REGULAR PAPER

Leaf developmental stage modulates metabolite accumulationand photosynthesis contributing to acclimation of Arabidopsisthaliana to water deficit

Ilektra Sperdouli • Michael Moustakas

Received: 28 January 2014 / Accepted: 10 March 2014

� The Botanical Society of Japan and Springer Japan 2014

Abstract We examined whether young and mature

leaves of Arabidopsis thaliana in their response to mild

water deficit (MiWD) and moderate water deficit (MoWD),

behave differentially, and whether photosynthetic accli-

mation to water deficit correlates with increased proline

and sugar accumulation. We observed that with increasing

water deficit, leaf relative water content decreased, while

proline and sugar accumulation increased in both leaf-

developmental stages. Under both MiWD and MoWD,

young leaves showed less water loss and accumulated

higher level of metabolites compared to mature leaves.

This, leaf age-related increase in metabolite accumulation

that was significantly higher under MoWD, allowed young

leaves to cope with oxidative damage by maintaining their

base levels of lipid peroxidation. Thus, acclimation of

young leaves to MoWD, involves a better homeostasis of

reactive oxygen species (ROS), that was achieved among

others by (1) increased sugar accumulation and (2) either

increased proline synthesis and/or decreased proline

catabolism, that decrease the NADPH/NADP? ratio,

resulting in a higher level of oxidized state of quinone A

and thus in a reduced excitation pressure, and by (3)

stimulation of the photoprotective mechanism of non-

photochemical quenching, that reflects the dissipation of

excess excitation energy in the form of harmless heat, thus

protecting the plant from the damaging effects of ROS.

Keywords Chlorophyll fluorescence � Excitation

pressure � Non-photochemical quenching � Oxidative

stress � Proline � Soluble sugars

Abbreviations

ETR Electron transport rate

Fv/Fm Potential (maximum) quantum yield of PSII

photochemistry

LWC Leaf relative water content

MDA Malondialdehyde

MiWD Mild water deficit

MoWD Moderate water deficit

NPQ Non-photochemical quenching

PSI, PSII Photosystem I, Photosystem II

PPFD Photosynthetic photon flux density

QA Quinone A

qP Photochemical quenching

ROS Reactive oxygen species

SE Standard error

SWC Soil volumetric water content

UPSII Actual (effective) quantum yield of PSII

photochemistry

Introduction

Soil water deficit is one of the most important factors

limiting crop productivity worldwide, causing several

effects on plant metabolic processes, including water

relations, nutrient uptake and metabolism, and photosyn-

thesis (Shinozaki et al. 2003; Xiong et al. 2012). Under

water deficit, plants close stomata to reduce transpiration

and, due to stomatal closure, CO2 diffusion to the

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10265-014-0635-1) contains supplementarymaterial, which is available to authorized users.

I. Sperdouli � M. Moustakas (&)

Department of Botany, School of Biology, Aristotle University

of Thessaloniki, University Campus, 54124 Thessaloniki, Greece

e-mail: moustak@bio.auth.gr

123

J Plant Res

DOI 10.1007/s10265-014-0635-1

chloroplasts decreases significantly, which down-regulates

carbon assimilation. As a result, the consumption of

reduction equivalents (NADPH) decreases, causing an

increase in NADPH/NADP? ratio, generating an oversup-

ply of reduction equivalents (Selmar and Kleinwachter

2013). Over reduction of components within the photo-

synthetic electron transport chain in the chloroplasts cannot

be balanced by availability of the electron acceptor

NADP? and electrons are captured by O2 instead of

NADP?, resulting in the generation of reactive oxygen

species (ROS) (Vankova et al. 2012).

Plants can acclimate to water deficit under some cir-

cumstances by accumulating osmolytes and proteins that

act as osmoprotectants, antioxidants, and/or stress signals

specifically involved in stress tolerance (Kubis et al. 2014;

Pinheiro et al. 2011; Shinozaki and Yamaguchi-Shinozaki

2007). Compatible solutes can counteract negative dehy-

dration effects on the cell structure and enzyme functioning

and detoxify ROS (Kavi Kishor et al. 1995; Kubis et al.

2014; Moustakas et al. 2011; Nanjo et al. 1999; Yoshiba

et al. 1997).

Positive correlations between the capacities for proline

accumulation and drought or salinity tolerance of different

varieties of the same species have been reported (De Ronde

et al. 2004; Hare and Cress 1997; Hayat et al. 2012; Kubis

et al. 2014; Sperdouli and Moustakas 2012b; Yamada et al.

2005). Proline accumulation in plant tissue is the result of a

decrease in proline degradation, increase in proline biosyn-

thesis, decrease in protein synthesis or proline utilization, or

increased hydrolysis of proteins (Hare et al. 1999). Proline

biosynthesis in the chloroplasts under water deficit consumes

NADPH and decreases the NADPH/NADP? ratio, thus

reducing ROS production (Vankova et al. 2012). Under

water deficit conditions, metabolic pathways that decrease

the NADPH/NADP? ratio resulting to reduced ROS pro-

duction are thus stimulated (De Ronde et al. 2004; Hare and

Cress 1997; Selmar and Kleinwachter 2013; Vankova et al.

2012). Proline signaling has been found to stimulate a sol-

uble sugar signaling pathway (Rasheed et al. 2011; Sperdouli

and Moustakas 2012b) and vice versa (Hu et al. 2012). Apart

from proline protection from ROS, sugar protection against

oxidative stress seems to be partly due to activation of spe-

cific ROS scavenging systems, with consequent reduction of

oxidative damage (Pinheiro and Chaves 2011).

Chlorophyll fluorescence measurements have become

widely used to study the functioning of the photosynthetic

apparatus and are a powerful tool to study the plant’s

response to water deficit (Massacci et al. 2008; Sperdouli

and Moustakas 2012a, b; Woo et al. 2008).

Previous works have shown that MiWD affected pho-

tosystem II (PSII) functioning, while under MoWD con-

ditions, photosynthetic acclimation was observed,

suggesting that PSII activity does not decrease in a

drought-dependent way (Sperdouli and Moustakas 2012a,

b). However, compared to the increasing knowledge about

the enhanced accumulation of certain metabolites, under

water deficit, revealed by studies using Arabidopsis, little

attention has been paid to the differential behavior of

young and mature leaves to water deficit. Young leaves of

A. thaliana were found to acclimate better to water deficit

by dissipating the excess excitation energy (Sperdouli and

Moustakas 2012c, 2014), and this seems to be an important

mechanism in order to avoid possible photodamage to PSII

under water deficit (Jung 2004; Sperdouli and Moustakas

2014). Thus, in the present study, we evaluated whether

differences in the tolerance of A. thaliana young and

mature leaves, to MiWD and MoWD, correlate with dif-

ferential metabolite accumulation of proline and sugars. In

summary, the overall hypothesis was that young leaves of

A. thaliana would show higher photosynthetic acclimation

to water deficit than mature leaves and thus would accu-

mulate more proline and sugars.

Materials and methods

Plant material, growth conditions, and water deficit

treatment

Arabidopsis thaliana ecotype Columbia (Col-0) plants were

grown in a growth chamber (EF7, Conviron, MN, Canada)

with controlled environmental conditions under a long day

photoperiod 14 h/10 h, with 40 ± 5 %/60 ± 5 % humidity,

temperature 22 ± 1 �C/19 ± 1 �C and light intensity of

120 ± 20 lmol photons m-2 s-1. Water deficit was

imposed on 4 week-old Arabidopsis plants (nine to ten

leaves per plant) by withholding water for a period up to

10 days. All plants, i.e. well watered plants (control plants)

and plants under water deficit, were sampled at the same day

for analysis. Two categories of water deficit, besides well

watered, were characterized: MiWD and MoWD (Sperdouli

and Moustakas 2012b). Watering of MoWD plants was

stopped 10 days before sampling, of MiWD plants 6 days

before sampling while control plants were watered 3 h

before sampling. The two developmental leaf stages that

were examined were fully developed mature leaves and

developing young leaves. As young leaves were assigned

those in the center of the leaf rosette with 1.5–2 cm length,

while the average length of mature leaves in the rosette was

4.1 ± 0.5 cm (Sperdouli and Moustakas 2014).

Soil and leaf water status

Soil volumetric water content (SWC) in m3 m-3 was

measured with a 5TE (Decagon Devices, Pullman, WA,

USA) soil moisture sensor, coupled to a ProCheck

J Plant Res

123

(Decagon Devices, Pullman, WA, USA) read out device.

Plant water status was determined by measuring leaf water

content (LWC) by the electronic moisture balance (MOC-

120H, Shimadzu, Tokyo, Japan) as previously described by

Sperdouli and Moustakas (2012a), using the formula:

(FW - DW)/DW 9 100 % where FW refers to fresh

weight and DW refers to dry weight.

Lipid peroxidation measurements

The level of lipid peroxidation in A. thaliana mature leaves

and developing young leaves from each treatment/control was

measured as malondialdehyde (MDA) content determined by

reaction with 2-thiobarbituric acid reactive substances as

described by Moustakas et al. (2011), according to Heath and

Packer (1968). Tissue was homogenized in 0.3 % TBA in

10 % trichloracetic acid at 4 �C and centrifuged for 10 min at

10,000g. The concentration of MDA was calculated from the

difference of the absorbance at 532 and 600 nm spectropho-

tometrically (PharmaSpec UV-1700; Shimadzu, Tokyo,

Japan), using the extinction coefficient of 155 mmol-1 cm-1,

by the formula, MDA = A532 - A600/155 9 40, and

expressed as nmol (MDA) g dry weight (DW) -1.

Proline and sugar determination

A. thaliana young and mature leaves from each treatment/

control, were cut into small pieces, weighed, placed sepa-

rately in glass vials containing 10 ml of 80 % (v/v) ethanol,

and heated at 60 �C for 30 min. The extract was then fil-

tered and diluted with 80 % (v/v) ethanol up to 20 ml. The

concentration of free proline was determined in this extract

as described by Moustakas et al. (2011), following the

acid-ninhydrin reagent method (Bates et al. 1973). Proline

concentration was determined using calibration curve

with standard dilutions of L-proline and the formula:

proline = [(A518 ? 0.0013)/(7.2665) 9 (15)] g-1 sample-1,

and expressed as lmol g DW-1.

Ethanol extracts from each treatment/control of A. tha-

liana young and mature leaves, as used for proline assay,

were diluted with 80 % (v/v) ethanol for the assay of

sugars, as described previously by Moustakas et al. (2011).

Briefly, the diluted extracts were added, drop-by-drop, in

2 ml anthrone reagent test tubes in an ice bath and left to

mix the content. Fully mixed samples were incubated in a

water bath at 90 �C for 15 min and cooled, and absorbance

was read at 625 nm using a PharmaSpec UV-1700 spec-

trophotometer (Shimadzu, Tokyo, Japan). Soluble sugars

were determined using calibration curve with standard

dilutions of sucrose and the formula: sugars =

[(A625 ? 0.0099)/(7.8109) 9 (15 9 3)] g-1 sample-1, and

expressed as lmol g DW-1.

Chlorophyll fluorescence measurements

Chlorophyll fluorescence was measured at room tempera-

ture in dark-adapted (20 min) A. thaliana young and

mature leaves using an imaging-PAM fluorometer (Walz,

Effeltrich, Germany), as described by Sperdouli and

Moustakas (2012b). Five areas of interest were selected,

one in the center of the leaf, two in the outer zone of the

front and two in the outer zone of the back of the leaf. First

Fo (minimum chlorophyll a fluorescence in the dark) and

Fm (maximum chlorophyll a fluorescence in the dark)

values were measured with dark-adapted samples, from

which the potential (maximum) quantum yield derived (Fv/

Fm). Fm was obtained with a saturating pulse of white light

(2,400 lmol photons m-2 s-1, 800 ms). This was fol-

lowed by exposure at 130 lmol photons m-2 s-1 with

repetitive measurements of F0o (minimum chlorophyll a

fluorescence in the light) and F0

m (maximum chlorophyll

a fluorescence in the light) every 20 s, from which auto-

matically values of other chlorophyll fluorescence param-

eters were calculated by the Imaging Win software. The

actinic illumination of 130 lmol photons m-2 s-1 was

selected to match that of the growth light of A. thaliana

plants, and low enough to avoid photoinhibition. In the

presence of actinic illumination, the fluorescence yield Fs

(steady state chlorophyll a fluorescence) was measured.

The calculated parameters included the photochemical

quenching, qP, a measure of the fraction of open PSII

reaction centers, representing the redox state of quinone A

(QA), the primary electron acceptor of PSII, and it was

calculated as (F0m–Fs)/(F

0m–F

0o) (Genty et al. 1989). The

NPQ parameter, which was calculated as (Fm–F0m)/F

0m,

estimates the non-photochemical quenching that reflects

heat dissipation of excitation energy in the antenna system

(Bilger and Bjorkman 1990). The relative PSII electron

transport rate (ETR) was calculated as c 9 UPSII (actual

quantum yield of photochemistry) 9 PPFD (photosyn-

thetic photon flux density); for the coefficient c, the value

of 0.42 was assumed [considering equal distribution of

photons between PSII and PSI (0.5) multiplied by the

homogeneous absorption factor of 0.84, that contains

assumptions on leaf absorption of PPFD] (Schreiber et al.

1994).

Statistical analysis

Each treatment/control was analyzed with six replicates. A

standard error (SE) was calculated and data were expressed

in mean ± SE of six replicates. Chlorophyll fluorescence

measurements represent averaged values (n = 6) from two

independent experiments with three leaf samples (each

with five areas of interest) from three different plants, per

J Plant Res

123

treatment per experiment. One-way ANOVA (StatView

computer package) was carried out and means were sepa-

rated using Duncan’s least significant ranges (LSR) at

95 %. For the analysis of the interaction of leaf age

(mature, young) and water deficit treatment (Control,

MiWD, MoWD) a two-way analysis of variance using the

StatView-ANOVA computer package (Abacus Concepts,

Inc Berkley, CA, USA) was performed. A linear regression

analysis was also performed (Sperdouli and Moustakas

2012b, c).

Results

Soil and leaf water status

In the two categories of water deficit, besides well watered,

that were examined water deficit was induced gradually by

withholding water. In MiWD, SWC was 66–68 % of

control (well watered), while in MoWD 50–52 % (Fig. S1).

The LWC did not differ in fully hydrated young and mature

leaves, but it was significantly higher (P \ 0.05) in young

leaves under both MiWD and MoWD (Fig. 1a). LWC

decreased from about 94 % (fully hydrated) to approxi-

mately 84 and 79 % in mature leaves under MiWD and

MoWD, respectively, but in young leaves to approximately

88 and 82 %, respectively.

Differential oxidative damage in young and mature

leaves

Symptoms of oxidative stress were observed in mature

leaves during both MiWD and MoWD, as reflected by

MDA accumulation, an indicator of the extent of lipid

peroxidation, due to ROS formation. In young leaves

symptoms of oxidative stress were observed only during

MiWD, while during MoWD young leaves were able to

cope with oxidative damage, as it was evident by almost

the same lipid peroxidation level compared to control

young leaves (Fig. 1b). During MoWD, symptoms of

oxidative stress in both leaf types were less than during

MiWD. The higher oxidative stress was observed in mature

leaves during MiWD.

Differential susceptibility to photoinhibition in young

and mature leaves during water deficit

Susceptibility to photoinhibition under water deficit stress

in A. thaliana leaves was determined as changes in the Fv/

Fm ratio (Fig. 1c). Under MiWD, Fv/Fm decreased by

18 % in mature leaves, while in young leaves decreased by

6 %, compared to their controls. However, during MoWD,

the decrease of Fv/Fm in both leaf types was lower (9.4 %

in mature leaves and only 2 % in young leaves compared to

their corresponding controls) (Fig. 1c).

Leaf developmental stage influences metabolite

accumulation during water deficit

The free proline content of A. thaliana leaves increased in a

dose response to water deficit (Fig. 2a). Proline content

Fig. 1 Changes in leaf water content, the level of lipid peroxidation,

and the maximum quantum yield of PSII (Fv/Fm) in mature and young

leaves of A. thaliana subjected to control, MiWD and MoWD

conditions. a Leaf water content. b The level of lipid peroxidation

measured as MDA production. c The maximum quantum yield of

PSII (Fv/Fm). Bars represent standard errors (n = 6) and different

lowercase letters indicate significant difference at 0.05

J Plant Res

123

was 7.042 and 5.956 lmol g DW-1 in control mature and

young leaves, respectively; increased under MiWD 34- and

47-fold, respectively; and increased under MoWD 55- and

78-fold, respectively, showing a higher accumulation of

proline in young leaves (Fig. 2a).

The sugar content of A. thaliana leaves increased less

compared to proline accumulation (Fig. 2b). The sugar

content of MiWD and MoWD mature leaves increased by

23 and 36 %, respectively, while in young leaves increased

by 61 and 86 %, under MiWD and MoWD, respectively.

Thus, a higher increase of sugar content in young compared

to mature leaves under both water deficit treatments was

observed (Fig. 2b).

Fig. 2 Changes in free proline and soluble sugar contents in mature

and young leaves of A. thaliana under MiWD and MoWD conditions.

a Free proline content. b Soluble sugar content. c The relationships

between free proline and soluble sugar contents in mature and young

leaves of A. thaliana subjected to control, MiWD and MoWD

conditions. Bars represent standard errors (n = 6). Different lower-

case letters in a and b indicate significant difference at 0.05

Table 1 Differences of the parameters LWC, proline and sugars

between different leaf-age (young, mature) samples from plants

subjected to different water deficit treatments (Control, MiWD,

MoWD)

Parameter YL vs ML

Control MiWD MoWD

LWC ns *** ***

Pro ns ns ***

SS ns ns *

ns not significant

* Significant differences (P \ 0.05)

** Significant differences (P \ 0.01)

*** Significant differences (P \ 0.001)

Fig. 3 Relationship of leaf water content with free proline accumu-

lation and soluble sugar accumulation in mature and young leaves of

A. thaliana subjected to MiWD and MoWD conditions. a The

relationship of leaf water content with proline accumulation. b The

relationship of leaf water content with soluble sugar accumulation.

Bars represent standard errors (n = 6)

J Plant Res

123

Relationship between proline and sugar accumulation

Sugar accumulation in young and mature leaves under both

water deficit treatments was tightly correlated

(R2 = 0.91622, P \ 0.0001) with the magnitude of proline

accumulation (Fig. 2c).

Relationship between leaf’s water content

and metabolite accumulation

While there was a significant difference in LWC between

young and mature leaves during MiWD, this was not

associated with any significant difference between young

and mature leaves in either proline or sugar accumulation

(Table 1). However, under MoWD the significant higher

LWC in young leaves was associated with a significant

difference between leaf age in both proline and sugar

accumulation (Table 1). LWC exhibited significantly neg-

ative linear correlation with both proline (Fig. 3a) and

sugar (Fig. 3b) content. Proline accumulation under MiWD

and MoWD was negatively correlated (R2 = 0.86371,

P \ 0.0001) with LWC in both young and mature leaves

(Fig. 3a). Sugar accumulation was also negatively corre-

lated (R2 = 0.63848, P \ 0.0001) with the magnitude of

LWC in both leaf types (Fig. 3b).

Young leaves under water deficit retain higher electron

transport rate and non-photochemical quenching

compared to mature leaves

The non-photochemical quenching that reflects heat dissi-

pation of excitation energy in the antenna system (NPQ)

remained much higher in young leaves under MiWD and

MoWD compared to mature leaves (Fig. 4a). Under

MoWD, NPQ decreased in young leaves by approximately

30 % compared to controls, while in mature leaves by

approximately 56 % compared to their control (Fig. 4a).

The relative PSII electron transport rate (ETR) was lower in

control young leaves compared to control mature leaves, but

under MiWD it was higher in young leaves (Fig. 4b). Under

MoWD, young leaves could retain an ETR not statistically

different to control young or mature leaves (Fig. 4b).

Young leaves under water deficit maintain a more

oxidative state of quinone A compared to mature leaves

The fraction of open PSII reaction centers, qP, that is the

redox state of QA, was not different in control young and

mature leaves (Fig. 4c). Under MiWD the redox state of QA

was more reduced in both leaf types. Under MoWD, young

leaves could maintain a more oxidised state of QA com-

pared to mature leaves, that was not significantly different

compared to control young and mature leaves (Fig. 4c).

Relationships of oxidative damage to photochemical

efficiency and excitation pressure

The level of lipid peroxidation, measured by MDA accu-

mulation, reflects ROS formation and corresponds to the

oxidative damage. Under both water deficit treatments

oxidative damage was negatively correlated with both the

maximum quantum yield of PSII photochemistry (Fv/Fm)

(R2 = 0.9636, P \ 0.0001) (Fig. 5a) and the redox state of

Fig. 4 Changes in NPQ, relative PSII ETR, and photochemical

quenching (qP) of the fraction of open PSII reaction centers from

mature and young leaves of A. thaliana subjected to control, MiWD

and MoWD conditions. a NPQ. b relative PSII ETR. c qP that is a

measure of the redox state of quinone A (QA). Bars represent standard

errors (n = 6) and different lowercase letters indicate significant

difference at 0.05

J Plant Res

123

QA, (qP) (R2 = 0.91809, P \ 0.0001) (Fig. 5b), that is a

measure of the excitation pressure on PSII.

Discussion

Water deficit increases the production of ROS in chloro-

plasts during photosynthesis, inhibiting the photosynthetic

processes (Pinheiro and Chaves 2011). An increase in ROS

levels can cause severe oxidation of cellular components

inducing redox status changes (Jubany-Marı et al. 2010).

To counteract water deficit, several physiological responses

and processes are induced or accelerated, such as the

accumulation of compatible solutes, e.g. proline (Yoshiba

et al. 1997) and sugars (Muller et al. 1996).

Young and mature leaves of A. thaliana plants showed

significant differentiation of LWC during both water deficit

treatments. Under MoWD the significant differentiation of

LWC between young and mature leaves was associated to

the significant differentiation of sugar and proline content

between them (Table 1). In mature leaves under both

MiWD and MoWD, symptoms of oxidative damage were

observed, as reflected by MDA accumulation, an indicator

of the extent of lipid peroxidation, and also by the Fv/Fm

values below 0.75 (0.662 and 0.734 under MiWD and

MoWD, respectively), which are generally indicative of

damage to PSII photochemistry (Juvany et al. 2012; Ta-

kahashi and Murata 2008).

Jung (2004) reported that young and mature leaves of

four-week-old Arabidopsis plants behaved differently

when exposed to water deficit. He concluded that under

water deficit, mature leaves suffer more oxidative stress

than young leaves. Recently, Juvany et al. (2013) pointed

out the importance of elucidating the mechanisms that

maintain an adequate cellular ROS balance that allows

growth and prevents oxidative damage in young leaves.

Studies of transcriptional regulation of genes involved in

proline synthesis confirmed developmental regulation at

gene level of proline synthesis and degradation (Verbrug-

gen and Hermans 2008). Thus, the higher accumulation of

proline in young than mature leaves under both MiWD and

MoWD, is due either to a higher induction of proline

synthesis gene D1-pyrroline-5-carboxylate reductase at the

early developmental stages of Arabidopsis (Hua et al.

1997; Nakashima et al. 1998), and/or to a higher catabo-

lism of proline in mature leaves, due to enhanced expres-

sion of proline dehydrogenase during the later

developmental stages (Funck et al. 2010; Nakashima et al.

1998).

The fluorescence quenching, predominantly caused by

photochemical and energy-dependent mechanisms, is

strongly influenced by the utilisation of NADPH and ATP

in photosynthesis (Krause and Somersalo 1989). High

reduction state of QA indicates excess excitation energy

and thus an imbalance between energy supply and demand.

Under such conditions singlet oxygen, 1O2, and hydrogen

peroxide, H2O2, are released (Dietz and Pfannschmidt

2011). Thus, the more oxidized redox state in young leaves

compared to mature leaves under MoWD would be

expected to alleviate the accumulation of excited species

within PSII, thereby reducing 1O2 formation (Munne-

Bosch et al. 2001).

In both young and mature leaves under MiWD, the

highest decreases of ETR and NPQ were measured,

accompanied by the most reduced redox state of QA, thus

with the highest fraction of closed PSII reaction centers and

high excitation pressure. In addition, under MiWD both

leaf types had the higher quantum yield of non-regulated

non-photochemical energy loss. Under water deficit con-

ditions, light in excess of what can be used in photosyn-

thesis increases, resulting to photoinhibition (Galmes et al.

2007; Sperdouli and Moustakas 2012a).

The response of A. thaliana young and mature leaves to

MiWD and MoWD, fits the ‘‘Threshold for Tolerance

Fig. 5 Relationships of the level of lipid peroxidation (MDA

formation) with the maximum quantum yield of PSII (Fv/Fm) and

photochemical quenching (qP) in the fraction of open PSII reaction

centers from mature and young leaves of A. thaliana subjected to

control, MiWD and MoWD conditions. a The relationship of the level

of lipid peroxidation with the maximum quantum yield of PSII (Fv/

Fm). b The relationship of the level of lipid peroxidation with

photochemical quenching (qP) that is a measure of the excitation

pressure on PSII. Bars represent standard errors (n = 6)

J Plant Res

123

Model’’, with a lag time required (during MiWD) for the

induction of a tolerance mechanism (Barcelo and Pos-

chenrieder 2002; Sperdouli and Moustakas 2012a). This

lag-time was required for a substantial increase of proline

and sugar accumulation that was achieved only during

MoWD in young leaves.

Acclimation of young leaves to MoWD involves ROS

homeostasis that results to a higher level of oxidized state

of QA, which alleviates the accumulation of excited spe-

cies within PSII, thereby reducing ROS formation (Munne-

Bosch et al. 2001; Vankova et al. 2012). Increased sugar

accumulation in young leaves under MoWD has contrib-

uted to activation of ROS scavenging systems, with con-

sequent reduction of oxidative damage (Pinheiro and

Chaves 2011; Smeekens et al. 2010). Transgenic Arabi-

dopsis plants overexpressing LcMYB1 gene, show

enhanced expression levels of D1-pyrroline-5-carboxylate

synthase and accumulated greater amounts of proline and

sugars and less MDA under salt stress (Cheng et al. 2013).

Acclimation of young leaves to MoWD is also achieved by

stimulation of photoprotective mechanisms such as NPQ

(Havaux et al. 2000; Neubauer and Yamamoto 1992). NPQ

reflects the dissipation of excess excitation energy in the

form of harmless heat, thus protecting the plant from the

damaging effects of ROS (Hideg et al. 2008). It seems that

photoprotection was achieved in young leaves under

MoWD, since NPQ was regulated in such a way that the

remained open fraction of PSII reaction centers was non

significantly different to controls (Lambrev et al. 2012),

thus young leaves under MoWD had the same excitation

pressure to controls. It is concluded that under MoWD, a

significant higher proline and sugar accumulation in young

leaves, contributed in maintaining their base levels of lipid

peroxidation. ROS homeostasis of young leaves during

MoWD resulted to acclimation, that was achieved among

others by (1) increased sugar accumulation and (2) either

increased proline synthesis that converts excess NADPH

into NADP? and/or decreased proline catabolism, resulting

to a higher level of oxidized state of QA, and by (3)

stimulation of the photoprotective NPQ mechanism that

protects the plant from the damaging effects of ROS.

Young leaves of MoWD A. thaliana plants possessed not

only an ETR almost the same with controls, but also the

same excitation pressure to controls. Thus, we can con-

clude from our results that photosynthetic acclimation of

young leaves to water deficit is correlated to increased

proline and sugar accumulation.

Acknowledgments We are grateful to Prof. Dr. Stefanos Sgardelis

(Department of Ecology, Aristotle University of Thessaloniki) for the

help in statistical analysis. This work was supported by the European

Fund of Regional Growth and the Hellenic General Secretariat for

Research and Technology under the project No. 09FR47 to Michael

Moustakas.

References

Barcelo J, Poschenrieder C (2002) Fast root growth responses, root

exudates, and internal detoxification as clues to the mechanisms

of aluminium toxicity and resistance: a review. Environ Exp Bot

48:75–92

Bates IS, Waldren RP, Teare ID (1973) Rapid determination of free

proline for water stress studies. Plant Soil 39:205–207

Bilger W, Bjorkman O (1990) Role of the xanthophyll cycle in

photoprotection elucidated by measurements of light-induced

absorbance changes, fluorescence and photosynthesis in leaves

of Hedera canariensis. Photosynth Res 25:173–186

Cheng L, Li X, Huang X, Ma T, Liang Y, Ma X, Peng X, Jia J, Chen

S, Chen Y, Deng B, Liu G (2013) Overexpression of sheepgrass

R1-MYB transcription factor LcMYB1 confers salt tolerance in

transgenic Arabidopsis. Plant Physiol Biochem 70:252–260

De Ronde JA, Cress WA, Kruger GHJ, Strasser RJ, Van Staden J

(2004) Photosynthetic response of transgenic soybean plants,

containing an Arabidopsis P5CR gene, during heat and drought

stress. J Plant Physiol 161:1211–1224

Dietz KJ, Pfannschmidt T (2011) Novel regulators in photosynthetic

redox control of plant metabolism and gene expression. Plant

Physiol 155:1477–1485

Funck D, Eckard S, Muller G (2010) Non-redundant functions of two

proline dehydrogenase isoforms in Arabidopsis. BMC Plant Biol

10:70

Galmes J, Abadia A, Medrano H, Flexas J (2007) Photosynthesis and

photoprotection responses to water stress in the wild-extinct

plant Lysimachia minoricensis. Environ Exp Bot 60:308–317

Genty B, Briantais JM, Baker NR (1989) The relationship between

the quantum yield of photosynthetic electron transport and

quenching of chlorophyll fluorescence. Biochim Biophys Acta

990:87–92

Hare PD, Cress WA (1997) Metabolic implications of stress-induced

proline accumulation in plants. Plant Growth Regul 21:79–102

Hare PD, Cress WA, Van Staden J (1999) Proline synthesis and

degradation: a model system for elucidating stress-related signal

transduction. J Exp Bot 50:413–434

Havaux M, Bonfils JP, Lutz C, Niyogi KK (2000) Photodamage of the

photosynthetic apparatus and its dependence on the leaf devel-

opmental stage in the npq1 Arabidopsis mutant deficient in the

xanthophyll cycle enzyme violaxanthin de-epoxidase. Plant

Physiol 124:273–284

Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A

(2012) Role of proline under changing environments. A review.

Plant Signal Behav 7:1456–1466

Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts

I. Kinetics and stoichiometry of fatty acid peroxidation. Arch

Biochem Biophys 125:189–198

Hideg E, Kos P, Schreiber U (2008) Imaging of NPQ and ROS

formation in tobacco leaves: heat inactivation of the water–water

cycle prevents down-regulation of PSII. Plant Cell Physiol

49:1879–1886

Hu M, Shi Z, Zhang Z, Zhang Y, Li H (2012) Effects of exogenous

glucose on seed germination and antioxidant capacity in wheat

seedlings under salt stress. Plant Growth Regul 68:177–188

Hua XJ, Van de Cotte B, Van Montagu M, Verbruggen N (1997)

Developmental regulation of pyrroline-5-carboxylate reductase

gene expression in Arabidopsis. Plant Physiol 114:1215–1224

Jubany-Marı T, Munne-Bosch S, Alegre L (2010) Redox regulation of

water stress responses in field-grown plants. Role of hydrogen

peroxide and ascorbate. Plant Physiol Biochem 48:351–358

Jung S (2004) Variation in antioxidant metabolism of young and

mature leaves of Arabidopsis thaliana subjected to drought.

Plant Sci 166:459–466

J Plant Res

123

Juvany M, Muller M, Munne-Bosch S (2012) Leaves of field-grown

mastic trees suffer oxidative stress at the two extremes of their

lifespan. J Integr Plant Biol 54:584–594

Juvany M, Muller M, Munne-Bosch S (2013) Photo-oxidative stress

in emerging and senescing leaves: a mirror image. J Exp Bot

64:3087–3098

Kavi Kishor PB, Hong Z, Miao GH, Hu CAA, Verma DPS (1995)

Overexpression of D1-pyrroline-5-carboxylate synthetase

increases proline production and confers osmotolerance in

transgenic plants. Plant Physiol 108:1387–1394

Krause GH, Somersalo S (1989) Fluorescence as a tool in photosyn-

thesis research: application in studies of photoinhibition, cold

acclimation and freezing stress. Phil Trans R Soc Lond

323:281–293

Kubis J, Floryszak-Wieczorek J, Arasimowicz-Jelonek M (2014)

Polyamines induce adaptive responses in water deficit stressed

cucumber roots. J Plant Res 127:151–158

Lambrev PH, Miloslavina Y, Jahns P, Holzwarth AR (2012) On the

relationship between non-photochemical quenching and photo-

protection of photosystem II. Biochim Biophys Acta

1817:760–769

Massacci A, Nabiev SM, Pietrosanti L, Nematov SK, Chernikova TN,

Thor K, Leipner J (2008) Response of the photosynthetic

apparatus of cotton (Gossypium hirsutum) to the onset of drought

stress under field conditions studied by gas-exchange analysis

and chlorophyll fluorescence imaging. Plant Physiol Biochem

46:189–195

Moustakas M, Sperdouli I, Kouna T, Antonopoulou CI, Therios I

(2011) Exogenous proline induces soluble sugar accumulation

and alleviates drought stress effects on photosystem II function-

ing of Arabidopsis thaliana leaves. Plant Growth Regul

65:315–325

Muller J, Boller T, Wiemken A (1996) Pools of non-structural

carbohydrates in soybean root nodules during water stress.

Physiol Plantarum 98:723–730

Munne-Bosch S, Jubany-Marı T, Alegre L (2001) Drought-induced

senescence is characterized by a loss of antioxidant defences in

chloroplasts. Plant Cell Environ 24:1319–1327

Nakashima K, Satoh R, Kiyosue T, Yamaguchi-Shinozaki K,

Shinozaki K (1998) A gene encoding proline dehydrogenase is

not only induced by proline and hypoosmolarity, but is also

developmentally regulated in the reproductive organs of Arabi-

dopsis. Plant Physiol 118:1233–1241

Nanjo T, Kobayashi M, Yoshiba Y, Sanada Y, Wada K, Tsukaya H,

Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K (1999)

Biological functions of proline in morphogenesis and osmotol-

erance revealed in antisense transgenic Arabidopsis thaliana.

Plant J 18:185–193

Neubauer C, Yamamoto H (1992) Mehler-peroxidase reaction

mediates zeaxanthin formation and zeaxanthin-related fluores-

cence quenching in intact chloroplasts. Plant Physiol

99:1354–1361

Pinheiro C, Chaves MM (2011) Photosynthesis and drought: can we

make metabolic connections from available data? J Exp Bot

62:869–882

Pinheiro C, Antonio C, Ortuno MF, Dobrev PI, Hartung W, Thomas-

Oates J, Ricardo CP, Vankova R, Chaves MM, Wilson JC (2011)

Initial water deficit effects on Lupinus albus photosynthetic

performance, carbon metabolism, and hormonal balance: meta-

bolic reorganization prior to early stress responses. J Exp Bot

62:4965–4974

Rasheed R, Wahid A, Farooq M, Hussain I, Basra SMA (2011) Role

of proline and glycinebetaine pretreatments in improving heat

tolerance of sprouting sugarcane (Saccharum sp.) buds. Plant

Growth Regul 65:35–45

Schreiber U, Bilger W, Neubauer C (1994) Chlorophyll fluorescence

as a non-intrusive indicator for rapid assessment of in vivo

photosynthesis. In: Schulze ED, Caldwell MM (eds) Ecophys-

iology of photosynthesis. Series ecological studies, vol 100.

Springer, Berlin, pp 49–70

Selmar D, Kleinwachter M (2013) Stress enhances the synthesis of

secondary plant products: the impact of stress-related over-

reduction on the accumulation of natural products. Plant Cell

Physiol 54:817–826

Shinozaki K, Yamaguchi-Shinozaki K (2007) Gene networks

involved in drought stress response and tolerance. J Exp Bot

58:221–227

Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory

network of gene expression in the drought and cold stress

responses. Curr Opin Plant Biol 6:410–417

Smeekens S, Ma J, Hanson J, Rolland F (2010) Sugar signals and

molecular networks controlling plant growth. Curr Opin Plant

Biol 13:274–279

Sperdouli I, Moustakas M (2012a) Spatio-temporal heterogeneity in

Arabidopsis thaliana leaves under drought stress. Plant Biol

14:118–128

Sperdouli I, Moustakas M (2012b) Interaction of proline, sugars, and

anthocyanins during photosynthetic acclimation of Arabidopsis

thaliana to drought stress. J Plant Physiol 169:577–585

Sperdouli I, Moustakas M (2012c) Differential response of photo-

system II photochemistry in young and mature leaves ofArabidopsis thaliana to the onset of drought stress. Acta Physiol

Plant 34:1267–1276

Sperdouli I, Moustakas M (2014) A better energy allocation of

absorbed light in photosystem II and less photooxidative damage

contribute to acclimation of Arabidopsis thaliana young leaves

to water deficit. J Plant Physiol 171:587–593

Takahashi S, Murata N (2008) How do environmental stresses

accelerate photoinhibition? Trends Plant Sci 13:178–182

Vankova R, Dobra J, Storchova H (2012) Recovery from drought

stress in tobacco. An active process associated with the reversal

of senescence in some plant parts and the sacrifice of others.

Plant Signal Behav 7:19–21

Verbruggen N, Hermans C (2008) Proline accumulation in plants: a

review. Amino Acids 35:753–759

Woo NS, Badger MR, Pogson BJ (2008) A rapid, non-invasive

procedure for quantitative assessment of drought survival using

chlorophyll fluorescence. Plant Methods 4:27

Xiong J, Zhang L, Fu G, Yang Y, Zhu C, Tao L (2012) Drought-

induced proline accumulation is uninvolved with increased nitric

oxide, which alleviates drought stress by decreasing transpiration

in rice. J Plant Res 125:155–164

Yamada M, Morishita H, Urano K, Shiozaki N, Yamaguhi-Shinozaki

K, Shinozaki K, Yoshiba Y (2005) Effects of free proline

accumulation in petunias under drought stress. J Exp Bot

56:1975–1981

Yoshiba Y, Kiyosue T, Nakashima K, Yamaguchi-Shinozaki K,

Shinozaki K (1997) Regulation of levels of proline as an

osmolytes in plants under water stress. Plant Cell Physiol

18:1095–1102

J Plant Res

123