PHYSIOLOGIA PLANTARUM 96: 722-726. 1996 c.pyrtsk, s ™,,™/osi. «a,,»n™ 1996Primed in Denmark - ail rights reserved SSSN 003i-9^17

Effect of sulphate on photosynthesis in greenhoese-growntomato plants

H. L. Xu, J. Lopez, F. Rachidi, N. IVemblay, L. Gauthier, Y. Desjardins and A. Gosselin

Xu, H. L., Lopez, J,, Rachidi, F,, Tremblay. N,, Gauthier, L., Desjardins, Y, and Gosse-lin, A, 1996. Effect of sulphate on photosynthesis in greenhouse-grown tomato plants- Physiol. Plant. 96: 722-726.

Sulphate accumulates in the rhizosphere of plants grown in hydroponic systems. Toavoid such sulphate accumulation and promote the use of environmentally sound hy-droponic systems, we examined the effects of four sulphate concentrations (O.i, 5.2,10.4 and 20.8 mM) on photosynthesis, ribulose-l,5-bisphosphate carboxylase/oxygen-ase (Rubisco, EC 4.1.1.39) activities and related physiological processes in green-house-grown tomato plants (Lycopersicon esculentum Mill. cv. Trust). The lowest sul-phate concentration (0.1 TnM) significantly decreased photosynthetic capacity (Pc) andRubisco activities on a leaf area basis. This result was supported by our data for drymatter per plant, which was low for plants in the 0.1 mM treatment. The photosynthe-sis-related variables such as leaf conductance, chlorophyll and soluble protein werelowest for the O.I mM treatment. Both total Rubisco activity and the activated ratiowere reduced with this treatment. However, Rubi,sco activities expressed per g of pro-tein or per g of chlorophyll were not significantly affected. These results suggest thatsulphur deficiency depressed Pt- by reducing the amount of both Rubisco an"d chloro-phyll and by causing an inactivation of Rubisco, The ratio of organic sulphur vs or-ganic nitrogen (S/N) in plants of the 0.1 nuM treatment was far below the normal val-ues. This low S/N ratio might be accountable for the negative effect of low sulphateon Prand plant growth. P , and dry matter were not affected until sulphate concentra-tion in the nutrient solution reached a high level of 20.8 mM.

Key words - Lycopersicon esculentum, photosynthesis, Rubisco, sulphate, tomato,

H. L. Xu, J. Lopez, L. Gauthier, Y. Desjardins and A. Gosselin (corresponding author,fax I-418-6567056), Horticulture Research Centre, Faculty of Agriculture and Food,Laval Univ., PQ, GJK 7P4, Canada; F. Rachidi, Meknes National Agrlculiure Insti-tute, Maroc;N. Tremblay, Agriculture and Agri-Food Canada, Si-Jean sur-RichelieuPQ, J3B 3E6, Canada.

Introduction Althotigh some of the reports have described the ef-fects of sulphur on growth and yield, there have been

The use of greenhouse hydroponic culture as a modem very few studies on the physiological effects of sulphateapproach to plant production has spread throughout the concentration on tomato plants. Indeed, no study ofworld in recent years. In practice, some ions, especially greenhouse-grown tomato has been conducted on the ef-sulphate, accumulate in the rhizosphere of plants grown feet of sulphate concentration on photosynthesis and rib-in hydroponic systems (Sonneveld and Van de Burg ulose-l,5-bisphosphatecarboxyiase/oxygenase (Rubisco),!99I, H. Zekki, L. Gauthier and A. Gosselin, unpub- the key enzyme in photosynthesis. The optimal amountlished data). Sulphate is an accompanying ion in K, NH4, of sulphate is not well known. It is therefore necessary toMg, Ca and Fe fertilisers. High concentrations of sul- examine the effects of both sulphur deficiency and ex-phate in the nutrient solution have been linked to leaf cess in the rhizosphere on the physiology of tomatochlorosis (Boivin 1990), blossom end rot (Nukaya et al. plants. The present study was aimed at determining the1992) and growth reduction of tomato (Cerda et al. effect of sulphate concentration on photosynthesis,1984). Rubisco activities and related physiological variables.

Received 11 July, 1995: revised 13 November, 1995

"^22 Physiot. Plant. 96, 1996

Tab. 1. Sulphate concentration, mineral composition, pH and EC inUie nutrient solutions of four sulphate treatments.

Element

Sulphate

NitrogenPhosphorusPotassiumCalciumMagnesiumIronManganeseZineBoronMolybdenumSodiumChloride

pHEC

S-!

0.1

16.01.69.09.04.00.07O.OI0.0070.020.00071.6

12.2

6.03,6

Concentration (niAf)

S-2

5.2

17.01.69.09.04.00.070.010,0070,020,00073,88,1

6,03,8

S-3

10.4

16.01.69.09.04.00.070.010.0070.020.00074.01.5

6.03.5

S ^

20.8

15.01.6

13.713.76.00.070.010.0070.020.00075,93,8

6,04,8

Abbreviations - EC, electrical conductivity of the nutrient solu-tion: gL, leaf conductance: K s, Michaelis-Menten constant: Pc,photosynthetic capacity: PN, net photosynthetic rate: QUE,quantum use efficiency: RD, dark respiration rate: Rubisco, ribu-lose-3,.5-bisphosphate carboxylase/oxygenase: RuBP, ribulose-1,5-bisphosphate: TL, leaf water potential.

Materials and methodsPlant material and treatments

Tomato plants {Lycopersicon esculentum Mill. cv. Trust)were sown on 24 December, 1993, in small cubes ofrockwool and were subsequently transplanted into largerrockwool blocks (7.5 x 7.5 x 6.5 cm; Grodan, Milton,Canada) on 19 January, 1994. Supplemental lighting of100 jtmol m"'s~' was supplied by high pressure sodiumlamps (BSHPCC-1000-120, Lumiponic, Inc., Montreal,PQ, Canada) to transplants over a 16-h photoperiod dur-ing the growing period. The seedlings were transplantedinto large rockwool blocks (15 x 90 x 7.5 cm [Grodan])at a density of three plants per m" in the greenhouse. Be-fore treatments started, plants were irrigated with a0,5x nutrient solution (10,2 N, 1.3 P; 4.9 K, 3,5 Ca, 1.2Mg, 0.007 Fe, 0.01 Mn, 0.003 Zn, 0,001 Cu, 0.01 B,0,0006 Mo, 0.8 Na, 0.9 C! and 1.4 SOf in mM). From24 February onward, plants were treated with four sul-phate concentrations in the nutrient solutions preparedwith distilled water (0.1, 5.2, 10.4 and 20.8 mM; Tab. 1).The same nutrient solution was used for the whole ex-periment period. There were no large fluctuations in nu-trient concentration around the root since the solutionwas drained (not recycled) and replaced by new solutionevery day. Plants were arranged in a randomised com-plete block design with four replications. Each experi-mental unit contained two rows of 9 plants. Plants wereirrigated four times a day (0600, 1000, 1400 and 1800 h)from a drip irrigation system controlled by a lysimetrictimer (Industries Hamois, Quebec, PQ, Canada), Drain-age (runoff) volume varied between 20 and 25% of theirrigated solution volume.

Measurement and analysis of photosynthesis

Photosynthesis was measured by a gas phase oxygenelectrode system (Hansatech LD2, Hansatech Instru-ments Ltd, Kings Lynn, Norfolk, UK) according toWalker (1987). A whole leaf blade was cut under waterin the greenhouse and immersed in water in a shallowpack. A leaf disc with an area of 10 cm"' was cut fromthe leaf blade under water and set in the leaf chamber formeasurement. The temperature of the leaf sample cham-ber was controlled at 20° C by circulating water from atemperature-controlled water bath (Model-LTD6 GrantInstruments Ltd, Cambridge, UK). Measurements wereconducted at different levels of photosynthetic photonfiux (PPF) to obtain a light-response curve. A rectangu-lar hyperbolic model, F-^ = Pc KM i {I + KM i )"' - RD,was used to fit the photosynthetic light-response curve(Xu et al. 1994), where P^ is the net photosynthetic rateat a given PPF (i); Pc is the gross photosynthetic capac-ity at the maximum PPF and the value of P c - RD is theasymptote of the light-response cur\'e; KM is the Michae-lis-Menten constant and equals the reciprocal of the PPFat which P^ = 0.5Pc; RD is the dark respiration rate. Thevalue of PCKM shows the maximum quantum use effi-ciency when PPF tends to 0.

Determination of Rubisco activities

Two leaf discs each with an area of 10 cm" were excisedas one sample from the top leaflet of the 5th leaf in thesun at midday and immediately put into liquid nitrogen.The frozen leaf discs were ground itito fine powder inliquid nitrogen and extracted with 5 ml of a buffer solu-tion containing 50 mM Bicine (pH 8.2), 20 mM KCl, 5mM dithiothreitol (DTT), 0.1 mM sodium ethylendi-amitietetraacetate (Nai-EDTA), 2% (w/v) polyvinyl-pyr-rolidone (PVF), 0.1 mM phenylmethylsulphonyl fluo-ride (PMSF) and 5 pM leupetin. Half of the extract wasremoved for initial activity. The retnainder was used formeasurements of total activity. Initial Rubisco activitywas measured at 23°C by injecting 50' [ll of leaf extractinto an assay buffer (400 jjl) containing 100 mM Bicine(pH 8.2), 20 mM MgCU, 5 mM DTT and 200 pMNa'^COj. The reaction was terminated after 30 s by add-ing 200 ttl of 2 M HCl. The samples were dried at 90°Cfor 3 h and then the acid-stable '*C was determined byliquid scintillation counting. Total Rubisco activit)' wasdetermined in a similar way except that the assay bufferwas incubated at 23 °C for 5 min before 50 fil of 20 mMribulose-1,5-bisphosphate (RuBP) was added (Jiang etal. 1993, Sharkey 1986).

Measarement of soluble protein, chlorophyll, nitrogen andsulphur contents

Protein content was measured by the Bradford (1976)method. Chlorophyll content was measured with 80%acetone as the extraction agent according to Hokujoh

Physiol, Planl. 96, 1996 723

Tab. 2. Effecss of sulphate concentration {mM) in the nutrient solution on Pc, KM, QUE, photosynthetic rate at 680 (imol m ' s ' of PPF(Psso). RD and dry matter of greenhouse-grown tomato plants. *, **, and NS mean significances at P < 0.05, P < 0.01 and no significance,respectively. The valaes followed by the same letter are not significantly different from each other at /• < 0.05.

Sulphate

(miif)

0.15.2

10.420.8

LinearQuadraticCubic

Pc(jimol m ^ K ')

16.1a20.4b21.0b17.6ac

NS

NS

(lO^m'smol ')

0.99a1.23b1.25b1.27b

**

NS

QUE(mol mol ')

0.159a0,251b0,260b0,223b

P.»o

(jimol t

12.9a16.4b17.0b14.0ac

*

NS

RD

-1.66a-2.36b-2.15b-2.10b

: :

DM

(g plant-')

84,2a96.1b94.3b97.ib

NSNS

and Ishitstika (1988). Leaf nitrogen content was tnea-sured by automatic colourimetry (TRAACS 800, Bran-Luebbe, Inc., New York, NY, USA) at the Agricultureand Agri-Food Canada Laboratory at St-Jean sur-Riche-lieu, Quebec, Canada, Sulphur content of leaves wastneasured by atotnic absorption spectrophotometry(Model ICAP 9000, Jarrel-Ash, Waltham, MA, USA) atthe Rock Forest Laborator)' of the Quebec Ministry ofAgriculture, Fisher}' and Food.

Measurements of leaf conductance in situ and leaf waferpotential

Leaf conductance for vapour diffusion in situ of the 5thleaf in the sun at midday was measured with a porometer(Hansatech Delta AP-4) in undisturbed conditions. Theleaf conductance for vapour diffusion was converted toleaf conductance for CO, diffusion by a coefficient of0.6. Leaf water potential was measured according toTurner (1988) by a pressure chamber (Model 3000, Soil-moisture Equipment Corp., Santa Barbara, CA, USA).The 5th leaf was cut with five leaflets attached, immedi-ately put into a poly vinyl bag and then placed in thepressure chamber. The rate of pressure application wasrelatively fast at the beginning of the measurement andthen slowed to 0.01 MPa s"' when the presstire was closeto the level of leaf water potential. When water exudedfrom the xylem and saturated the cut end of the petiole,pressure application was stopped. The negative value of

the balance pressure was then assumed to be the leaf wa-ter potential.

Statistical analysis

Data were analysed using the general linear model pro-cedures of the statistical analysis system (SAS 1985).Linear, quadratic and cubic models were assessed forsignificance to show the response trend to increasingsulphate concentration (Little and Hill 1978). TheWaller-Duncan comparison test was also performed toshow the difference among treatments.

Results and discussionPhotosynthetic capacity, quantum use efficiencj and darkrespiration

Pc was decreased by 23% with the lowest concentra-tion of sulphate (0.1 mM) compared to that at 10.4 mM(Tab. 2). The photosynthetic rate measured at tlie PPFof 680 pmol m"* s"' (Peso) showed the same trend as thatof Pc. However, when sulphate concentration increasedfrom 10.4 to 20.8 mM, Pc and Ptso decreased slightly(Tab, 2). The KM and the quantum use efficiency(QUE=KMPC) were also the lowest in plants of the 0.1mM treatment. Among the other three treatments, therewas no significant difference in K^, although QUE de-creased slightly when sulphate concentration was in-creased from 10.4 to 20.8 mM (Tab. 2). Dark respira-tion rate (RD) was low for the 0.1 mM treatment, but

Tab. 3. Effects of sulphate in the nutrient solution {m.¥) on gL, ^i., chlorophyll content (Chi), soluble protein content, leaf content of nitro-gen (N) and sulphur (S) of greenhouse-grown tomato plants. *, **, and NS mean significances at P < 0,05, at P < 0.01 and no significance,respeclively. The values followed by the same letter are not significantly different from each other at P < 0.05.

Sulphate

{mM)

0,15.2

10.420.8

LinearQuadraticCubic

(MPa)

-0.55a-0.61b-0.63b-0.65b

NSNS

gl.

( l O ' m ' s " ' )

0,86a1,14bI,30b1.34b

*

NSNS

Chlfl

215a273h273b259b

*

cmt

(mgm-^)

77a121b128b127b

NS

CUa+b

292a394b401b386b

***

Chlfl/b

2.7a2.2b2.1b2.0b

**

NS

Protein

(g r

5.52a6.69b6,94b6,92b

NS

Total N

n-)

1.38a1.56b1.55b1.69b

:

Protein N

• ( % )

64.1a68.8b71.6b65.6ac

NS

NS

S

(mg m *)

52a381b426b447b

NS

724 Physiol, Plant, 96, 1996

Tab. 4. Effects, of sulphate concentration in the nutrient solution on Rubisco activities of greenhouse-grown tomato plants.

Sulphate(mM)

Rubisco activities (timol OT S ') Activated ratio

Perm-of LA Per g of protein Per g of Chi

0.1MM

10.420.8

LinearQuadraticCubic

Initial

40.4a50.3b51.8b49.3b

^*

NS

Total

53.1a57.8b62.4b60.2b

NS

IniuaJ

7.31a7.56a'7.47a7.12a

NSNSNS

TotaJ

9.60a8.64a9.00a8.69a

NSNSNS

Initial

138a128b129b128b

MSNS

Total

182a147b159b156b

*

76.1a87.0b83.1bc8L9c

* ? ) '

imNS

was not significantly different atnong the other threetreatments (Tab, 2).

Plant growth

Dry matter per plant was significantly lower in sulphtir-deftcient plants (Tab. 2). However, there was no differ-ence in plant dry weight atnong the other three treat-nnents. Although Pc was slightly lower with 20.8 mMsulphate cotnpared to that with 10.4 rrLW. dry matter perplant was not lower with 20.8 lnM than with 10.4 mM.

Leaf conductance and leaf water potential

Leaf conductance for CO, diffusion ( g j was the lowestfor tiie 0.1 mM treatment (Tab. 3), which was consistentwith the lowest Pc in this treattnent. However, gL wasnot lower at 20.8 than at the 10,4 mM treatment, as wasPc. These restilts suggest thai low PQ in the high sulphatetreatment was not attributable to stomatal functions.Leaf water potential (fi) was slightly higher in plants ofthe 0.1 mM treatment than in plants of the other threetreatments (Tab. 3), Although the electrical conductivitywas relatively high in the nutrient solution of the 20.8mM treatment, T L was not significantly lower than thatin plants treated with 5.2 mM and 10.4 nM sulphate.

Leaf content of chlorophyll, soluble protein, nitrogen andsulphur

Chlorophyll a and b ievels were lowest in the 0,] mMtreatment and this was consistent with the values of Pc(Tab. 3), Low chlorophyll levels were confirmed by theyellow leaf colour in 0.1 mM treatment (data nolshown). There have been other reports showing leaf de-coloration by sulphur deficiency although chlorophyllwas not quantified (Cerda et al. 1984, Rennenberg1984). Direct eifects of sulphur on chlorophyll synthesishave not been demonstrated.

Soluble protein content in the leaf and total leaf nitro-gen content were lowest for the 0.1 tnM treatment. Theratio of soluble protein niiiogen to total nitrogen wasalso lower for the 0.1 mM treatment than for tbe others(Tab. 3). In tomato leaves, organic sulphur is 17 ± 4% of

total sulphur under a normal sulphate supply, while or-ganic nitrogen is 80% of total nitrogen (Cerda et al.1984). By calculation, in our experiment, the ratio of or-ganic sulphur vs organic nitrogen (S/N) reached 0.052for the 5.2 tnM ti'eatinent. However, this ratio in leavesat the 0.1 mM treatment was only 0.008, which was farbelow the documented normal values.

Rubisco activities

Initial and total Rubisco activities based on leaf areawere the lowest for the 0.1 mM treatment (Tab, 4). Notonly total Rubisco activity but also the activated (initial/total) ratio was reduced with this treatment. However,Rubisco activities expressed per g of protein or per g ofchlorophyll were not low in this treatment as comparedto those of the other three.

hi the present experiment, unfortunately, we did notmeasure the exact amount of Rubisco protein using anti-bodies. However, soluble protein content shows the rela-tive value of the amount of Rubisco because 50-75% ofsoluble protein is Rubisco (Hartman and Harpel 1994).Sulphur deficiency decreased P^ possibly through a re-duction in synthesis of soluble protein and chlorophylland an inactivation of Rubisco since Rubisco activitiesexpressed per g of protein or per g of chlorophyll werenot lower for the 0.1 rruV/ treatment than for the otherthree. Ferreira and Teixeira (1992) have reported that forLemna plants sulphur starvation caused a decrease inprotein synthesis and a preferential degradation ofRubisco. As documented, besides the requirement of el-emental sulphur for protein structure, protein synthesisalso needs an appropriate ratio of S/N (Rennenberg1984), A low S/N ratio in the presettt study might ac-count for the negative effect of low sulphate on Pc,Rubisco activit}', protein content and plant growth. Re-sults of the treatment effect of high sulphate concentra-tion showed that tomato plants are tolerant to excessivesulphate to a very high level. The slight negative effectof high sulphate concentration on tomato photosynthesiswas possibly not related to high electrical conductivity-induced water stress and might be attributed to an indi-rect effect of the osmotic stress or to a specific effect ofsulphur.

Physiol.,Piant. 96.1996 725

Acknowledgments - The authors thank Dr Serge YeUe as, well asDr Binh Nguyen-G. Savita Visal, Cinta Sebastia-H and SimonRoy for their technical advice and assistance. Research was sup-ported by Quebec Ministry of Education, Quebec Ministry ofAgriculture,, Natural Science and Engineering Research Councilof Canada, and Osram Sylvania Co. Ltd. Laval University con-tribution CRH-169,

ReferencesBoivin, C. 1990. La recirculation des solutions nutritives en cul-

ture sur laine de roche. - Cahier des conferences. 9emejoumee d'information sur les cultures abritees. pp. 76-80.SB 414-64-J86. Quebec, Canada,

Bradford, M. M. 1976, A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of prolein-dye binding. - Anal. Biochem. 72: 248-254,

Cerda, A,, Martinez, F. & Caro, M, 1984. Gro,wth of tomatoplants as influenced by sulphate and phosphorus concentra-tions in the root media. - Vlth Int. Colloq. OptimizationPlant Nutr. 1: 109-115,

Ferreira, R, B. M. & Teixeira, A. R. N. 1992. A preferential deg-radation of ribulose-bisphosphate carboxylase without plantdeath, - J . Biol. Chem, 267: 7253-7257.

Hartman, F. C. & Harpel, M. R. 1994. Structure, function, regu-lation, and assembly of D-ribQiose-1,5-bisphosphate carbox-ylase/oxygenase, -Annu . Rev. Bioehem, 63: 197-234.

Hokujou, Y. & Ishitsuka. J, 1988. Experimental Methods inCrop Physiology. - Jpn. Assoc, Agric. Technol, (Tokyo), pp,337-346. ISBN 4-88979-019-5,

Jiang, C. Z,, Rodermel, S. R, & Shihles, R, M, 1993. Photosyn-thesis, Robisco activity and ainounl, and their regulation bytranscription in senescing soybean leaves. - Plant Physiol.]01: 105-112.

Liule, M. T, & Hill, F. J. 1978, Agricultural Experimentation,Design and Analysis. - John Wiley and Sons, Inc., NewYork, NY. p, 294. ISBN 0-471-05372-4,

Nukaya, A., Voogt, W. & Sonneveld, C. 1992. Effects of NO,,S64 and Cl ratios on tomatoes grown in a recireulation sys-tem, - Acta Hortic, 303: 91-98.

Rennenberg, H. 1984. The fate of excess sulphur in higherplants. - .Annu. Rev. Plant Physiol. 35: 121-153.

SAS, 1985. SAS User's Guide: Statistics, Version 5. - SAS In-stitute, h ic , Caiy, NC. ISBN 0-917382-66-8.

Sharkey, T. D, 1986. Regulation of ribulose-I,5-bisphosphatecarboxylase activity in response to changing partial pressureof O, and lighl in Phaseolus vulgaris. - Plant Physiol. 81:788-791.

Sonneveld. C. & Van de Burg, A. M. M. 199!. Sodium chloridein fruit vegetable crops in soilless culture. - Neth, J. Agric.Sci. 39: 115-122.

Turner, N. C. 1988. Measurement of plant water status by thepressure chamber technique, - Irrig. Sci. 9: 115-122.

Walker, D. 1987. The Use ofthe Oxygen Electrode and Fluores-cence Probe in Simple Measurements of Photosynthesis. -Oxygraphics, Norfolk, pp, 1-16. ISBN 1-870-23200-3.

Xu, H. L,, Gauthier, L. & Gosselin, .A. 1994. Photosynthetic re-sponses of greenhouse tomato plants to high solution electri-cal conductivity and low soil water content. - J. Hortic. Sci.69:821-832.

Edited by H. E. Sommer

726 Physiol, Plant. 96, 1996