elements have also been identified in the promoters of GPP and GLDH
in rice [68].
Int. J. Mol. Sci. 2023,24, 2537 5 of 17
It was also proposed that the VTC3 dual protein kinase/protein phosphatase is in-
volved in signal transduction to adjust Asc levels in response to light and temperature
changes [
69
]. VTC3 is probably located in the chloroplast, and it was suggested that the
control exerted by VTC3 is post-transcriptional and does not alter the transcript levels of
Asc biosynthesis genes in Arabidopsis [69].
The conversion of L-galactono-1,4-lactone to Asc in the mitochondria is light and
photosynthesis dependent because photosynthetic inhibitors prevent it [
52
]. H
2
O
2
pro-
duced upon stress effects may inactivate selectively and reversibly Arabidopsis GLDH by
oxidizing a cysteine residue (Cys-340) [
70
]. GLDH is protected from inactivation both by
L-galactono-1,4-lactone and Asc; therefore, their availabilities and the level of H
2
O
2
may
affect the rate of Asc biosynthesis in vascular plants.
Bryophytes, including Brachytecium velutinum,Marchantia polymorpha, and Physcomitrium
(formerly Physcomitrella)patens, possess Asc content slightly less than seed plants, in the
range of 0.3 to 2 µmol/g [71,72]. Sodeyama et al. [72] presented evidence on the Smirnoff-
Wheeler pathway as a source of Asc, whereas the D-galacturonate pathway did not seem to
contribute to Asc biosynthesis in P. patens. Interestingly, two VTC2 paralogs are functional
in P. patens, and, in contrast to higher plants, they are both equally responsible for Asc
biosynthesis. Furthermore, the light-induced fluctuation of the transcript level of the two
VTC2 genes was comparable to AtVTC2. Additionally, DCMU treatment diminished VTC2
expression and Asc content, as observed earlier in Arabidopsis. Thus, VTC2 expression is a
crucial control point of Asc biosynthesis in P. patens. Interestingly, knockout mutants with
low Asc content exhibited restricted side branch growth in their protonemata; this may be
caused by multiple factors related to the various physiological functions of Asc.
Green alga grown under benign conditions contain about 100-fold less Asc than
vascular plants, i.e., in the range of 100 to 500
µ
M [
73
75
]. Asc is synthesized via the
Smirnoff-Wheeler pathway in green algae; however, its regulation significantly differs
compared to plants. It has been shown that in Chlamydomonas reinhardtii, the expression of
the VTC2 gene is induced by H
2
O
2
and
1
O
2
, resulting in a strong increase in Asc content. On
the other hand, photosynthesis is not directly required for Asc biosynthesis. Additionally,
in contrast to plants, there is no circadian regulation of Asc biosynthesis, and C. reinhardtii
lacks negative feedback regulation by Asc in the physiological concentration range. These
mechanisms enable a rapid and manifold increase in Asc content upon various stress
treatments, including light stress and sulfur deprivation [74,76,77].
3. Interaction between Photosynthetic and Mitochondrial Electron Transport in Relation
to Asc Biosynthesis
Photosynthetic electron transport rates (ETR) and NADPH levels are only slightly
affected in vtc2 mutants containing about 20% Asc in comparison with wild-type Arabidop-
sis plants when grown under moderate or low light conditions [
35
,
44
,
78
,
79
], whereas the
photosynthetic ETR of vtc2 mutants is diminished at high light [
44
]. The vtc2 mutants
have a slightly lower stomatal conductance, which may be related to a regulatory effect
of Asc [
79
,
80
]; this, however, is compensated by a larger stomatal number and increased
RuBisCO content. Therefore, the overall CO
2
assimilation rate is affected by Asc deficiency
only in high light, but not under normal growth conditions in Arabidopsis [79,81].
The last step of Asc biosynthesis, the conversion of L-galactono-lactone into Asc, is
catalyzed by GLDH in the mitochondria [
82
,
83
]. GLDH is a protein of 58 kDa, located at
Complex I in the mitochondrial intermembrane space, tightly tethered to the membrane
through protein-protein interactions [
84
] (Figure 1). Complex I is organized in two arms:
the matrix arm transfers electrons from NADH to ubiquinone, and the membrane arm is
responsible for proton translocation [
85
]. In addition to being essential for Asc biosynthesis,
GLDH has a non-enzymatic role in the assembly of the membrane arm of Complex I [
86
].
This function is likely to be independent of the role of GLDH in Asc biosynthesis because
the vtc2-1 Arabidopsis mutant accumulates wild-type levels of Complex I [86].
Int. J. Mol. Sci. 2023,24, 2537 6 of 17
During the oxidation of L-galactono-1,4-lactone to Asc by GLDH, electrons are fed
into the mitochondrial electron transport chain via cytochrome c [
5
,
87
], a soluble redox-
active heme protein that transfers electrons from Complex III to Complex IV [
88
] (Figure 1).
Cytochrome c knockdown mutants of Arabidopsis had a 60% decrease in GLDH activity
without affecting the Asc content, showing that low cytochrome c levels are enough under
normal growth conditions to sustain Asc biosynthesis [89].
Plant mitochondria also have an alternative oxidase (AOX) pathway, taking electrons
directly from the ubiquinone pool without the contribution of the cytochrome c pathway
(Figure 1). Bartoli et al. [
90
] found that AOX-overexpressing Arabidopsis lines accumu-
lated more Asc than wild-type plants, particularly at high light. Higher throughput in the
cytochrome c pathway would require a larger pool of electron acceptors for L-galactono-
1,4-lactone oxidation; therefore, an enhanced capacity of the AOX pathway may favor
Asc biosynthesis by maintaining the cytochrome c pool in a more oxidized state. This is
particularly relevant under high light conditions to prevent over-reduction of the mitochon-
drial electron transport chain and, at the same time, to enhance Asc biosynthesis to protect
the cells against the damaging effect of ROS [
90
]. These results show that integration of
L-galactono-1,4-lactone oxidation and mitochondrial electron transport chain activity via
cytochrome c could coordinate Asc biosynthesis and respiration [88].
It has also been shown that the conversion of L-galactono-1,4-lactone depends on the
photosynthetic electron transport chain because DCMU and dibromothymoquinone could
effectively inhibit the Asc content increase upon L-galactono-1,4-lactone treatment [
52
].
This result indicates the occurrence of a crosstalk between photosynthetic and respiratory
electron transport chains (Figure 1).
It has been suggested that Asc may be a signal connecting the metabolisms of chloro-
plast and mitochondria [
37
,
91
] based on observations on transgenic tomato plants an-
tisensed in mitochondrial malate dehydrogenase (mdh). When grown under long-day
conditions, these mdh lines had reduced tricarboxylic acid (TCA) cycle activity without
affecting respiration, and intriguingly, CO
2
assimilation rates and carbohydrates were
slightly enhanced compared to wild-type plants, and an approx. fourfold increase in
Asc content occurred [
92
]. This was explained by an upregulated flux through GLDH in
the mdh lines and a higher capacity to use L-galactono-lactone as a respiratory substrate,
thereby suggesting that GLDH can effectively act as an alternative electron donor in cases
where flux through the TCA cycle is impaired [
91
,
92
]. However, contradicting results were
obtained under short-day conditions [
91
] and in Arabidopsis mdh mutants [
93
]. On the
other hand, Asc feeding to isolated leaf discs also resulted in increased photosynthesis
rates, further suggesting an Asc-mediated link between the energy-generating processes of
respiration and photosynthesis.
In summary, these results suggest that the interaction between chloroplasts and mito-
chondria acts as a vital determinant of the light-dependent regulation of Asc biosynthesis
in plants and that Asc may act as a metabolic regulator between the energy systems of
the mitochondria and chloroplasts [37]. However, the mechanistic details and the sensing
system remain to be explored.
4. Ascorbate Is an Alternative, ‘Emergency’ Donor to Photosystem II
Asc is a weak reductant that has the potential to reduce amino acid radicals, such as
tyrosine and tryptophan [
94
]. This feature makes it capable of donating electrons to Tyr
Z+
in PSII with inactive oxygen-evolving complexes (OEC). This was initially demonstrated
in vitro
on TRIS-washed, UV-B-irradiated, and heat-treated isolated thylakoids [
95
97
] and
later on heat-treated intact leaves [98,99].
Heat stress results in the removal of the extrinsic proteins and the release of Ca-
and Mn-ions from their binding sites, resulting in the inactivation of OEC [
100
,
101
]. It
has been shown that electron donation from Asc to Tyr
Z+
occurs in heat-stressed leaves;
thus, Asc is a naturally occurring electron donor that can replace water, the terminal
electron donor of PSII [
99
] (Figure 1). With the aid of chlorophyll afluorescence induced
Int. J. Mol. Sci. 2023,24, 2537 7 of 17
by short (5-ms) light pulses it was shown that the halftime of electron donation from
Asc to PSII is in the range of 25 ms in wild-type Arabidopsis leaves and about 55 ms in
Asc-deficient vtc2 mutants [
78
,
99
]. This alternative electron transport occurs in Arabidopsis,
pea, barley, Marchantia polymorpha,Nephrolepis exaltata,C. reinhardtii, etc.; thus, it appears
to be ubiquitous in the plant kingdom [
99
]. The electron transfer rate from Asc to PSII
depends on the species and their physiological state, which is most probably related to the
availability of Asc in the lumen (probably in the range of a few mM, [102]).
Isolated photosynthetic samples with inactivated OECs are extremely susceptible
to illumination. The impaired electron donation from the OEC results in the accumula-
tion of highly oxidizing radicals, including P680
+
, Tyr
Z+
, and superoxide and hydroxyl
radicals [
103
,
104
], leading to a rapid inactivation and degradation of PSII reaction cen-
ters [
105
,
106
]. This type of photodamage is called weak light or donor-side-induced
photoinhibition. By using intact leaves of wild-type, Asc-overproducing (miox4) [
107
],
and Asc-deficient Arabidopsis mutants (vtc2-3) [
6
] subjected to heat stress (40
C, 15 min),
it was demonstrated that the continuous electron flow from Asc to PSII alleviates PSII
photoinactivation [
78
]. Gradual inactivation of PSII charge separation activity occurred
on a time scale of tens of minutes, along with extensive protein degradation, including
probably the complete disassembly of PSII [
78
]. Besides the rate of photoinactivation, the
recovery rate from the photoinactivated state also depended on leaf Asc content. Thereby,
Asc contributes significantly to the ability of plants to withstand heat stress conditions and
aids recovery [78,108].
Asc may also act as an alternative electron donor in bundle sheath chloroplasts.
These are found in the so-called NADP
+
malic enzyme type species carrying out C4-
photosynthesis, such as maize and sorghum. The amount of PSII in bundle sheath chloro-
plasts is small, and their OECs have low activity. It was shown that Asc is an effective
electron donor for PSII in bundle sheath chloroplasts
in vivo
[
109
,
110
]. On the other hand,
since the number of PSII reaction centers is low, photosynthetic electron transport is moder-
ate, but it is sufficient to maintain PSI cyclic electron flow, ensuring thylakoid membrane
energization and ATP synthesis for the Calvin-Benson cycle [
109
,
110
]. Moreover, the re-
placement of water by Asc as a PSII electron donor also ensures low O
2
concentration
within the bundle sheath chloroplasts, diminishing the risk of competition of O
2
with CO
2
molecules for the catalytic sites of RuBisCO (reviewed by [111]).
Asc also donates electrons to PSI in isolated thylakoid membranes (see, e.g., [
112
]) and
in DCMU-treated bundle sheath cells isolated from maize leaves [
113
]. However,
in vivo
,
Asc was a far more effective electron donor for PSII than for PSI [99,109,110].
5. Ascorbate May Impair the Oxygen-Evolving Complex
When considering the physiological roles of Asc, it has to be taken into account that it
is a reductant, and therefore, its cellular concentration is to be maintained in a particular
range [
114
]. The basal Asc concentration in green algae is very low compared to higher
plants (approx. 60 to 100
µ
M in C. reinhardtii and 5 mM in Arabidopsis) [
74
,
75
,
77
,
115
]. It
was observed that upon sulfur deprivation of C. reinhardtii, Asc accumulates to the mM
range and that, in this range, Asc over-reduces the Mn cluster of OEC [
76
,
77
]. The exact
mode of action, i.e., as to which S-state is being inactivated, is not understood.
Once the Mn-cluster is over-reduced by Asc, it may continuously provide electrons
to Tyr
Z+
. However, the electron donation by Asc to PSII is relatively slow (halftime of
approx. 20 to 50 ms, [
99
]) in comparison with the rate of electron transfer from intact OECs
to Tyr
Z+
(halftime of about 0.1 to 1 ms; [
116
]); for this reason, Asc cannot entirely prevent
the accumulation of Tyr
Z+
and P680
+
upon illumination in sulfur-deprived
C. reinhardtii
cultures. Furthermore, strongly oxidizing species lead to donor-side induced photoinhibi-
tion, resulting in the relatively rapid degradation of PSII reaction center proteins, including
PsbA, CP43, PSBO, and possibly others [77].
The loss of PSII activity could be regarded as damage induced by sulfur deprivation.
However, upon downregulation of PSII activity, overexcitation and further photodamage
Int. J. Mol. Sci. 2023,24, 2537 8 of 17
are minimized. The metabolic changes downregulating photosynthetic activity and cell
proliferation may serve to preserve cellular sulfur content and avoid more substantial
damage [
77
]. On the other hand, the inactivation of OECs contributes to the establishment of
hypoxia enabling hydrogenase expression, and H
2
production will act as a safety valve for
photosynthetic electron transport [
117
]. Thereby, the damage imposed by sulfur limitation
is minimized, and the alga cells may recover if sulfur becomes available again [118].
Intriguingly, Asc inactivates the OEC in green algae when accumulated to the mM
range, but the same Asc concentration in vascular plants is physiological [
115
], which
enables a full operation of OEC activity. On the other hand, it was demonstrated earlier
that upon the chemical removal of the extrinsic OEC subunits in isolated PSII membranes,
bulky reductants, including Asc, could directly reduce the Mn-cluster [119].
There are notable differences between the extrinsic OEC proteins of vascular plants
and green algae [
120
,
121
]. Namely, in green algae, the Mn-cluster is shielded by a 33 kDa
PSBO subunit and two smaller subunits, PSBP and PSBQ, with some structural differences
and binding properties in comparison to vascular plants [
121
,
122
]. Moreover, in vascular
plants, an additional subunit, PSBR, is found in the vicinity of the Mn-cluster [
123
]. The
structural differences and the variance in binding properties of the extrinsic proteins may
be key in explaining why Asc reduces the Mn-cluster in algae but not in higher plants when
present in the same concentration range. It is also conceivable that, during evolution, as
cellular Asc concentration increased [
124
], the extrinsic proteins have evolved to protect
the OEC against the reducing effect of Asc present in the thylakoid lumen.
In Arabidopsis subjected to darkness for 24 h, the Mn-cluster becomes inactivated,
being one of the earliest effects of dark-induced senescence on photosynthesis [
125
]. Re-
markably, the extent of OEC inactivation was much weaker in Asc-deficient mutants
compared to wild-type plants, suggesting that Asc was responsible for the diminishment
of oxygen evolution. In a psbo1 knockout mutant, the compromised OEC activity was
further aggravated upon dark treatment, suggesting that the extrinsic proteins protect the
OEC against the reducing effect of Asc. In the absence of PSBR, only a slightly disturbed
photosynthetic activity was observed under normal growth conditions, whereas a strongly
diminished OEC activity was observed in the dark. A double psbo1 vtc2 mutant showed
a slightly milder photosynthetic phenotype than that of the single psbo1 mutant [
125
].
Thus, these results suggest that Asc over-reduces the Mn-complex in prolonged darkness.
This is probably enabled by a dark-induced dissociation of the extrinsic OEC subunits,
which would otherwise hinder the access of Asc to the Mn-cluster [
125
] (Figure 1) or,
hypothetically, it may be related to the lumen volume decrease in the dark [126].
Another example that Asc may negatively affect certain cellular processes was found
by Castro et al. [
127
]. Exogenous Asc concentrations above 30 mM caused cellular and
oxidative damage that were enhanced by high light. The high Asc concentration induced
H
2
O
2
accumulation, stomatal closure, and impairment in CO
2
assimilation, as well as
photosynthetic electron transport. Therefore, these data show that Asc concentration and
localization need to be highly controlled, particularly relevant when aiming at generating
crops with elevated Asc contents.
6. The Role of Ascorbate in Non-Photochemical Quenching
Excess light may lead to photooxidative stress involving the formation of ROS and
light damage (recently reviewed by, e.g., [
128
130
]). Non-photochemical quenching (NPQ)
of excitation energy is a complex photoprotection mechanism, including energy-dependent
(qE), zeaxanthin-related (qZ), state-transition-related (qT), and photoinhibitory (qI) compo-
nents (e.g., [131,132]).
The pH-regulated qE component is formed in response to light-intensity changes
within a few minutes. In vascular plants, activation of qE is mediated by PsbS that acts as a
lumen pH sensor and induces LHCII antenna rearrangement in a zeaxanthin-dependent
manner [133,134].
Int. J. Mol. Sci. 2023,24, 2537 9 of 17
The qZ component of NPQ is activated in the time range of 10 to 30 min, which
correlates with the formation of zeaxanthin by violaxanthin de-epoxidase (VDE) in the
lipid phase of the thylakoid membrane of vascular plants [
135
137
]. VDE belongs to the
lipocalin protein family, and it utilizes Asc as a co-substrate (Figure 1), providing the
reducing power for de-epoxidation [
138
]. At the pH-optimum of VDE of pH 5.0, the Km
value for Asc is approximately 1.0 mM [
138
,
139
], which is probably in the range of luminal
Asc concentration [
102
]. During the operation of the violaxanthin cycle, VDE attaches to the
luminal side of the thylakoid membrane following its pH-dependent activation [
140
142
].
The active VDE is probably a dimer, capable of binding violaxanthin and Asc [141,143].
The maximum VDE activity is several times faster than zeaxanthin epoxidase activ-
ity [
144
]. Consequently, zeaxanthin accumulates in strong light when a low lumen pH is
established. In contrast, zeaxanthin is epoxidized only when VDE activity is low, i.e., under
low light or in darkness. It was recently demonstrated that up-regulation of VDE, PsbS, and
zeaxanthin epoxidase in soybean significantly accelerated the violaxanthin cycle, leading
to faster induction and relaxation of NPQ. This increased the efficiency of CO
2
assimilation
and PSII electron transport in fluctuating light conditions and significantly improved the
biomass yield in field studies, demonstrating that the violaxanthin cycle plays a crucial role
in the regulation of photosynthesis, thereby relating to plant productivity [145].
Besides playing a role in NPQ, zeaxanthin may also contribute to photoprotection by
acting as an antioxidant and possibly by modulating thylakoid membrane
properties [146,147]
.
In addition, zeaxanthin may be required for the PSII repair cycle [148].
The physiological importance of Asc for zeaxanthin formation was demonstrated
in vivo
using vtc mutants of Arabidopsis: it was shown that they accumulate less zeax-
anthin in high light, and consequently, they have diminished and/or delayed NPQ
induction [6,45,78]
. In the miox4 Asc-overproducing mutant [
107
], a slightly higher NPQ
level was obtained than in the wild type [
78
], demonstrating that Asc may be limiting NPQ
formation [44].
In contrast to the vtc2 mutant of Arabidopsis, the C. reinhardtii vtc2 mutant (Crvtc2-1)
with strongly decreased Asc content performs normal violaxanthin de-epoxidation [
75
].
C. reinhardtii lacks a plant-type VDE and instead uses an unrelated enzyme belonging to
lycopene cyclases, called Chlorophycean VDE (CVDE). It is located on the stromal side of the
thylakoid membrane [
149
], and it does not require Asc for violaxanthin de-epoxidation [
75
].
On the other hand, a slow, H
2
O
2
-dependent NPQ component (probably qI) is enhanced
upon Asc-deficiency in C. reinhardtii [75].
Many green alga species, including Chlorella vulgaris, contain plant-type VDEs [
150
],
which are crucial for photoprotective NPQ. Thus, there is an evolutionary divergence
of photoprotective mechanisms among Chlorophyta [
151
]. Chromalveolate algae, such as
Phaeodactylum tricornutum, use the diadinoxanthin cycle instead of the violaxanthin cycle in
a fashion similar to vascular plants to support qE [
152
]. PtVDE converts the monoepoxide
diadinoxanthin to diatoxanthin, possibly involving violaxanthin as an intermediate [
153
].
The reaction requires Asc, though less than vascular plants [
154
]. On the other hand, vio-
laxanthin is also required for the biosynthesis of fucoxanthin and its derivatives, which are
the main light-harvesting pigments in Chromalveolate algae. The conversion of violaxanthin
to neoxanthin is catalyzed by the so-called violaxanthin de-epoxidase-like (VDL) protein,
which does not require Asc as a reductant. It was found that PtVDL is modulated only by
pH, whereas VDE activity is controlled on multiple levels, including the pH-dependent
affinity for Asc [155].
Mosses have plant-type VDE enzymes [
156
], which probably require Asc as a reductant.
The regulation of Asc biosynthesis in vascular plants and mosses is similar [
72
], but the
Asc-dependence of NPQ in mosses has not been investigated.
Int. J. Mol. Sci. 2023,24, 2537 10 of 17
7. Reactive Oxygen Species Management by Ascorbate in the Chloroplast
The best-known and most extensively discussed role of Asc is participation in ROS
management. Excellent recent reviews are available on this topic [
14
,
129
,
130
]; therefore,
only a few aspects related to photosynthesis are mentioned here.
Asc is essential for the enzymatic scavenging of ROS in the so-called Mehler reaction
or the water–water cycle (reviewed by e.g., [
14
,
157
], Figure 1). This reaction becomes
particularly relevant at high light intensities and/or when the Calvin-Benson cycle cannot
work at high speed, for instance under drought, cold, or salt stress. Under such conditions,
the outflow of electrons at the PSI acceptor side is inhibited, leading to a surplus of electrons;
therefore, ferredoxin reduces O
2
, and superoxide is produced. It is then reduced to H
2
O
2
by superoxide dismutase, which is reduced by APX to water. MDA can be directly reduced
back to Asc by PSI, and/or in the Asc-glutathione cycle in which MDAR and DHAR use
NADPH as reducing power [
158
]. Asc thereby participates in the mitigation of ROS. In
this respect, it is to be considered that ROSs are key signaling molecules that enable cells
to respond to changes in environmental conditions rapidly, and they integrate different
environmental signals to activate stress-response networks and defense mechanisms [
130
].
In addition to its function as a powerful antioxidant, Asc is also considered as being
a major player in redox homeostasis and part of the redox signaling network that regu-
lates plant responses to biotic stress [
14
,
159
,
160
]. Interestingly, however, an approx. 80%
decrease in Asc content led to only a minor increase in glutathione level when plants were
grown under standard laboratory conditions [
35
,
81
], though cellular glutathione levels are
redistributed and an approx. two-fold increase in chloroplastic glutathione concentration
was observed [
161
]. In addition, Asc peroxidase activity and the redox state of Asc are also
unaltered in vtc2 mutants, just as well as the photosynthetic activity and carotenoid content,
and only moderate changes occur at constant high light in these parameters [
33
,
35
]. Thus,
an approx. 80% reduction of cellular Asc content does not lead to photooxidative stress,
and apparently, Asc is in large excess in vascular plants, or its deficiency, are compensated
via various yet unexplored mechanisms.
8. Open Questions and Perspectives
Future work will be required to gain a complete overview of Asc functions within
the cell. In recent years, several functions of Asc have been discovered in addition to its
best-known role of mitigating ROS accumulation, and it is likely that the list will be further
expanded. For instance, Asc serves as a chaperone for 2-ODD, a versatile oxidative enzyme
group in plants, of which only a couple have been characterized [
162
], and it is conceivable
that Asc regulates the activities of some of them. In addition, the role of Asc as a metabolic
regulator and a key player in chloroplast-mitochondria signaling has been suggested [
37
],
which warrants experimental confirmation. Moreover, Asc transporters may also play a
key role in Asc metabolism and functions of which only two have been characterized on
a molecular level in vascular plants (AtPHT4;4 and AtDTX25 in the chloroplast envelope
membrane and the vacuole, respectively [
163
,
164
]). Therefore, future work needs to be
directed at exploring other essential Asc transporters both in vascular plants and algae.
Funding:
This work was supported by the National Research, Development, and Innovation Of-
fice (K132600), and the Lendület/Momentum Programme of the Hungarian Academy of Sciences
(LP2014/19).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
All data presented in this study are available within this article. There
are no special databases associated with this manuscript.
Acknowledgments:
I apologize to colleagues whose work I was unable to review. I thank Soujanya
Kuntam (BRC Szeged) for the careful proofreading.
Int. J. Mol. Sci. 2023,24, 2537 11 of 17
Conflicts of Interest: The author declares no conflict of interest.
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... Currently, only two Asc transporters have been molecularly characterized in plants, although there is biochemical evidence suggesting the existence of several others (Tóth, 2023). ...
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Ascorbate (Asc), commonly known as vitamin C, is a vital molecule for plant growth, development, and stress resilience. It is also known to play a crucial role in various physiological processes, including photosynthesis, cell division, and differentiation. This article thoroughly explores the processes governing the metabolism of Asc in plants and its roles in physiological functions. It lays down a robust theoretical groundwork for delving into Asc production, transportation, functions, and its potential applications in stress alleviation and horticulture. Furthermore, recent studies indicate that Asc plays a role in regulating fruit development and affecting postharvest storage characteristics, thereby influencing fruit ripening and resilience to stress. Hence, there is a growing importance in studying the synthesis and utilization of Asc in plants. Although the critical role of Asc in controlling plant redox signals has been extensively studied, the precise mechanisms by which it manages cellular redox homeostasis to maintain the equilibrium between reactive oxygen scavenging and cell redox signaling remain elusive. This gap in knowledge presents fresh opportunities to explore how the production of Asc in plants is regulated and how plants react to environmental stressors. Furthermore, this article delves into the potential for a comprehensive investigation into the essential function of Asc in fruits, the development of Asc-rich fruits, and the enhancement of postharvest storage properties.
... Unlike in vascular plants, Asc biosynthesis in Chlamydomonas is induced by reactive oxygen species [15,20] and it is not regulated by the circadian clock. Further, in Chlamydomonas, Asc is not required for zeaxanthin-and energy-dependent non-photochemical quenching [9]. Hence, with its varied physiological functions in green algae in comparison to vascular plants, and for the potential biotechnological application of Asc biosynthesis [4,5], it is essential to have a reliable method for Asc detection in live algal cells. ...
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Ascorbate (Asc) is an important antioxidant that also participates in various biological processes in plants such as hormone metabolism, stress response and signaling pathways. Asc is also a vital vitamin for human health and enriching its content through biofortification is a desirable objective. Therefore, reliable in situ methods for assessing Asc levels are essential. However, most of the existing fluorescent probes for Asc detection are limited to liquid samples, such as human sera or plant extract, or require sophisticated techniques and equipment for in-cell detection, such as photo-induced electron transfer or time-gated luminescence microscopy. Moreover, many of these probes are not cell wall permeable and cannot be used in plants or algal cells. In this article, we introduce a reaction-based, Asc probe – AP-Cyan, that can efficiently and qualitatively detect Asc in various microalgal cultures, including Chlamydomonas reinhardtii, Chlorella sorokianiana and Parachlorella kessleri. The probe is simple to use and produces fast results that can be observed with standard fluorescence microscopes with basic blue, green and red filters. The probe has an emission range (λem = 488 nm) that does not overlap with chlorophyll autofluorescence, making it suitable for algal cells. Thus, our probe offers a simple and powerful method to detect Asc in microalgal cells.
... The redox system, in a cross-talk with plant hormones, plays an important role in adjusting growth and development to the changes in environmental conditions (Kocsy et al. 2013;Considine and Foyer 2014;T oth 2023). During these alterations, the reactive oxygen species (ROS) can accumulate in excess, and their levels are controlled by the various antioxidants (Suzuki et al. 2012;Foyer and Hanke 2022). ...
Article
We assumed that miRNAs might regulate the physiological and biochemical processes in plants through their effects on the redox system and phytohormones. To check this hypothesis, the transcriptome profile of wild‐type Arabidopsis and lines with decreased ascorbate (Asc), glutathione (GSH), or salicylate (Sal) levels were compared. GSH deficiency did not influence the miRNA expression, whereas lower levels of Asc and Sal reduced the accumulation of 9 and 44 miRNAs, respectively, but only four miRNAs were upregulated. Bioinformatics analysis revealed that their over‐represented target genes are associated with the synthesis of nitrogen‐containing and aromatic compounds, nucleic acids, and sulphate assimilation. Among them, the sulphate reduction‐related miR395 – ATP‐sulfurylase couple was selected to check the assumed modulating role of the light spectrum. A greater induction of the Asc‐ and Sal‐responsive miR395 was observed under sulphur starvation in far‐red light compared to white and blue light in wild‐type and GSH‐deficient Arabidopsis lines. Sal deficiency inhibited the induction of miR395 by sulphur starvation in blue light, whereas Asc deficiency greatly reduced it independently of the spectrum. Interestingly, sulphur starvation decreased only the level of ATP sulfurylase 4 among the miR395 target genes in far‐red light. The expression level of ATP sulfurylase 3 was higher in far‐red light than in blue light in wild‐type and Asc‐deficient lines. The results indicate the coordinated control of miRNAs by the redox and hormonal system since 11 miRNAs were affected by both Asc and Sal deficiency. This process can be modulated by light spectrum, as shown for miR395.