Báo cáo khoa học: Sugar signalling and antioxidant network connections in plant cells pptx

Both glucose and sucrose are recognized as pivotal integrating regulatory molecules that control gene expression related to plant metabolism, stress resistance, growth and development [1

Sugar signalling and antioxidant network connections in plant cells

Mohammad Reza Bolouri-Moghaddam1, Katrien Le Roy2, Li Xiang2, Filip Rolland3and

Wim Van den Ende2

1 Department of Agronomy, Plant Breeding and Biotechnology, Faculty of Crop Science, Sari Agricultural Science and Natural Resources University, Iran

2 Laboratorium voor Moleculaire Plantenfysiologie, Katholieke Universiteit Leuven, Belgium

3 Laboratorium voor Functionele Biologie, Katholieke Universiteit Leuven, Belgium

Introduction

The ability of cells to perceive and correctly respond

to their microenvironment forms the basis of cellular

signalling Glucose is an important cellular nutrient

that also acts as a regulatory metabolite modulating

gene expression in yeast, animals and plants In plants,

sugars play important roles as both nutrients and

sig-nal molecules Both glucose and sucrose are recognized

as pivotal integrating regulatory molecules that control

gene expression related to plant metabolism, stress

resistance, growth and development [1–3] There is

great interest in dissecting the processes that are

involved in the sugar sensing and response pathways allowing plants to adapt to the constantly changing environment, but the sugars’ dual function as nutrients and signalling molecules significantly complicates anal-yses of the mechanisms involved [4] Although our knowledge of the detailed molecular mechanisms of sugar perception and signalling in plants is far from complete, hexokinase (HXK) and Snf1-related kinase 1 have already been identified as conserved sugar signal-ling components controlsignal-ling eukaryotic energy homeo-stasis, stress resistance, survival and longevity

Keywords

antioxidant; fructan; hexokinase; invertase;

mitochondria; phenolic compounds, ROS

scavenging; senescence; signalling; sugar

Correspondence

W Van den Ende, Laboratorium voor

Moleculaire Plantenfysiologie, Katholieke

Universiteit Leuven, Kasteelpark Arenberg

31, bus 2434, B-3001 Leuven, Belgium

Fax: +32 161967

Tel: +32 16321952

E-mail: wim.vandenende@bio.kuleuven.be

(Received 5 January 2010, revised 15

February 2010, accepted 2 March 2010)

doi:10.1111/j.1742-4658.2010.07633.x

Sugars play important roles as both nutrients and regulatory molecules throughout plant life Sugar metabolism and signalling function in an intri-cate network with numerous hormones and reactive oxygen species (ROS) production, signalling and scavenging systems Although hexokinase is well known to fulfil a crucial role in glucose sensing processes, a scenario is emerging in which the catalytic activity of mitochondria-associated hexoki-nase regulates glucose-6-phosphate and ROS levels, stimulating antioxidant defence mechanisms and the synthesis of phenolic compounds As a new concept, it can be hypothesized that the synergistic interaction of sugars (or sugar-like compounds) and phenolic compounds forms part of an inte-grated redox system, quenching ROS and contributing to stress tolerance, especially in tissues or organelles with high soluble sugar concentrations

Abbreviations

ABA, abscisic acid; ASC, ascorbic acid; AtHXK1, Arabidopsis thaliana hexokinase 1; GSH, glutathione; HXK, hexokinase; NDP-sugar, nucleoside diphosphate-sugar; MeJa, methyl jasmonate; mtHXK, mitochondrial hexokinase; PCD, programmed cell death; RFO, raffinose family oligosaccharides; ROS, reactive oxygen species; VHA-B1, one of three isoforms of the B subunit of the peripheral V1 complex in vacuolar-type H-ATPase.

Furthermore, invertase-related sugar signals seem to be

very important during plant defence reactions [5,6]

In conclusion, sugar signals integrate numerous

environmental and endogenous developmental and

metabolic cues and therefore operate in a complex

net-work with plant-specific hormone signalling and

stress-related pathways

The field of antioxidants has received much

atten-tion lately, both in fundamental and applied research,

but especially in the field of medicine (food

supple-ments) Because the toxic oxygen gas appeared in the

Earth’s atmosphere 2.2 billion years ago, aerobic

organisms such as animals and plants only survived

because they evolved antioxidant defence processes [7]

The need for oxygen for the efficient production of

energy (ATP) in mitochondria is in balance with the

necessity of controlling the level of reactive oxygen

species (ROS), such as the hydroxyl radical •OH; the

superoxide radical O2 ) and hydrogen peroxide H2O2,

which are always produced in the presence of oxygen

and particularly under stress In general, metabolic

rates appear to inversely correlate with stress

resis-tance and lifespan in a variety of organisms and this

has been attributed to oxidative stress [8], a deleterious

process caused by the overproduction of ROS that can

be important as a mediator of cell damage leading to

various disease states, senescence and aging in

humans, animals [9] and plants [7] In plants, high

photosynthetic activity in source leaves can induce the

accumulation of both soluble sugars and ROS

Intriguingly, however, sugar starvation can lead to

ROS accumulation as well [10] Enzymic and

nonenzy-mic antioxidants as endogenous protective mechanisms

work in a complex co-operative network to reduce the

cytotoxic effects of ROS in both plant and animal cells

[11–13]

Increased cellular oxidation is also a key feature of

leaf senescence [14], a process that seems to be

associ-ated with temporal high levels of soluble sugars

Although an involvement of metabolic signals in the

regulation of plant senescence has been demonstrated

in a range of studies [15, and references therein], the

exact mechanisms involved in the stimulation of the

ROS detoxification systems remain largely unresolved

So far, the protective effects of soluble sugars against

oxidative stress have been mostly attributed to

(indi-rect) signalling effects, triggering the production of

specific ROS scavengers [10,16] However, it was

recently proposed that soluble sugars, especially when

they are present at higher concentrations, might act as

ROS scavengers themselves [17] Here, the putative

roles of sugars and sugar metabolizing enzymes in

both sugar and ROS signalling pathways and their

co-operation with antioxidant networks in plant cells are presented A picture is emerging in which the activ-ity of HXK may control ROS production in plant organelles such as mitochondria and chloroplasts Also, the effect of these pathways on plant senescence

is discussed

Sugar signalling, a multifunctional network

Sugars not only fuel growth and development as car-bon and energy sources, but in addition have acquired important regulatory roles as signalling molecules [2,3] This is now particularly well established for glucose, the prime carbon and energy source in eukaryotic cel-lular metabolism HXK, the first enzyme in glucose catabolism, was identified as a genuine glucose sensor, with separable catalytic and signalling activities [18,19] (Fig 1) In addition, HXK-independent glucose and sucrose signalling pathways appear to be active in plants [20–24], but a sucrose sensor remains to be iden-tified (Fig 1) Although glucose signalling has been associated primarily with active cell division, respira-tion, cell wall biosynthesis and sugar-mediated feed-back regulation of photosynthesis (Fig 1), sucrose signalling appears to be specifically associated with anthocyanin production and with the regulation of storage- and differentiation-related processes [24–26] (Fig 1) In addition to the important post-transla-tional effects on metabolic enzyme activity, genome-wide expression profiling has recently uncovered the huge impact of carbon status on gene expression [27– 32] Diverse environmental stress conditions can cause cellular energy starvation, triggering metabolic repro-gramming through the Snf1-related kinase 1 pathway [33 and references therein]

Excess photosynthate is generally transiently stored

as starch in the chloroplast during the day, in part through sugar-mediated induction of gene expression and redox activation of ADP-glucose pyrophosphory-lase, a key enzyme in starch biosynthesis [34–36] Apart from the breakdown of sucrose by invertases (see below), the degradation of chloroplastic starch in leaf cells during the night (mainly via maltose and glucose export) and from plastids (amyloplasts) in starch-storing organs is also a putative source for glucose signals [25,37] Also, trehalose metabolism appears to play an important regulatory role in co-ordinating metabolism with plant growth and development [38] Apart from the breakdown of sucrose and reserve carbohydrates, the hydrolysis of cell wall polysaccharides might also generate sugar signals Several cell wall glycoside hydrolases are

upregulated under stress conditions such as darkness,

sugar depletion, senescence and infection [33,39,40]

The exact molecular mechanisms involved in sugar

signalling are still largely unknown, but mutant

screens have revealed a tight interaction with various

hormone signalling pathways [2] Glucose controls

abscisic acid (ABA) signalling and biosynthesis gene

expression [41] and HXK-dependent signalling

inter-acts positively and negatively with auxin and

cytoki-nin signalling, respectively [19] Glucose and ethylene

signalling converge at the level of EIN3 protein

stabil-ity [42]

Role of sucrose splitting enzymes

Sucrose is one of the most widespread disaccharides in

nature In higher plants it represents the major

trans-port compound bringing carbon skeletons from source

(photosynthetically active leaves) to sink tissues (roots,

young leaves, flowers, seeds, etc.) Invertases mediate

the hydrolytic cleavage of sucrose into glucose and

fructose (Fig 1) These enzymes are very well studied

in plants, fungi and bacteria For a long time, inverta-ses were believed to be absent in the animal kingdom Recently, however, at least two invertases have been discovered in the genome of Bombyx mori, but their exact functions remain unclear [43]

Plants possess three types of invertase: acid cell wall invertases (glycoside hydrolase family 32) located in the apoplast (i.e the continuum of cell walls of adja-cent plant cells), acid vacuolar invertases (glycoside hydrolase family 32) located in the vacuole and neu-tral⁄ alkaline invertases (glycoside hydrolase family 100) located in the cytoplasm, chloroplasts and mito-chondria [44–47] Neutral invertases can be inhibited

by glucose and fructose [48,49] Knock outs of two cytoplasmic neutral invertases in Arabidopsis [50] and the model legume Lotus japonicus [51] resulted in severely reduced growth rates It remains to be further explored whether the invertase exerts a direct effect on plant growth and development or an indirect effect via glucose signalling (Fig 1) Intriguingly, the nuclear

Fig 1 A hypothetical sugar–antioxidant network in plant cells Glucose (Glc) and the activity of (organellar) HXK take a central position in this scheme, as they emerge as important regulators of cytosolic ROS The HXK generated glucose-6-phosphate (Glc6P) and NDP-glucose boost the glycosylation of phenolic compounds, the synthesis of ASC and contribute to hormone homeostasis The released ROS is used as a signal

to stimulate the antioxidant defence system In parallel with HXK activity, HXK acts as a glucose sensor, controlling cell division and cell expansion, in concert with hormone and ROS signalling Next to glucose, sucrose can also be sensed and metabolized Invertases (INVs) can influence plant growth and development either directly (e.g by influencing source–sink relationships or by acting as a true regulatory protein)

or indirectly via sugar signalling (altering the hexose ⁄ sucrose ratio) Sucrose is sensed by an unknown sensor stimulating the accumulation of reserve compounds (e.g vacuolar fructan biosynthesis) and anthocyanins Both anthocyanins and phenolic compounds might be involved in the scavenging of cytosolic ROS, creating phenolic compound radicals (phenolic compounds•), which are reduced by ASC It is postulated that sugars at high concentrations (sucrose, fructans, sugar-like compounds) could directly scavenge ROS derived from excess H 2 O 2 entering the vacuole, producing sugar radicals (sugar • ) Vacuolar glycosylated phenolic compounds might assist in recycling the sugars from sugar radicals.

localization of AtCINV1, a well-characterized neutral

invertase of Arabidopsis, and its interaction with

a phosphatidylinositol monophosphate 5-kinase [52]

strongly suggest that (at least some) neutral invertases

can fulfil regulatory functions apart from their

cata-lytic function [53] It is becoming increasingly clear

that invertases can do much more than simply

hydro-lyse sucrose For instance, the LIN6 cell wall invertase

of tomato is considered to be a pivotal enzyme for the

integration of metabolic, hormonal and stress signals,

regulated by a diurnal rhythm [54] Expression of a

yeast-derived invertase in the apoplast under a

meri-stem-specific promoter caused accelerated flowering

and enhanced branching of the inflorescence and seed

yield, whereas a cytoplasmic localization of this

invert-ase resulted in delayed flowering and both reduced seed

yield and branching in Arabidopsis [55] In potato,

cyto-plasmic localization of yeast invertase was detrimental

to tuber yield, whereas the opposite was observed for

an apoplastic yeast invertase construct [56] Targeting

of this yeast invertase to plastids resulted in early leaf

senescence, consistent with reduced sucrose and

increased hexose levels in the leaves [57] These results

emphasize the importance of the exact source, nature

and location of the sugar signals [55] and the

impor-tance of invertases as modulators of the sucrose⁄

glucose ratio The sucrose⁄ glucose ratio might be more

important than the absolute sucrose and glucose

con-centrations, as suggested by different groups [58–63],

but this requires further investigation

Biotic or abiotic stresses and hormonal signals can

also induce cell wall invertase expression and sink

for-mation in leaf tissues [45] Sucrose synthase is also able

to split sucrose, but it generates fructose and

UDP-glucose instead of UDP-glucose It cannot be excluded that fructose-specific signalling pathways exist in plants [64] Intriguingly, some of the so-called cell wall inver-tases and sucrose synthases have lost their catalytic activities, and they may function as regulatory proteins [65–68], but this requires further investigation They are possibly involved in sucrose sensing processes

Soluble sugars as a part of an antioxidant system

ROS are continuously produced during mitochondrial respiration [69,70] and photosynthesis [71] The mito-chondrial source of ROS production is as important

in nonphotosynthesizing plant cells as it is in mamma-lian cells [69] Small soluble sugars and the enzymes associated with their metabolic pathways are widely believed to be connected to oxidative stress and ROS signalling [10,72–74] On the one hand, endogenous sugar availability can feed the oxidative pentose phos-phate pathway [10,75], creating reducing power for glutathione (GSH) production, contributing to H2O2 scavenging On the other hand, excess sugar produc-tion in source leaves by increased photosynthetic activities may result in the generation of excess cyto-solic H2O2, especially when the export of sugars from these leaves is hampered due to decreased sink strength under stress Therefore, it was proposed that sugars themselves, especially the longer water-soluble oligo- and polysaccharides, such as fructans, might be effective candidates for capturing ROS in tissues exposed to a wide range of environmental stresses [17] Fructans are known to protrude deep between the headgroups of the membranes (Fig 2) to stabilize

Stress

H2O2 Cytoplasm

POX OH

Vacuole

OH +

Fructan

O

Fructan radical

OH

Fig 2 A possible dual role for vacuolar

fruc-tans in the vicinity of the tonoplast under

stress Abiotic and biotic stresses can lead

to increased concentrations of cytosolic

H2O2, which can enter the vacuole via

diffu-sion and ⁄ or through aquaporins Vacuolar

fructans (blue) can insert deeply between

the headgroups of the tonoplastic

mem-branes, stabilizing them under stress Type

III peroxidases (green) associate intimately

with the inner side of the tonoplast

Peroxid-ases produce•OH radicals Fructans are

well positioned to scavenge these radicals,

a process in which fructan radicals and

water are formed Fructan radicals might be

generated back into fructans with the help

of phenolic compounds (see also Fig 1).

them [76] Under stress, excess cytoplasmic H2O2 can

diffuse through the tonoplast (aquaporins can assist

in this process), where tonoplast-bound class III

per-oxidases catalyse the reduction of H2O2 by taking

electrons to various donor molecules, such as phenolic

compounds, lignin precursors, auxin or secondary

metabolites However, the functioning of these

perox-idases is also accompanied by the production of •OH

and •OOH through the so-called hydroxylic cycle of

these enzymes [77,78] Vacuolar fructans are ideally

positioned to stabilize the tonoplast, but also to

temporarily scavenge the aggressive •OH and •OOH

radicals that are produced in the vicinity of these

membranes (Fig 2) In this process, the fructans (or

other sugars⁄ sugar-like compounds) are converted into

(less harmful) fructan radicals It has been proposed

that such sugar radicals could be recycled back into

sugars with the help of phenolic compounds or

antho-cyanins [17,79] Interestingly, the synthesis of phenolic

compounds⁄ anthocyanins can also be stimulated by

sugar-mediated signalling and metabolic pathways

(Fig 1) Synthetic oligosaccharides also show strong

antioxidant activity in vitro and counteract lipid

per-oxidation processes in mice [80] In conclusion, it can

be postulated that vacuolar sugars or sugar-like

com-pounds, present in the vicinity of the tonoplast and

interacting with this membrane (Fig 2), might fulfil

crucial roles in scavenging radicals and thus

prevent-ing lipid peroxidation by excess H2O2 produced under

stress conditions

Among a range of small sugars tested, sucrose

showed the strongest antioxidant capacity in vitro

[81–83], strongly suggesting that similar antioxidant

reactions with sucrose can also occur in planta At low

concentrations, sucrose might serve as a substrate or

signal for stress-induced modifications, whereas at

higher concentrations it may function directly as a

pro-tective agent (e.g in vacuoles of sugar beet and sugar

cane plants, co-operating with the classic, cytoplasmic

antioxidant systems) (Fig 1) [17]

Other important water-soluble carbohydrates derived

from sucrose (sucrosyl oligosaccharides) include the

raffinose family oligosaccharides (RFOs: a-galactosyl

extensions of sucrose), next to the fructans (b-fructosyl

extensions of sucrose) Sucrosyl oligosaccharides and

the enzymes associated with their metabolism might

interact indirectly with ROS signalling pathways

Recently, RFOs as well as galactinol have been

pro-posed to fulfil important roles in oxidative stress

protection in plants [81,84] and seeds [85–87]

Previ-ously, raffinose was shown to protect

photophos-phorylation and electron transport of chloroplast

membranes against freezing, desiccation and high

temperature stress [88], strongly suggesting that chloro-plastic RFOs might be operating as ROS scavengers The oxidized RFO radicals might be regenerated by ascorbic acid (ASC) or other reducing antioxidants, such as flavonoids [89] The overexpression of galacti-nol synthase (GolS1, GolS2, GolS4) and raffinose syn-thase in transgenic Arabidopsis plants increased the galactinol and raffinose concentrations and resulted in effective ROS scavenging capacity and oxidative stress tolerance [81] Moreover, lipid peroxidation was signif-icantly lower than in wild-type plants Furthermore, these transgenic plants exhibited higher photosystem II (PSII) activities compared with wild-type plants, and appeared to more tolerant under high light and chilling conditions [81]

Fructans might protect plants against freezing⁄ drought stresses by stabilizing membranes [90,91] Recent studies on transgenic plants carrying fructan biosynthetic genes [92–94] suggest that the enhanced tolerance of these plants is associated with the presence

of fructans Their reduced lipid peroxidation levels indicate that fructans, similar to RFOs, might also act directly as ROS scavengers Alternatively, fructans might work indirectly by stimulating other specific antioxidative defence mechanisms [17] Intriguingly, changes in fructan concentrations showed a close cor-relation with changes in ASC and GSH concentrations

in immature wheat kernels, strongly suggesting a con-nection with the well-known cytoplasmic antioxidant systems Therefore, fructans may form an integral part

of a more complex ROS scavenging system in fructan-accumulating plants It was proposed that glucose, produced by the vacuolar fructan initiator enzyme sucrose : sucrose 1-fructosyl transferase, after retrans-location to the cytoplasm could directly fuel biosynthe-sis of classical antioxidants [95,96], establishing a direct connection between vacuolar and cytoplasmic antioxidation mechanisms

Similarly, sugar alcohols (mannitol, inositol, sorbitol) also possess ROS scavenging capacities In tobacco, mannitol is believed to protect thioredoxin, ferredoxin, GSH and the thiol-regulated enzyme phosphoribulo-kinase against•OH radicals The targeting of mannitol biosynthesis to chloroplasts in transgenic tobacco plants resulted in an increased resistance to methyl viologen-induced oxidative stress It became clear that mannitol in the chloroplast does not reduce ·OH radical production, but that it increases the capacity to scavenge these radicals and protects cells against oxida-tive damages [97] Contrary to glucose, fructose and sucrose, mannitol, even at high concentrations, does not repress photosynthesis and its presence has no obvious harmful effects on plants [97,98]

A role for trehalose in protection against ROS has

also been demonstrated [99] This is particularly

important in micro-organisms, as only a few plants are

known to accumulate trehalose to a great extent [100]

Trehalose is capable of reducing oxidant-induced

mod-ifications of proteins during exposure of yeast cells to

H2O2[101] The ability of trehalose to reduce

intracel-lular oxidation during dehydration has been

demon-strated, especially when yeast cells were deficient in

superoxide dismutase [102] Moreover, as observed for

fructans, trehalose reduced the levels of lipid

peroxida-tion, suggesting an additional property of this sugar

for improving tolerance to water loss Also it was

shown that, in vitro, trehalose significantly reduces

oxi-dation of unsaturated fatty acids through a weak

inter-action with the double bonds [103] The regulated

overexpression of trehalose biosynthetic genes in

trans-genic rice plants produced increased amounts of

treha-lose in the shoot and conferred high levels of tolerance

to salt, drought and low-temperature stresses

Com-pared with nontransformed rice, several independent

transgenic lines exhibited sustained plant growth, less

photo-oxidative damage and a more favourable

mineral balance under stress conditions [104]

Implication of HXK in sugar signalling

and antioxidant activity

HXKs are ubiquitous proteins in all living beings,

catalysing the phosphorylation of glucose and fructose

[105] HXKs are not only essential in the first step of

glycolysis There is also evidence that some HXK

forms are involved in NDP-sugar synthesis [106]

HXKs appear to occur in two groups: one with plastid

signal peptides (type A) and one with N-terminal

membrane anchors (type B) [107], and have been

found in the cytosol, the stroma of plastids, the Golgi

complex [108–113] or associated with the

mitochon-drial membrane [114] The major glucose

phosphory-lating enzyme in the moss Physcomitrella patens is a

chloroplast stromal HXK [108]

In addition to its familiar role as a metabolic

enzyme, Arabidopsis thaliana HXK1 (AtHXK1) serves

as an intracellular glucose sensor This sensor is

pre-dominantly associated with mitochondria (mtHXK),

but was also reported to be found in the nucleus

[115,116] The nuclear AtHXK1 appears to be part of

a glucose signalling complex (Fig 3) that suppresses

the expression of photosynthetic genes The signalling

activity of AtHXK1 requires two unexpected partners:

VHA-B1 and RPT5B (Fig 3) VHA-B1 is one of the

three isoforms of the B subunit of the peripheral V1

complex in vacuolar-type H-ATPase and is responsible

for noncatalytic ATP binding RPT5B is a subunit of the 19S regulatory particle, which binds either end of the 20S proteasome to provide ATP dependence and the specificity for ubiquitinated proteins At a low glu-cose concentration, the target photosynthetic genes are expressed by a specific transcription factor At a high glucose concentration, glucose diffuses into the nucleus and binds to nuclear HXK1 This process probably triggers a conformational change in HXK1, which then acts as a transcriptional repressor together with VHA-B1 and RPT5B (Fig 3) In animals, the

Mond-oA⁄ Mlx complex is believed to monitor intracellular glucose-6-phosphate concentrations, translocating the complex to the nucleus when levels of this key metabo-lite increase However, nuclear localization of the MondoA⁄ Max-like X (Mlx) complex depends on the enzymatic activity of HXK [117] In yeast, glucose-dependent changes in gene expression utilize at least three mechanisms, including carbon catabolite repres-sion in which Saccharomyces cerevisiae hexokinase 2 (ScHXK2) has a nonmetabolic role in modulating specific transcriptional regulators [118] In maize, the inhibition of mtHXK by ADP, mannoheptulose and glucosamine as compared with the insensitive cytosolic HXK, has been considered as evidence for a putative role of mtHXK in hexose sensing [109] However, this interesting hypothesis awaits more direct demonstra-tion Such investigations may also clarify whether (and under which conditions) HXK is translocated from the mitochondrion to the nucleus, or mediates distinct signalling events depending on its subcellular distribu-tion [119]

The rice OsHXK5 and OsHXK6 proteins appear to have a similar dual localization and sensor function [120] In addition to HXKs, plants also contain several fructokinases, some of which might also be involved in sugar sensing [121] Surprisingly, the AtHXK1 mutant glucose insensitive 2is insensitive to glucose, but is still sensitive to fructose and sucrose [2]

The mitochondrial localization of the HXKs is thought to improve access to the ATP produced in res-piration for consumption by active metabolite fluxes through sucrose cycling, glycolysis and sugar nucleo-tide synthesis Consistently, an entire functional glyco-lytic metabolon appears to be associated with the outer mitochondrial membrane, allowing pyruvate to

be provided directly to the mitochondrion, where it is used as a respiratory substrate and glycolytic enzymes appear to associate dynamically with mitochondria in response to respiratory demand [122,123]

These observations suggest that mtHXK activity could

be involved in the regulation of both mitochondrial respiration and ROS production in plants, similar to

the key preventive antioxidant role of mtHXK through

a steady-state ADP recycling mechanism in rat brain

neurons [124] Similar to mammalian HXKs [125,126],

plant mtHXKs have also been reported to associate

with porin⁄ voltage-dependent anion channels [115],

but further research is needed to unravel the precise

interactions between plant mtHXKs,

voltage-depen-dent anion channels and the outer mitochondrial

membrane The presence of different hydrophobic

N-terminal sequences in mammalian and plant

mtHXKs suggests that these interactions might

sub-stantially differ between mammals and plants It was

demonstrated that HXKI and HXKII reduce the

intra-cellular levels of ROS and inhibit the mitochondrial

permeability transition pore in mammalian cells [127]

An authentic mtHXK activity, which is subject to

inhibition by ADP, was detected on potato tuber

(Solanum tuberosum) outer mitochondrial membranes

[128] A mtHXK activity with similar kinetics has been

described in pea leaves [129] The potato mtHXK is

much more sensitive to ADP inhibition in the micro-molar range with glucose as a substrate than with fruc-tose, suggesting a different affinity for these hexoses [128] Moreover, it was demonstrated that this mtHXK can contribute to a steady-state ADP recycling (ADP production by mtHXK, bound to the outer mitochon-drial membrane; ADP consumption through oxidative phosphorylation) that regulates H2O2 formation in the electron transport chain on the inner mitochondrial membrane Importantly, this mitochondrial ADP recy-cling mechanism led to a decrease in the mitochondrial membrane potential, whereas an inhibition of mtHXK led to an increase in H2O2 production Thus, mtHXK bound to the outer mitochondrial membrane can guide the ADP delivery to the F0F1ATP synthase enzyme via the adenine nucleotide transporter in an efficient chan-nelling to the mitochondrial matrix In conclusion, mtHXK activity plays a specific role in generating ADP to support oxidative phosphorylation, thereby avoiding an ATP synthesis-related limitation of

Plastid

Mitochondria

26S

proteasome

V-ATPase Vacuole

Glucose

Nucleus HXK1

RPT5B

VHA-B1

Fig 3 The AtHXK1 signalling complex driving the expression of photosynthetic genes in Arabidopsis, depending on the glucose concentra-tion (A) When the glucose concentration (white dots) is low, the target photosynthetic genes are turned on by a specific transcription factor (white) This activation may involve an unidentified coactivator complex (B) When glucose is in excess (for instance, under nitrate-deficient conditions or stress condition when invertases are activated, and glucose is not utilized in cells), glucose diffuses freely into the nucleus, where it binds to nuclear AtHXK1 Glucose binding triggers a conformational change in AtHXK1, which then acts together with VHA-B1 (blue) and RPT5B (yellow) as a transcriptional repressor Both VHA-B1 and RPT5B interact with the transcription factor, thereby linking AtHXK1 to DNA binding and suppressing gene expression The mechanism of the nuclear translocation of AtHXK1, VHA-B1 and RPT5B is still unknown This figure has been reproduced from [116] and reprinted with permission from AAAS.

respiration and subsequent H2O2 release in plants

[128] In maize roots, mtHXK activity is believed to be

directly linked to NDP-sugar synthesis (Fig 1), needed

for cellulose, phenylpropanoid and flavonoid

biosyn-thesis The attachment of mtHXK to the mitochondrial

membrane is absolutely necessary to perform its

preven-tive antioxidant activity, in both animal and plant

mito-chondria [124,127,128] Interestingly, both methyl

jasmonate (MeJa) and glucose-6-phosphate are known

to induce the detachment of mammalian mtHXK from

the outer mitochondrial membrane [124,130,131]

Glu-cose-6-phosphate had no such effect on plant

mito-chondria [128] The effect of MeJa on the detachment

of mtHXK from plant mitochondria has not yet been

reported However, MeJa addition to plants leads to

ROS accumulation, alterations in mitochondrial

move-ments and morphology, and cell death [132], similar to

that observed in animal cells [131], suggesting that

similar mechanisms might be operating in plant and

animal cells, but this remains to be demonstrated

Next to the important signalling function of HXKs,

as described above for AtHXK1, these new data

indi-cate that mtHXK activities might play a key role

as a regulator of ROS levels (Fig 1) mtHXKs could

respond rapidly to changes in the cellular demand for

glucose-6-phosphate, which is known to be a key

inter-mediate in several metabolic pathways sensitive to

ADP⁄ ATP ratios, including glycolysis, sucrose

synthe-sis, the pentose phosphate pathway, cellulose

biosyn-thesis and phenylpropanoid and flavonoid biosynbiosyn-thesis

(Fig 1) However, the links between glucose, HXK

and ROS control extend the processes described in

mitochondria Indeed, the HXK-derived

glucose-6-phosphate (Fig 1) can feed the l-galactose route in

the so-called Smirnoff–Wheeler pathway, leading to

biosynthesis of ASC [133], which has major

implica-tions in the well-known cytoplasmic ROS

detoxifica-tion processes, cell elongadetoxifica-tion and, possibly, cell

division [134] ASC works in close co-operation with

GSH to remove H2O2 via the Halliwell–Asada

path-way Moreover, ASC takes part in the regeneration of

a-tocopherol, providing extra protection of the

mem-branes [135] The NDP-sugars, produced by the

activ-ity of sucrose synthases (and other enzymes), can serve

as donor substrates for glycosyltransferases that

cata-lyse the glycosylation of most plant hormones (except

ethylene), contributing to hormone homeostasis [136]

Moreover, NDP-sugars are also important as donor

substrates for the glycosylation and stability of many

secondary metabolites, such as phenolic compounds

(Fig 1), contributing to increasing antioxidant abilities

[17] and assisting in recycling sugars from sugar

radicals (Fig 1) Cellular NDP-sugar concentrations

are often very low This includes that they are a limit-ing substrate for product synthesis, as was shown for (U⁄ A)DP-glucose-dependent starch biosynthesis in potato [137] and UDP-glucose-dependent based cellu-lose synthesis in Avena coleoptiles [138] Therefore, it can be postulated that an accumulation of phenolic compounds would also greatly depend on the cellular NDP-sugar concentrations, but this requires further investigation It is widely recognized that phenolic compounds are involved in the H2O2 scavenging cas-cade in plant cells [139], but the links with HXK activ-ities and sugar recycling processes have so far received little attention

Apart from glucose, fructose and sucrose, sugar alcohols, such as sorbitol, mannitol and myo-inositol, may also be transported into vacuoles [140,141] Anthocyanins, flavonoids and a wide array of conju-gated endogenously synthesized toxic or xenobiotic compounds typically accumulate in the vacuole and several of these substances cross the tonoplast via ATP-binding cassette (ABC)-type carriers [142,143] Phenolic compounds and fructans (or other vacuolar, sugar-like compounds) might operate in a synergistic way to scavenge excess H2O2(Figs 1 and 2)

Taken together, glucose and HXK take a central position and a dual role in both sugar signalling and antioxidant networks, with important links to ASC biosynthesis and, via NDP- glucose production, to the synthesis of glycosylated phenolic compounds (Fig 1) High concentrations of vacuolar compounds (sugars or sugar-like compounds) could also form an integral part

of this antioxidant network (Fig 1)

Senescence – a link between sugar signalling and ROS production pathways

In addition to aging, plants are characterized by a highly specific process termed leaf senescence After a period of active photosynthesis, the leaf’s contribution

to the plant diminishes and the leaf then enters its final stage of development: senescence This highly regulated process is characterized by the loss of chlorophyll, the breakdown of macromolecules and the massive remo-bilization of nutrients to other parts of the plant [15] Senescence can be triggered by multiple developmental and environmental signals Drought, darkness, leaf detachment and the hormones ABA and ethylene induce leaf yellowing [144] Cytokinins, on the other hand, can delay plant senescence, and studies with the AtHXK1 mutant glucose insensitive 2 show that sugars and cytokinins work antagonistically [19] Interestingly, cytokinin-induced cell wall invertase expression is an

essential downstream component of

cytokinin-medi-ated local delay (green islands) of leaf senescence [58]

Leaf senescence is accompanied by considerable

changes in cellular metabolism and gene expression

[145] Several of these senescence-associated genes

encode transporters of sugar, peptides, amino acids

and transporters that could participate in the substrate

and nutrient mobilization that occurs as part of the

senescence programme [146] However, micro-array

analysis has revealed significant differences in gene

expression between dark⁄ starvation-induced and

devel-opmental senescence [147]

ROS play an important role in the response of

plants to biotic and abiotic stress, plant cell growth,

regulation of gene expression, stomatal opening,

hor-mone signalling and programmed cell death (PCD)

[148–153] This multifaceted role for ROS indicates a

tight control of their production and accumulation

lev-els The observation that ROS may mediate both ABA

signalling and ABA biosynthesis [154] suggests that the

feedback regulation of ABA biosynthetic genes by

ABA may be mediated in part by ROS through a

pro-tein phosphorylation cascade [155]

Senescence-associ-ated genes are expressed in response to increases in the

tissue contents of ROS [14] Increased cellular

oxida-tion is a key feature of leaf senescence [14] PCD may

be triggered by enhanced ROS levels and cellular

oxi-dation [156–158] Genetic evidence suggests that ROS

do not trigger PCD or senescence by causing

physico-chemical damage to the cell, but rather act as signals

that activate pathways of gene expression that lead to

regulated cell suicide events [152,153] An imbalance

between ROS production and antioxidant defence can

lead to an oxidative stress condition Increased levels

of ROS may be a consequence of the action of plant

hormones, environmental stress, pathogens, altered

sugar levels and fatty acids [159–162] and ROS may in

turn induce ROS scavengers and other protective

mechanisms (Fig 1) [163]

For leaves, evidence supports a role for sugar

accu-mulation in the initiation and⁄ or acceleration of

senes-cence However, the regulation of senescence or aging

may respond to different metabolic signals in

hetero-trophic plant organs and heterohetero-trophic organisms

[164] van Doorn [165] questioned whether sugar

increases or decreases cause leaf senescence, and

con-cluded that there is not enough hard evidence to

dem-onstrate a causative relationship The long-lasting

debate on this matter probably reflects the complexity

of senescence regulation, with sugars being only one

factor The initial trigger of processes leading to cell

death probably needs to be searched for in

mitochon-dria [132] Apart from the actual concentrations of

glucose and sucrose within mitochondria and their sur-roundings, it becomes increasingly clear that the activ-ity of mtHXK is essential to regulate ROS levels in mitochondria (Fig 1) Although a temporal ROS over-shoot, via ROS signalling, leads to an increased pro-duction of antioxidants contributing to ROS scavenging and stress tolerance, it can be speculated that a more drastic ROS overshoot could serve as an initial trigger of senescence Consistent with this hypothesis and the central importance of mtHXK activity, it was recently demonstrated that MeJa addi-tion, perhaps acting by releasing mtHXK from the outer membrane, results in four sequential processes: (a) ROS accumulation, (b) alterations in mitochondrial movements and morphology, (c) photosynthetic dys-function and (d) cell death [132]

The Arabidopsis hypersenescing mutant hys1 pro-vides the clearest link between sugar, senescence and stress signalling The repressive effect of sugars on photosynthetic gene expression and activity and the correlation between HXK expression and the rate of leaf senescence [19,21] are indicative of an important role for HXK-dependent sugar signalling in leaf senescence Similar to the effects of targeting the yeast invertase to chloroplasts, an accelerated senes-cence was observed during overexpression of HXK [166] HXK overexpression in tomato plants impaired growth and photosynthesis, and induced rapid senescence in photosynthetic tissues [167] It was also demonstrated that mtHXKs play a role in the control of PCD in Nicotiana benthamiana [168] HXK dislocation from mitochondria leads to the closure of voltage-dependent anion channels and sub-sequent mitochondrial swelling and cell death in mammalian cells [169] HXK binding to the mito-chondria inhibits apoptosis [169] Increasing glucose phosphorylation activity by mtHXK may reduce apoptosis through both the inhibition of mitochon-drial permeability transition and more efficient glucose metabolism due to their better access to ATP [127]

In conclusion, plant mtHXKs might act as a glucose sensor (regulatory function) and, through its catalytic activity, as a crucial regulator of ROS levels in mito-chondria (Fig 1) Similar functions could be postu-lated for chloroplastic HXKs

All abiotic stresses generate ROS, potentially leading

to oxidative damage affecting crop yield and quality Next to the well-known classical antioxidant mecha-nisms, sugars and sugar metabolizing enzymes come into the picture as important new players in the defence against oxidative stress Therefore, sugar metabolizing enzymes, such as organellar HXKs, form

promising targets to improve crop yield, stress

toler-ance and longevity in plants

Conclusion

Sugars are finally being recognized as important

regu-latory molecules with both signalling and putative

ROS scavenging functions in plants [2] and other

organisms [170–172] The exact source, nature and

location of the sugar and nonsugar signals, such as

ROS and hormones, are important to provide an

inte-grative regulatory mechanism controlling various

func-tions of a plant cell The responses to sugars and

oxidative stress are not only linked (Fig 1), but sugars

also affect scores of stress-responsive genes [27] Apart

from acting as signals, we hypothesize that vacuolar

sugars or sugar-like compounds, possibly in

combina-tion with phenolic compounds, form a so far

unrecog-nized vacuolar redox system acting in concert with the

well-established cytoplastic antioxidant mechanisms

Acknowledgements

WVdE, KLR, LX and FR are supported by grants

from FWO Vlaanderen

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