P

Ma

Ra

b

c

J

a

ARRAA

KPSPPO

I

pra2fidme

h0

Journal of Plant Physiology 177 (2015) 1–10

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l h om epage: www.elsev ier .com/ locate / jp lph

hysiology

odification of non-stomatal limitation and photoprotection due to Knd Na nutrition of olive trees

an Erela,c, Uri Yermiyahua, Alon Ben-Gala, Arnon Dagb, Or Shapirac, Amnon Schwartzc,∗

Institute of Soil, Water and Environmental Sciences, Gilat Research Center, Agricultural Research Organization, Bet-Dagan, IsraelInstitute of Plant Sciences, Gilat Research Center, Agricultural Research Organization, Bet-Dagan, IsraelThe Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, Food and Environment, The Hebrew University of

erusalem, Rehovot, Israel

r t i c l e i n f o

rticle history:eceived 8 December 2014eceived in revised form 6 January 2015ccepted 8 January 2015vailable online 15 January 2015

eywords:otassiumodiumhotosynthetic capacityhotorespirationlive trees

a b s t r a c t

Potassium (K) is an essential macronutrient shown to play a fundamental role in photosynthetic processesand may facilitate photoinhibition resistance. In some plant species, sodium (Na) can partially substitutefor K. Although photosynthetic enhancement has been well established, the mechanisms by which Kor Na affects photosynthesis are not fully understood. Olive (Olea europaea L.) trees were previouslyshown to benefit from Na nutrition when K is limiting. In order to study the effect of K and Na onphotosynthetic performance, we measured gas exchange and chlorophyll fluorescence in young olivetrees supplied with either K, Na or no fertilizer, and subjected to manipulated levels of CO2, O2 andradiation. Light and CO2 response curves indicate substantially superior photosynthetic capacity of K-sufficient trees, while Na substitution generated intermediate results. The enhanced performance ofK, and to a lesser extent, Na-supplied trees was found to be related mainly to modification of non-stomatal limitation. This indicates that K deficiency promotes inhibition of enzymatic-photochemicalprocesses. Results indicate lower chlorophyll content and altered Rubisco activity as probable causes ofphotosynthetic impairment. Potassium deficiency was found to diminish photoprotection mechanismsdue to reduced photosynthetic and photorespiratory capacity. The lower CO2 and O2 assimilation rate inK-deficient trees caused elevated levels of exited energy. Consequently, non-photochemical quenching,

an alternative energy dispersion pathway, was increased. Nonetheless, K-deficient trees were shown tosuffer from photodamage to photosystem-II. Sodium replacement considerably diminished the negativeeffect of K deficiency on photoprotection mechanisms. The overall impact of K and Na nutrition playsdown any indirect effect on stomatal limitation and rather demonstrates the centrality of these elementsin photochemical processes of photosynthesis and photoprotection.

© 2015 Elsevier GmbH. All rights reserved.

ntroduction

Potassium, the most abundant univalent cation in plant tissues,lays a central role in physiological processes including osmoticegulation, enzyme activation, electrochemical balance, phloemnd xylem transport and stress signaling (Shabala and Pottosin,014; Wakeel, 2013). Consequently, for superior physiological per-ormance, K is needed in relatively high quantities. Soil K is limitingn many agricultural areas (Zörb et al., 2014) leading to various

eficiency phenomena characterized by retarded cell growth, accu-ulation of simple carbohydrates (Cakmak et al., 1994; Marschner

t al., 1996), low chlorophyll (Chl) content (Bednarz and Oosterhuis,

∗ Corresponding author. Tel.: +972 8 9489917; fax: +972 8 9489069.E-mail address: Schwartz@agri.huji.ac.il (A. Schwartz).

ttp://dx.doi.org/10.1016/j.jplph.2015.01.005176-1617/© 2015 Elsevier GmbH. All rights reserved.

1999), reduced photosynthetic activity (Barnes et al., 1995; Bednarzand Oosterhuis, 1999) and eventual reduced growth and pro-ductivity (Römheld and Kirkby, 2010). Potassium deficiency alsocan reduce plant resistance to various biotic and abiotic stresses(Cakmak, 2005; Zörb et al., 2014).

The effect of K deficiency on photosynthetic reactions is well-established (for comprehensive reviews see: Amtmann and Rubio,2012; Pettigrew, 2008; Zörb et al., 2014) but the mechanism of K-driven photosynthetic inhibition is not fully understood (Zörb et al.,2014). Since K is involved in many physiological processes thereare several possible means of impairment of the photosyntheticrate (A). The first likely mechanism is reduced stomatal and/or

mesophyll conductance, as frequently found in response to K defi-ciency (Peaslee and Moss, 1968; Peoples and Koch, 1979; Terryand Ulrich, 1973). Secondly, K is necessary for the establishmentof an alkaline pH in the chloroplast stroma, required for optimal

2 ant Ph

ptar(mZvetsroomTdc

rcsemcctota(eptcp(caAp(pdiiutEsi2

pNlateeicfmcr

R. Erel et al. / Journal of Pl

hotosynthetic activity (Wu and Berkowitz, 1992). Thirdly, due tohe key role of K in assimilate transport, K-deficient plants tend toccumulate high levels of soluble sugars (Cakmak et al., 1994) thusestricting carbon fixation through feedback inhibition processesHuber, 1984). Fourthly, reduced Chl content in K-deficient plants

ay limit photosynthetic carbon fixation (Battie-Laclau et al., 2014;hao et al., 2001). Lastly, is the general impairment of enzyme acti-ation or synthesis (Zörb et al., 2014), specifically of Rubisco (Jint al., 2011). In several key studies, the inhibitory effect of K deple-ion was shown to be indirect, stemming mainly from elevatedtomatal resistance (Zhao et al., 2001). In alfalfa, K deficiency waseported to have no direct negative effect on photosystem (PS) Ir II activity (Peoples and Koch, 1979). Hence, the inhibitory effectf K deficiency on carbon assimilation may often be related to sto-atal limitation (Peaslee and Moss, 1968; Peoples and Koch, 1979;

erry and Ulrich, 1973; Tsonev et al., 2011) making it difficult toetermine the main direct deleterious effect of K deficiency on netarbon assimilation.

The olive (Olea europaea L.) originated in the eastern Mediter-anean basin and has spread to many areas of the world with aomparable climate. These growing areas are characterized by dryummers with relatively high temperature and radiation, consid-rably above the optimal range for photosynthesis. Excess lightay lead to photoinhibition of the photosynthetic reaction asso-

iated with PS-II. Drought, which leads to a reduction in stomatalonductance and decreased CO2 assimilation, may also exacerbatehe inactivation of PS-II (Lawlor and Tezara, 2009). Photoinhibitionccurs when the rate of D1 protein breakdown of PS-II exceedshe rate of its repair (Lichtenthaler and Burkart, 1999). Excessbsorbed light leads to production of reactive oxidative speciesROS) as byproducts of photosynthetic electron (e−) flow (Müllert al., 2001). ROS and excess e− flow are highly hazardous to planterformance, and may lead to temporary or permanent damageo the photosynthetic reaction centers, membranes and the entirehloroplast. To avoid light-driven photodamage, numerous photo-rotection mechanisms of energy use and dissipation have evolvedTakahashi and Badger, 2011). Since K deficiency is frequently asso-iated with a reduced rate of carbon assimilation, K-deficient plantsre likely to be more susceptible to photodamage (Cakmak, 2005).lternative pathways for excess absorbed energy might be dissi-ation through non-photochemical quenching (NPQ) such as heatMüller et al., 2001) or by photochemical means through photores-iration (Takahashi and Badger, 2011). Although photorespirationirectly competes with carbon assimilation and consumes energy,

t is also effective in photoprotection (Kozaki and Takeba, 1996) ast provides an alternative acceptor for excess e−. Plants growingnder harsh environmental conditions were found to have par-icularly efficient NPQ and mechanisms for respiration protection.vidence demonstrates that the photorespiratory pathway plays aubstantial role in photoprotection, particularly in species originat-ng from stressful environments (Eppel et al., 2013; Ort and Baker,002).

While K is unquestionably an essential macronutrient, somelant species are capable of at least partially replacing K witha. The beneficial effects of Na on plant growth and yield have

ong been acknowledged, specifically under conditions of low Kvailability (Marschner, 2012). Comprehensive reviews regardinghe potential beneficial function of Na are available in Subbaraot al. (2003), Wakeel et al. (2011), and more recently, Kronzuckert al. (2013). Most studies focusing on the beneficial function of Nandicate substitution of K in osmoregulation as the major positiveontribution of Na in C3 species (Kronzucker et al., 2013). Thus, Na

unction is often assumed to be fairly limited to vacuoles of cells in

ature tissues (Marschner, 2012). None the less, studies of intacthloroplasts demonstrated Na replacement of K in stormal pHegulation and thus maintaining photosynthetic reactions (Huber

ysiology 177 (2015) 1–10

and Maury, 1980; Kaiser et al., 1980; Wu and Berkowitz, 1992).Sodium ability to generate stromal alkalization indicates Na/Hantiport in the chloroplast. More recently, ion channel in the chloro-plast envelope were found to be equally permeable for Na and K andmay also contribute to stormal alkalization and CO2 assimilation(Pottosin et al., 2005).

Notwithstanding Na replacement of K in intact chloroplast, it isnot fully understood to what extent Na can replace K in its role inphotosynthesis processes. Sugar beet is a species found to highlyutilize Na, but Na in its leaves did not replace K in Chl formation(Marschner and Possingham, 1975). Conversely, Chl content andRubisco activity in Eucalyptus leaves increased following Na appli-cation compared to the unfertilized control, but remained lowerthan K-treated trees (Battie-Laclau et al., 2014). Furthermore, thephotosynthetic capacity of K and Na-treated trees was frequentlyhigher than that of the unfertilized control, indicating modifica-tion of both stomatal and non-stomatal limitations by nutritionalstatus. Similarly, in cacao (Theobroma cacao) plants, an increas-ing proportion of Na replacement of K was shown to be beneficialfor net carbon assimilation (A). While novel, these findings fail topresent any further understanding regarding mechanisms drivingthe superior A.

Olive trees are grown extensively in the Mediterranean region,frequently on shallow, infertile soils. Hence, adaptive mechanismsevolved that led to high efficiency of mineral acquisition and uti-lization (Fernandez-Escobar, 2010). Potassium is considered tobe particularly important in olive cultivation (Fernandez-Escobar,2010), however, the assumption of a high K requirement lackssolid scientific evidence. In a four-year study using an inert perlitesubstrate, growth and fruit production of olives were not signif-icantly affected by fertigation with nutrient solutions containingsix K levels ranging from 0.20 to 5.20 mM (Erel et al., 2013). Fur-ther investigation revealed that trees which were irrigated withlow levels of K accumulated high levels of Na. The presence of Nain a K-deficient irrigation solution was further found to result inenhanced growth, superior photosynthetic rate, higher leaf starchconcentration and reduced deficiency symptoms compared to Kand Na-deficient plants (Erel et al., 2014). Photosynthetic rate washighest in plants receiving K in the irrigation solution, slightly lowerwhen only Na was applied and lowest in unfertilized trees. Conse-quently, Erel et al. (2014) indicated olive as a species capable ofpartly replacing K by Na and suggested that Na plays a positive rolein photosynthetic reactions when olive is exposed to insufficient K.

The objectives of the present study were to examine the roleof K and Na substitution on photosynthetic and photoprotectionprocesses in olive by measuring combined gas exchange and Chl afluorescence under manipulated CO2, light intensity and O2 levels.

Materials and methods

Experimental design and treatments

Detailed descriptions of growth conditions and the treatmentsetup are provided in Erel et al. (2014). In brief: one year-old olivetrees (Olea europaea L.) var. Barnea were planted in 60-L containersfilled with perlite substrate and grown in a greenhouse. After twomonths of acclimation, trees were pruned to 65-cm single shootsand differential nutrient solution application was initiated in May2012. The experiment was designed in random blocks with sixreplicates per treatment. Mineral concentrations of K and Na andnomenclature are given in Table 1. The −K−Na treatment con-

tained no K or Na, the +Na treatment contained 2.4 mM Na and no Kand the +K treatment contained 2.5 mM K and 2.8 mM Na. All othernutrients were applied to all treatments at uniform concentrations.Average measured mineral concentrations of specific elements

R. Erel et al. / Journal of Plant Ph

Tab

le

1Po

tass

ium

(K) a

nd

sod

ium

(Na)

con

cen

trat

ion

in

irri

gati

on

solu

tion

s,

and

con

cen

trat

ion

s

of

min

eral

s

and

chlo

rop

hyl

l in

leav

es

of

oliv

e

tree

s

grow

n

for

10

mon

ths

wit

h

the

foll

owin

g

irri

gati

on

solu

tion

s:

2.48

mM

K

and

2.84

mM

Na

(+K

),

0.08

mM

K

and

2.48

mM

Na

(+N

a)

and

0.09

mM

K

and

0.13

mM

Na

(−K

−Na)

. Th

e

trea

tmen

t

effe

ct

was

test

ed

by

one-

way

AN

OV

A

(Tu

key–

Kra

mer

mu

ltip

le

com

par

ison

s

test

);

dif

fere

nt

lett

ers

ind

icat

e

stat

isti

cal s

ign

ifica

nce

(n

=

6).

Min

eral

in

irri

gati

on

(mM

)

Leaf

mac

ron

utr

ien

ts

(%)

Leaf

mic

ron

utr

ien

ts

(mg

kg−1

)

Ch

loro

ph

yll

(�g

cm−2

)To

tal D

W(g

tree

−1)

K

Na

K

Na

P

N

Cl

Mg

Fe

Zn

Mn

Cu

+K

2.48

±

0.20

2.84

±

0.31

1.01

A0.

01B

0.13

A1.

41B

0.19

A0.

13C

47A

10.5

A17

.4A

3.1A

48.1

A36

55A

+Na

0.08

±

0.03

2.42

±

0.15

0.20

B0.

19A

0.13

A1.

59B

0.20

A0.

16B

41A

8.8A

9.4B

2.8A

41.2

A30

45B

−K−N

a

0.09

±

0.03

0.13

±

0.04

0.19

B0.

03B

0.14

A1.

81A

0.21

A0.

22A

46A

10.0

A10

.3B

3.2A

26.5

B24

70C

ysiology 177 (2015) 1–10 3

were: Ca: 2.0 ± 0.1 mM, Mg: 1.2 ± 0.2 mM, NH4: 0.27 ± 0.04 mM,NO3: 4.6 ± 0.1 mM and P: 0.30 ± 0.02 mM. The pH was maintainedin the range of 5.7–5.8. Target concentrations were achieved byproportionally dissolving commercial grade salts of: Ca(NO3)2,NH4H2PO4, KCl, MgSO4, Mg(Cl)2 and NaCl. Micronutrients (Fe, Mn,Zn, Cu, Mo and B) were applied at a rate of 0.1 mL L−1 of commercialsolution (“Koratin + B”, Fertilizers and Chemicals Ltd., Israel). Foreach treatment, salts were dissolved in desalinated water withinitial electrical conductivity < 50 �S m−1. The described solutionswere applied to trees daily via an automatic drip irrigation systemat a rate of ∼30% over actual tree-scale evapotranspiration. Mea-surements were performed 12–14 months after initiation of theexperiment, between May and July 2013.

Biomass, minerals and chlorophyll (Chl)

Leaf analysis was performed in May 2013 according to the pro-tocol formerly used for olive described in Erel et al. (2013). Uponconcluding the trials, plants were removed, dried and weighed.Chlorophyll content was determined using the dimethyl sulfoxide(DMSO) extraction method following Barnes et al. (1992). In May2013, young, fully-developed leaves showing no necrotic symp-toms were sampled and transferred on ice to the nearby lab. Sevenspecimens of 0.18 cm−2 disks (100–150 mg FW) from three leavesper plant were cut and placed in vials containing 5 ml DMSO. Thevials were placed in a water bath pre-heated to 70 ◦C for 85 min incomplete darkness. Vials were left to cool to room temperature andanother 5 ml of DMSO was added. The absorbance of the extractionwas measured at wavelengths of 665 and 648 nm.

Gas exchange and electron transport rate (ETR)

In general, measurements were performed using a portablegas-exchange system equipped with a leaf chamber fluorom-eter (LI-6400-40 LI-COR, Inc., Lincoln, NE). Unless otherwisestated, chamber conditions were set to environmental CO2concentration of 400 �mol mol−1, photosynthetic photon flux den-sity (PPFD) of 1,000 �mol mol m−2 s−1 (10% blue), humidity of15 mmol H2O mol−1, air flow of 300 �mol s−1 and block temper-ature of 25 ◦C. Measurements were taken on clear days between09:00 and 12:00.

Two sets of photosynthetic response to intercellular CO2 con-centration (A/Ci curve) were performed. Initially, A/Ci curves weremeasured at ambient O2 concentration on six trees per treatment,with three measurements per tree, by manipulating chamber CO2concentration from 400 �mol mol−1 to 200, 50, 150, 300, 400, 600,900, and 1200. The photosynthetic parameters, Vcmax, J and TPU,acquired from A/Ci, were fitted using a tool supplied by Sharkeyet al. (2007). Stomatal (Ls) and non-stomatal (Lns) limitation werederived from A/Ci as described by Farquhar and Sharkey (1982) asfollows:

LS = ACi=400 − ACa=400

ACi=400(1)

where ACi=400 is the CO2 assimilation rate at intercellular CO2 con-centration (Ci) of 400 �mol mol−1 and ACa=400 is A at the ambientCO2 concentration of 400 �mol mol−1. The non-stomatal limitation(Lns) is the assimilation rate at Ci = 400 �mol mol−1 relative to treat-ment +K. Two months later, another set of A/Ci curves were madewhile altering O2 concentration in order to estimate photorespi-ration. The second set was performed on three selected trees per

treatment at six CO2 concentrations (400, 200, 50, 600, 1000, and1600) and two O2 levels (2% and 21%). The non-respiratory condi-tions were set up by connecting the air inlet to a pressurized airtank containing 2% O2 as described in Sperling et al. (2014).

4 ant Ph

m1at

mpea(

E

wtJ

P

ilrbput

J

J

J

R

wmobpwe

C

wupMaaii6tsliswq(a

plant biomass, Fv/Fm, NPQ, qI and qE were analyzed by one-wayANOVA (Tukey–Kramer multiple comparisons test) using JMP 7statistical software (SAS Institute, Cary, NC). Photosynthetic param-eters, Vcmax, J and TPU, obtained from A/Ci as described by Sharkey

Fig. 1. Photos of representative twigs, six months after exposure to irrigation solu-tions containing: 2.48 mM K and 2.84 mM Na (+K), 0.08 mM K and 2.48 mM Na (+Na)and 0.09 mM K and 0.13 mM Na (−K−Na).

-5

0

5

10

15

20

25

30

35

40

45

0 200 400 600 80 0 100 0

A( µmol

m-2s-1)

Ci (ppm)

+K+Na-K-Na

R. Erel et al. / Journal of Pl

The photosynthetic response to PPFD (light curve) was deter-ined for three trees per treatment by decreasing PPFD from

600 �mol m−2 s−1 down to 1200, 800, 600, 400, 200, 100, 50, 25nd 0. Measurements were taken only when leaf stomatal conduc-ance was higher than 0.2 mol m−2 s−1.

For all response curves, following stabilization of CO2 and sto-atal conductance (approximately 1–2 min), the light saturation

ulse was illuminated and gas-exchange and fluorescence param-ters were logged. First, steady state florescence (Fs) was loggednd, immediately after saturation pulse the maximal fluorescenceFm

′) was logged. ETR was calculated according to:

TR = F ′m − FS

F ′m

× 0.5 × PPFD × 0.875 (2)

here 0.5 is the proportion of light harvesting by PS-II and 0.875 ishe commonly-accepted leaf absorbance coefficient (Maxwell andohnson, 2000).

hotorespiration

Calculations used for photorespiration are thoroughly describedn Valentini et al. (1995). In brief, total e− flow (JT) was calcu-ated from fluorescence data obtained from both A/Ci and lightesponse curves. Photosynthetic electron flow partitioning, to car-oxylation (JC) and to oxidation (JO), as well as CO2 production byhotorespiration (Rl), were evaluated using A/Ci response curvesnder photorespiratory and non-photorespiratory conditions usinghe following relationships:

T = ETR = JC + JO (3)

C = 13

[JT + 8 (A + RD)] (4)

O = 23

[JT − 4 (A + RD)] (5)

l = 112

JC [JT − 4 (A + RD)] (6)

here A and RD (day respiration) were determined by gas exchangeethods as described above and ETR (JT) was derived from the flu-

rescence measurements. To validate the model, the relationshipetween A and ETR under non-photorespiratory conditions waslotted and confirmed. JC and JO from the light response curvesere also calculated according to Valentini et al. (1995) assuming

− flow of 4 mol mol−1 CO2.

hlorophyll a fluorescence

Relaxation kinetic of Chl fluorescence quenching was madeith a OS5P modulated fluorometer (Opti-Sciences, Hudson, NH)sing puddle model parameters. The general description of theerformed kinetic of Chl fluorescence quenching is described byüller et al. (2001); calculations were according to Lichtenthaler

nd Burkart (1999). Specific instrument operation conditions weres follows: the red modulated source was used at a modulationntensity of 8 and the detector gain was set to 4. Saturation pulsentensity was set to PPFD of 12,190 �mol m−2 s−1 at a flash rate of0 s. A halogen lamp was used as an actinic light source and seto PPFD of 1422 �mol m−2 s−1. A day before performing the mea-urements plants were placed in a dark room for ∼14 h. Initially,eaves received a saturation pulse to determine maximal and min-mal dark-adapted fluorescence (Fm and F0). Immediately after theaturation pulse, actinic light was turned on and quantum yield

as determined every minute until reaching steady state. Subse-

uently maximal F at steady state in actinic light (Fm′) was recorded

standard deviation of Fm′ to trigger next step was set to 1%). There-

fter, actinic light turned off while continuing frequent saturation

ysiology 177 (2015) 1–10

pulses upon steady state, and then recording maximal F after rapidrelaxation (Fmr).

Maximum quantum yield of PS-II (Fv/Fm) was calculated as fol-lows:Fv

Fm= Fm − F0

Fm(7)

NPQ was calculated after reaching steady state at the followingphotosynthetic radiation:

NPQ = Fm − Fm′

Fm′(8)

The NPQ components, energy-dependent quenching (qE) andphotoinhibitory quenching (qI), were calculated following relax-ation with the actinic light off.

qE = Fmr − Fm′

Fm′(9)

qI = Fm − Fmr

Fm′(10)

Data analysis

Effects of treatments on leaf minerals and Chl concentrations,

Fig. 2. A/Ci response curves of trees grown with irrigation solutions containing:2.48 mM K and 2.84 mM Na (+K), 0.08 mM K and 2.48 mM Na (+Na) and 0.09 mM Kand 0.13 mM Na (−K−Na). Measurements were conducted on 7th–16th of May 2013.Each point represents the average ± S.D. of six trees × three measurements (n = 18).

R. Erel et al. / Journal of Plant Physiology 177 (2015) 1–10 5

-5

5

15

25

35

45

0 20 0 40 0 60 0 80 0 100 0 120 0 140 0

A(µmolm

-2s-1 )

Ci (µmol mol-1)

-K-Na

-5

5

15

25

35

45

0 20 0 40 0 60 0 80 0 100 0 120 0 140 0

A(µmolm

-2s-1 )

+Na

-5

5

15

25

35

45

0 200 400 600 800 1000 1200 1400

A(µmolm

-2s-1 )

+K21%2%

0

40

80

120

160

200

0 20 0 40 0 60 0 80 0 100 0 120 0 140 0

J T(µmol

m-2s-1)

Ci (µmol mol-1)

-K-Na

0

40

80

120

160

200

0 200 40 0 60 0 80 0 100 0 120 0 140 0J T(µmolm

-2s-1)

+Na

0

40

80

120

160

200

0 20 0 40 0 60 0 80 0 100 0 120 0 140 0

J T(µmolm

-2s-1)

+K

F photoO K anN repres

eSt

R

L

eim1((A−0CbmnC

ig. 3. Assimilation and JT response curves of olive trees to internal CO2 under non-2, filled symbols). Trees were grown with irrigation solutions containing: 2.48 mMa (−K−Na). Measurements were taken from 18th to 24th of July 2013, each point

t al. (2007), were analyzed by one-way ANOVA as described above.tomatal (Ls) and non-stomatal (Lns) limitations were transformedo arcsine prior to ANOVA.

esults

eaf mineral and chlorophyll concentrations

The concentrations of K and Na in leaves were highly depend-nt on their concentrations in the irrigation solutions. When therrigation solution contained 2.5 mM K and 2.8 mM Na (+K treat-

ent), their concentrations in the dry matter of leaf tissues were.01% and 0.01%, respectively. When irrigated with K-deficient0.08 mM) nutrient solution but in the presence of 2.4 mM Natreatment +Na), leaf K decreased to 0.20% and Na rose to 0.19%.t low concentrations of K (0.09 mM) and Na (0.13 mM, treatmentK) in the nutrient solution, their concentrations were 0.19% and.03%, respectively. Of the other measured macronutrients N, P,l and Mg only the concentrations of N and Mg were affected

y treatments, being significantly higher in −K−Na. Of the foureasured micronutrients, Fe, Zn, Mn and Cu, only Mn was sig-

ificantly affected by treatment showing higher values in +K. Leafhl density decreased significantly when no K or Na were applied

respiratory conditions (2% O2, hollow symbols) and under ambient conditions (21%d 2.84 mM Na (+K), 0.08 mM K and 2.48 mM Na (+Na) and 0.09 mM K and 0.13 mMents the average ± S.D. of three trees times six leaves (n = 18).

(26.5 �g cm−2 compared to 41.2 and 48.1 �g cm−1 in response toNa and K application, respectively (Table 1). The chemical Chl anal-ysis was supported by visual leaf chlorosis of the K-deficient plantswhich was largely compensated by Na (Fig. 1). By the end of theexperiment, the total produced biomass was lowest in −K−Na,significantly higher in response to Na addition and highest in +K.

A/Ci curve under photorespiratory environment

The response of net photosynthetic rate (A) to intercellular CO2concentration (Ci) varied among treatment (Fig. 2). At just aboutany given Ci level, A was notably lower for −K−Na while +K showedthe highest A values, slightly but consistently higher than those of+Na. The average photosynthetic Rubisco capacity per unit leaf area(Vcmax) was 146, 134 and 111 �mol m−2 s−1 for +K, +Na and −K−Na,respectively, the latter being significantly lower than +K (Table 2).The potential electron transport rate per unit leaf area (J) was alsohighest for +K, slightly lower for +Na, and significantly lowest for−K−Na. In accordance with Vcmax and J, triose-phosphate utiliza-

tion (TPU) was also highest for +K and lowest for −K−Na. The A/Cicurves allow us to evaluate the reduction in A caused by stomatalconductance (Ls) by comparing A when Ci = 400 �mol mol−1 to A atenvironmental CO2 of 400 �mol mol−1. The results show a 35–37%

6 R. Erel et al. / Journal of Plant Physiology 177 (2015) 1–10

Table 2Photosynthetic Rubisco capacity (Vcmax), potential electron transport rate (J) andtriose phosphate utilization (TPU) acquired from fitting the A/Ci response curvefollowing Sharkey et al. (2007), and stomatal (Ls) and non-stomatal (Lns) limita-tion to photosynthesis calculated from the CO2 response curve. Key to treatments:2.48 mM K and 2.84 mM Na (+K), 0.08 mM K and 2.48 mM Na (+Na) and 0.09 mMK and 0.13 mM Na (−K−Na). Treatment effect was tested by one-way ANOVA(Tukey–Kramer multiple comparisons test); different letters indicate statistical sig-nificance (n = 18).

Treatment Vcmax J TPU Ls Lns

(�mol m−2 s−1) (%)

+K 146A 209A 14.2A 34.6A 0B

r−lat

A

c5mObo+tdnlretoCl

P

eoleaAe(uttodi

R

aaP

Fig. 4. Electron flow to carboxylation (JC , solid symbols) and to photorespiration(JO, hollow symbols) (A) and proportion of photorespiration in total e− flow (B) oftrees grown with irrigation solutions containing: 2.48 mM K and 2.84 mM Na (+K),0.08 mM K and 2.48 mM Na (+Na) and 0.09 mM K and 0.13 mM Na (−K−Na). Datafrom A/Ci curves measured in July 2013 were calculated and transformed accordingto Valentini et al. (1995).

Fig. 5. Assimilation rate (A) and JT (B) of olive trees in response to photosynthetic

+Na 134AB 182A 12.9AB 37.2A 11.8B

−K−Na 111B 149B 11.2B 28.3A 45.8A

eduction in A for +K and +Na due to stomatal limitation whileK−Na had somewhat lower stomatal limitation. The non-stomatal

imitation of A caused by nutritional status (compared to +K) reveals drastic reduction of 46% when no K and Na were applied, whilehe addition of solely Na resulted in a mere 12% reduction (Table 2).

/Ci curve under non-photorespiratory environment

A second set of A/Ci curves was constructed under two O2 con-entrations: 21% and 2%. In general, within the lower range of Ci,0–400, A was considerably higher in a non-respiratory environ-ent as expected due to the restriction of photorespiration (Fig. 3).nce Ci increased further, A under +K and +Na was comparableetween the two O2 levels. Under −K−Na, A was higher through-ut the Ci range under low O2. Under photorespiratory conditions,K showed the typical e− flow curve in response to Ci. JT con-inuously increased up to Ci of ∼400 �mol mol−1 and graduallyecreased thereafter indicating reduced photorespiration. Underon-photorespiratory conditions the JT response curve closely fol-

owed the A curve, i.e. a sharp linear increase in the lower Ciange which reached saturation around 300 �mol mol−1. The gen-ral response of +Na to Ci and O2 level was somewhat similar tohat described for +K, however the magnitude of the effect of O2n JT was reduced. As for −K−Na, with the exception of the lowesti data point, the JT response to Ci was nearly identical for both O2

evels.

artitioning of noncyclic photosynthetic electron flow

The A/Ci response curve under the two O2 levels was used tostimate e− flow to carboxylation (JC) and respiration (JO). Through-ut the Ci range, JC and JO were highest in +K, lower in +Na andowest in −K−Na (Fig. 4A). The only exception was at the high-st Ci point at which +K and +Na had similar carboxylation ratesnd all treatments had very low and comparable respiration rates.t the lowest Ci data point (∼70 ppm) the ratio of JO to the total− flow (JT) was high (64–65%) and similar among treatmentsFig. 4B). As Ci increased, the proportion of JO decreased contin-ously. The decrease attenuated in the order −K−Na > +Na > +Khroughout the range of Ci, i.e. +K had a higher ratio of e− flowo photorespiration when Ci exceeded 70 �mol mol−1. For Ci levelsf 260–300 �mol mol−1, typical under natural environmental con-itions, the ratio of e− flow to photorespiration was: 40, 32 and 27%

n +K, +Na and −K−Na, respectively.

esponse to light

The general response of the CO2 assimilation rate to PPFD was nonlinear asymptotic increase with values reaching a maximumt a light intensity above 600–800 �mol m−2 s−1 (Fig. 5A). At lowPFD, between 0 and 200 �mol m−2 s−1, A was fairly comparable

photon flux density (PPFD). Trees were grown with irrigation solutions containing:2.48 mM K and 2.84 mM Na (+K), 0.08 mM K and 2.48 mM Na (+Na) and 0.09 mM Kand 0.13 mM Na (−K−Na). Measurements were taken from 10th to 17th of July 2013;each point represents the average ± S.D. of four leaves × three trees (n = 12).

ant Ph

aeboa

E

rtalcsTa42ooC(vdwr1

F

Iwni(hnt

D

S

KSoB1ptsadnspHriiIt

R. Erel et al. / Journal of Pl

mong the three treatments. Above 200 �mol m−2 s−1, A was high-st for +K, lower for +Na and lowest for −K−Na. The difference in Aetween +K and the other treatments increased with PPFD through-ut the measured range. Total JT responded to PPFD similarly withn even more strongly pronounced treatment effect (Fig. 5B).

lectron flow to carboxylation and respiration in response to light

The general response of JC and JO was comparable to thateported for JT (Fig. 4B). Specifically, JC and JO were not affected byreatments up to 400 �mol m−2 s−1, while from 400 �mol m−2 s−1

nd onwards, +K had superior JC and JO followed by +Na while theowest values were recorded under −K−Na. For +K, JO increasedontinuously with PPFD, while for +Na and −K−Na, JO reachedaturation at PPFD of 800 and 600 �mol m−2 s−1, respectively.he proportion of e− flow to photorespiration (JO/JT) was lownd did not differ significantly when PPFD was lower than00 �mol m−2 s−1. Above 400, JO/JT was constant for −K−Na, at1–24%. JO partitioning was higher for +Na, reaching a plateauf 26–28% at PPFD of 800 and highest in +K, reaching a plateauf 33% only at PPFD of 1200 �mol m−2 s−1. Similarly to JO, theO2 produced by photorespiration (Rl) was negligible at low PPFD0–40 �mol m−2 s−1) and increased towards saturation with ele-ated PPFD. At high PPFD, Rl was constantly highest for +K while theifference in RI between the +Na and −K−Na treatments increasedith PPFD. Above 400 �mol m−2 s−1, RI of the +Na and −K−Na

eached saturation while for +K it continued to increase until200 �mol m−2 s−1.

luorescence kinetics

In dark-adapted leaves, the maximum quantum yield of PS-I (Fv/Fm) was significantly higher for +K than for −K−Na,

hile +Na provided intermediate results (Fig. 7). Conversely,on-photochemical quenching (NPQ) was lowest for +K and signif-

cantly higher for −K−Na. Both the energy-dependent quenchingqE) and photo-inhibitory quenching (ql) components of NPQ wereighest for −K−Na and lowest for +K (Fig. 7). While qE was not sig-ificantly affected, qI was significantly higher for −K−Na comparedo +K.

iscussion

tomatal (Ls) and non-stomatal (Lns) limitation

The present study clearly demonstrates the positive influence of nutrition on photosynthetic capacity of the olive (Figs. 2 and 5).imilarly, numerous studies have indicated an inhibitory effectf K deficiency on CO2 assimilation (e.g. (Barnes et al., 1995;attie-Laclau et al., 2014; Bednarz and Oosterhuis, 1999; Huber,985; Peoples and Koch, 1979; Terry and Ulrich, 1973). Variousossible mechanisms have been suggested to explain the pho-osynthetic impairment: stomatal regulation, establishment of atromal H+ gradient, protein synthesis, enzyme activation andssimilate transport (Amtmann and Rubio, 2012). The mechanismriving down-regulation of photosynthesis under K deficiency isot fully understood (Zörb et al., 2014). Furthermore, although Naubstitution was shown to mitigate the effect of K deficiency onhotosynthesis (Battie-Laclau et al., 2014; Gattward et al., 2012;ampe and Marschner, 1982; Subbarao et al., 1999) knowledge

egarding the role of Na in maintaining high photosynthetic capac-

ty is scarce and limited to C4 species. It is assumed that Na may benvolved in osmotic regulation of plant cells (Wakeel et al., 2011).n a previous study, K-deficient olive trees responded positivelyo addition of Na to the irrigation solution (Erel et al., 2014). The

ysiology 177 (2015) 1–10 7

greater photosynthetic performance of Na-supplied trees was dueto reduction of both stomatal and non-stomatal limitation.

Interpreting the role of K, including distinguishing between sto-matal and non-stomatal limitation of photosynthesis is difficultusing the common, simple instantaneous measurement of A. Thatis mainly due to the fact that K deficiency often impairs stoma-tal conductance as well as A (Marschner, 2012; Peaslee and Moss,1968; Peoples and Koch, 1979; Terry and Ulrich, 1973). Evaluat-ing the A/Ci response curve enables direct estimation of importantparameters related to the photosynthetic process, regardless ofstomatal conductance. Parameters obtained from A/Ci responsecurves in the current study indicate that the three major charac-teristics of assimilation response to intercellular CO2 were highestwhen the irrigation solution contained sufficient K and signifi-cantly lower when K and Na were deficient. Those characteristicsof photosynthetic activity, Vcmax, J and TPU, did not change sig-nificantly when Na replaced K in the irrigation medium (Table 2).Hence, the inhibitory effect of K deficiency under these condi-tions is shown to be mainly a result of non-stomatal limitation,i.e. impairment of photosynthetic reactions rather than failure toregulate stomatal movement. Subsequent to suboptimal K and Nalevels, non-stomatal limitation was 45.8% in K-deficient trees. Thereplacement of K by Na significantly moderated the photosyntheticimpairment to a mere 11.8%. Stomatal limitation, however, was notsignificantly modified by nutritional status. Direct, Lns inhibition ofphotosynthetic processes by K shortage has also been suggested forChinese hickory (Carya cathayensis) and Eucalyptus trees (Battie-Laclau et al., 2014; Jin et al., 2011). In Eucalyptus leaves showingdeficiency symptoms, the photosynthetic parameters Vcmax, J andTPU were impaired along with a pronounced effect on stomatalconductance (gs) (Battie-Laclau et al., 2014). In olive, diurnal mea-surements also revealed a tendency for reduced gs in response tolow levels of K and Na (Erel et al., 2014). In the current study, K andNa levels had only a marginal effect on Ls, indicating that the rate ofCO2 diffusion in the gas phase has only a small effect on the rate ofcarbon fixation. Apparently, photosynthetic capacity was reducedby K deficiency which led to a decline in A, while addition of Napartially compensated for the lack of K.

We therefore conclude that, in the olive, although K and Nashortage may reduce gs as often shown, the negative effect on thephotosynthetic apparatus causes the bulk reduction in the rate ofcarbon assimilation. Similar conclusions were made by Zhao et al.(2001) for cotton and Jin et al. (2011) for Chinese hickory. Bednarzet al. (1998) studied the effect of K on A in cotton and concludedthat Ls was the main factor limiting A under conditions of moderateK deficiency, while under more severe K deficiency A was primarilylimited by a reduction of Lns.

Photosynthetic performance

In the present study the photosynthetic potential, as reflectedin A/Ci and light curves, was highly and consistently superior whenK was available in sufficient amounts, while substitution of K by Nawas efficient in maintaining a large proportion of the photosyn-thetic capacity. Photosynthetic rate at natural CO2 environmentand high light radiance was 12.1, 17.3 and 20.4 �mol m−2 s−1 for−K−Na, +Na and +K, respectively, i.e. in the absence of K, assimila-tion rate was nearly half of K-sufficient trees. Comparable resultswere obtained by diurnal measurement of A under different Klevels (Erel et al., 2014). Such dramatic reduction in assimilationunder natural conditions may lead to carbon starvation and limitdevelopment of K-deficient tree as indicates from the lower starch

concentration found in leaves of K-deficient olive trees (Erel et al.,2014).

Under low, unsaturated light intensities, A was not affectedby nutritional level (Fig. 5). The positive effect of Na on A is in

8 ant Physiology 177 (2015) 1–10

arecai

Lltcla(NswNnamElJ

Almtope2imebaCaR

P

tdeistm

patf(iJadph2f

Fig. 6. Electron flow to carboxylation (JC , solid symbols) and to photorespiration(JO , hollow symbols) (A), Proportion of photorespiration in total e− flow (B) andCO2 production by photorespiration (Rl) (C) of trees grown with irrigation solutions

R. Erel et al. / Journal of Pl

greement with some previous studies on Eucalyptus, cacao anded beet (Battie-Laclau et al., 2014; Gattward et al., 2012; Subbaraot al., 1999). In other plant species however, Na was not able toompensate for K deficiency to mitigate impairment of A (Terrynd Ulrich, 1973). More frequently, Na is known for its adversenfluence on the photosynthetic process (Wakeel, 2013).

A few mechanisms may lead to enhanced A by K and Na. Ass was shown to have only a minor effect, the main causes areikely to be metabolic-enzymatic processes. The first clear nutri-ional effect of K and Na on Lns was the positive response of Chlontent (Table 1, Fig. 1). Previous studies have reported reduction ofeaf Chl in response to K deficiency (Jin et al., 2011; Zhao et al., 2001)s well as the beneficial effect of Na on leaf Chl in Eucalyptus treesBattie-Laclau et al., 2014). Very often, Chl is closely related to leaf, however in the current study chlorotic, K-deficient leaves had a

urprisingly high leaf N concentration (Table 1). Furthermore, Mgas significantly higher and Fe was not reduced when both K anda were deficient. This implies that the chlorosis was not a result ofutritional disorder but due to either Chl synthesis impairment or

higher rate of Chl degradation. Chlorosis caused by K deficiencyay be due to oxidative damage caused by ROS (Cakmak, 2005).

ither way, low Chl level in relation to K and Na deficiency is highlyikely to contribute to A impairment (Amtmann and Rubio, 2012;in et al., 2011).

Low leaf Chl content is probably not the sole reason for low in K-deficient plants. Subbarao et al. (2000) found lower Chl in

ow-K leaves of Red beet with no effect on A. Strong evidence forechanistic Lns impairment of A is the consistently lower assimila-

ion parameters in K-depleted plants (Table 2, Fig. 2). Impairmentf Vcmax, J and TPU indicates down-regulation of key enzymaticrocesses of CO2 assimilation. Similarly, previous studies indicatednzymatic impairment under K deficiency (Battie-Laclau et al.,014; Jin et al., 2011). Another potentially important K-driven

nhibitor of A is feedback inhibition resulting from assimilate accu-ulation due to the role of K in assimilate transport (Cakmak

t al., 1994; Pettigrew, 2008). However, in olive, total soluble car-ohydrates (SCH) were not modified by K level (Erel et al., 2014)nd hence, feedback inhibition of photosynthesis is questionable.onversely, low K status has been frequently associated with SCHccumulation and A depletion in other plant species (Amtmann andubio, 2012; Battie-Laclau et al., 2014; Gerardeaux et al., 2010).

hotorespiration and photoprotection

In a detailed review paper, Cakmak (2005) discussed the essen-ial role of K nutrition in suppressing ROS production and oxidativeamage. This is achieved mainly by the maintenance of high A andfficient distribution of assimilates such that photosynthetic e− fluxs controlled and the creation of ROS is prevented. In the currenttudy, A certainly was superior in response to K and Na nutrition;he question at hand is to what extent does K and Na nutrition

odify photoinhibition?The answer lies within two parallel processes: non-

hotochemical quenching (NPQ, mainly dissipated as heat)nd photochemical processes (mainly photorespiration). Pho-orespiration facilitates an alternative photochemical pathwayor excited energy exceeding the capacity for CO2 assimilationKozaki and Takeba, 1996). The current study reveals that undernsufficient K and Na conditions, total e− flow and, even more so,O, were extremely low (Fig. 4A). Under stressful conditions, suchs excessive light, the photorespiration rate is expected to riseue to excess absorbed energy (Kozaki and Takeba, 1996). Hence,

lants adapted to harsh environments characteristically haveigh photorespiration capacity (Rivero et al., 2009; Sperling et al.,014). A continuous increase of photorespiration with PPFD wasound here only in response to high K level (Fig. 6). In K-deficient

containing: 2.48 mM K and 2.84 mM Na (+K), 0.08 mM K and 2.48 mM Na (+Na) and0.09 mM K and 0.13 mM Na (−K−Na). Data from light curves measured in July 2013were calculated and transformed according to Valentini et al. (1995).

trees, JO and Rl increased only up to a certain point beyond whichthe photorespiration rate remained unchanged. Saturation inphotorespiration under high PPFD is expected to impair photo-protection in K-deficient trees. This dramatic effect of nutrition onthe magnitude of photorespiration implies that K, and to a lesserextent Na, elevates photochemical protective mechanisms againstphotodamage and enhance plant performance under high PPFDconditions by reducing ROS and photoinhibition.

In addition to the photochemical pathway of photoprotection,NPQ plays a substantial role in dissipation of excited energy. Mea-suring the relaxation kinetics of chlorophyll fluorescence providesinsights into patterns of NPQ energy dispersion (Lichtenthaler andBurkart, 1999). In date trees exposed to high irradiance most ofthe absorbed energy was directed to NPQ (Sperling et al., 2014)implying the significance of NPQ as a defensive mechanism inplants well-adapted to stressful environments. In olives sufferingfrom K and Na deficiency the magnitude of NPQ was significantlyenhanced (Fig. 7). Furthermore, both components of NPQ – qI

(‘photoinhibition’) and qE (‘thermal energy dissipation’) – werealleviated when K was deficient. The enhanced NPQ energydissipation process found under combined K and Na deficiencyprobably compensates for the decrease in photochemical defense

R. Erel et al. / Journal of Plant Physiology 177 (2015) 1–10 9

BAB

A

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

+K +N a -K-Na

NPQ

A

A A

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

+K +N a -K-Na

qE

BAB

A

0.0

0.5

1.0

1.5

2.0

2.5

+K +N a -K-Na

qI

AAB

B

0.70

0.72

0.74

0.76

0.78

0.80

0.82

+K +N a -K-Na

Fv/Fm

F high Ne of thr( ifican

pPte(tTbt

oreimncp

C

rtttpprtRn

cp

ig. 7. Parameters of fluorescence of trees supplied with: no K and Na (−K−Na),

xposure to darkness in July 2013. Each bar represents the average ± standard errorTukey–Kramer multiple comparisons test); different letters indicate statistical sign

rocesses as mentioned above. However, under conditions of highPFD and temperature (a common occurrence in the olive habitat),he effectiveness of energy removal by NPQ is impaired (Eppelt al., 2013). Indeed, Fv/Fm, as a reliable indicator for photodamageLichtenthaler and Burkart, 1999), shows that significant pho-oinhibition is likely to damage PS-II in K-deficient trees (Fig. 7).he lower Fv/Fm in K-deficient trees indicates that the rate ofreakdown of the D1-protein of PS-II exceeds the ability to repairhe photodamage in these trees (Takahashi and Badger, 2011).

To summarize, the photoprotection mechanism of K-deficientlive trees was altered by three main processes: (a) significanteduction of CO2 assimilation, resulting in higher excess exitednergy; (b) reduced photorespiration, the alternative photochem-cal pathway; and (c) an increase in NPQ, a complementary

echanism. The overall balance of these processes results in sig-ificant photodamage to K-deficient plants. Sodium was found toonsiderably modify the negative effect of K deficiency on photo-rotection mechanisms.

onclusions

Inhibition of photosynthesis by poor K nutrition, previouslyeported for numerous other plant species, was demonstrated inhe present study for olive. Photosynthetic impairment was showno be a consequence of both stomatal and non-stomatal limita-ion. However, the extent of non-stomatal limitation was moreronounced, indicating inhibition of enzymatic-photochemicalrocesses resulting from K deficiency. Probable reasons for theeduced photosynthetic capacity are related to lower Chl con-ent and altered enzymatic processes, specifically Rubisco activity.eplacement of K by Na was shown to considerably moderate the

egative effect of suboptimal K.

Olive trees suffering from K deficiency were found to have lowerapability of photoprotection and thus were more susceptible tohotoinhibition. The negative impact of low K was shown to be

a (+Na) and high K (+K) irrigation solutions. Data were collected following 14-hee trees × four leaves (n = 12). The treatment effect was tested by one-way ANOVAce.

related to reduced CO2 assimilation capacity as well as limited pho-torespiration capacity. The lower CO2 and O2 assimilation capacityelevated the amount of excess exited energy and consequentlythe importance of NPQ as an alternative energy dispersion path-way. Nonetheless, K-deficient trees were shown to suffer fromsignificant photodamage to the photosystem. As for photosyntheticcapacity, Na replacement considerably modified the negative effectof K deficiency on photoprotective mechanisms.

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