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EXOGENOUS APPLICATION OF POTASSIUMDIHYDROGEN PHOSPHATE CAN ALLEVIATETHE ADVERSE EFFECTS OF SALT STRESSON SUNFLOWERMuhammad Saeed Akram a & Muhammad Ashraf ba Department of Botany, University of Agriculture, Faisalabad,Pakistanb Department of Botany and Microbiology, King Science University,Riyadh, Saudia Arabia

Version of record first published: 29 Apr 2011.

To cite this article: Muhammad Saeed Akram & Muhammad Ashraf (2011): EXOGENOUS APPLICATIONOF POTASSIUM DIHYDROGEN PHOSPHATE CAN ALLEVIATE THE ADVERSE EFFECTS OF SALT STRESS ONSUNFLOWER, Journal of Plant Nutrition, 34:7, 1041-1057

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Journal of Plant Nutrition, 34:1041–1057, 2011Copyright C© Taylor & Francis Group, LLCISSN: 0190-4167 print / 1532-4087 onlineDOI: 10.1080/01904167.2011.555585

EXOGENOUS APPLICATION OF POTASSIUM DIHYDROGEN

PHOSPHATE CAN ALLEVIATE THE ADVERSE EFFECTS OF SALT

STRESS ON SUNFLOWER

Muhammad Saeed Akram1 and Muhammad Ashraf2

1Department of Botany, University of Agriculture, Faisalabad, Pakistan2Department of Botany and Microbiology, King Science University, Riyadh, Saudia Arabia

� A greenhouse experiment was conducted to examine whether foliarly applied potassium + phos-phorus (K + P) in the form of monopotassium phosphate (KH2PO4) could mitigate the adverseeffects of salt stress on sunflower plants. There were two levels of root-applied salt [0 and 150 mMof sodium chloride (NaCl)], and varying levels of KH2PO4 [(NS (no spray), WS (spray of wa-ter), 5 + 4, 10 + 8, 15 + 12, and 20 + 16 mg g−1 K + P, pH 6.5] applied foliarly to 18-dayold non-stressed and salt stressed sunflower plants. Salt stress adversely affected the growth, yield,photosynthetic capacity, and accumulation of mineral nutrients in the sunflower plants. However,varying levels of foliar applied KH2PO4 proved to be effective in improving growth and yield ofsunflower under salt stress. The KH2PO4 induced growth in sunflower was found to be associatedwith enhanced photosynthetic capacity, water use efficiency and relative water contents.

Keywords: KH2PO4, supplementary K + P, salt stress, mineral nutrients, photosyntheticcapacity

INTRODUCTION

Nutritional imbalance is one of the major adverse effects of salt stress onplants (Ashraf, 1994; Munns, 2005). High amounts of ions such as sodium(Na+) and chloride (Cl−) in the root zone reduce the uptake of someessential nutrients such as nitrogen (N), phosphorus (P), potassium (K),calcium (Ca2+) and magnesium (Mg2+) in plants, resulting in poor plantgrowth (Marschner, 1995; Hu and Schmidhalter, 2005). For example, saltstress caused a significant reduction in N content in different crops such asgreen bean (Pessarakli, 1991), sunflower (Ashraf and Sultana, 2000), tomato(Flores et al., 2000), and Bruguiera parviflora (Parida and Das, 2004).

Received 6 March 2008; accepted 25 July 2009.Address correspondence to M. Ashraf, Department of Botany, University of Agriculture, Faisalabad

041, Pakistan. E-mail: ashrafbot@yahoo.com

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1042 M. S. Akram and M. Ashraf

Similarly, a considerable reduction in the uptake of K+, Ca2+, and Mg2+

was found in Atriplex griffithii var. stocksii due to sodium chloride (NaCl)stress (Khan and Ungar, 2000). A significant reduction in leaf K+ was alsoobserved in the salt stressed plants of sugar beet (Ghulam et al., 2002). De-ficiencies in phosphorus and potassium due to salt stress were observed intomato (Adam, 1991), and cucumber (Sonneveld and Kreij, 1999).

A number of earlier studies show that such salt-induced nutritional defi-ciencies can be overcome by the exogenous application of nutrients eitherthrough the root zone or as a foliar spray. For example, salt-induced re-duction in uptake and translocation of K+ by plants could be alleviated bythe supplementary K+ applied to the growth medium, e.g., in tomato (Sattiand Lopez, 1994; Song and Fujiyama, 1996), maize (Botella et al., 1997),sunflower (Delgado and Sanchez-Raya, 1999), and bean and sunflower(Benlloch et al., 1994).

Grattan and Grieve (1999) were of the view that despite the additionof K fertilizer to the soil, K in soil solution remains relatively low. Thus,it is not feasible to completely correct Na-induced K deficiencies by theaddition of K fertilizers to soil. Under such circumstances, foliar feedingwith macro-nutrients is beneficial in overcoming nutritional deficiencies, ashas already been examined in different crops e.g., in cotton (Howard, 1993;Howard et al., 1998), citrus (Calvert, 1969), avocado (Sing and McNeil,1992), and ‘French’ prune trees (Southwick et al., 1996). Oosterhus (1998)was of the view that foliar feeding of a nutrient may actually promote theroot absorption of the same nutrient. The other beneficial effect of foliarfeeding is that it can reduce the quantity of fertilizer applied to the soil.Furthermore, foliar-applied nutrients are frequently absorbed and utilizedby the plant.

There are some reports which show that foliarly applied K along with Pmitigate the deleterious effects of salt stress on growth and yield of differentcrops. For example, foliar spray of monopotassium phosphate (KH2PO4)alleviated the deficiencies of both P and K in salt stressed strawberry (Kayaet al., 2001a), and tomato (Satti and Al-Yahyai, 1995). Kaya et al. (2001b)found that foliar spray of KH2PO4 alleviated the salt-induced adverse effectson the growth of spinach under saline conditions by correcting Na-inducedK deficiency as well as substantially improving K+/Na+ ratio. Foliarly appliedKH2PO4 was also found to be effective in delaying senescence and increasinggrain yield in winter wheat during hot and dry summers (Sherchand andPaulsen, 1985; Batten et al., 1986). A marked increase in barley yield wasreported due to foliar application of P (Qaseem et al., 1978). Benbella andPaulsen (1998) have shown that foliar application of 5 to 10 kg KH2PO4 ha−1

after anthesis increased grain yield of wheat by up to 1 Mg ha−1.All these reports led us to hypothesize that foliar application of KH2PO4

could mitigate the deleterious effects of salt stress on sunflower plants, par-ticularly under saline conditions, which usually restrict nutrient availability

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Dihydrogen Phosphate and Salt Stress 1043

to plants. Thus, the primary objective of the present study was to uncoverthe physiological phenomena responsible for sustaining growth of KH2PO4

supplied sunflower plants exposed to salt stress.

MATERIALS AND METHODS

The seed of sunflower (Helianthus annuus L.) cv. ‘SF -187’, originallyfrom Monsanto, St. Louis, MO, USA was obtained from the Regional Officeof Pakistan Seed Council, Faisalabad, Pakistan. The experiments were con-ducted under greenhouse conditions during spring 2005 in the BotanicGarden, Department of Botany, University of Agriculture, Faisalabad,Pakistan (latitude 31◦30 N, longitude 73◦10 E and altitude 213 m). Dur-ing the experimentation, the environmental conditions were as follow: dayand night temperatures 30.6 ± 5.1◦C, and 18.3 ± 7.6◦C, respectively, relativehumidity (RH) 35.9 ± 6.5, and the day-length from 10 to 11 h. Achenes ofsunflower were surface sterilized in 5% sodium hypochlorite solution for 10min. before further experimentation. Ten sunflower achenes were directlysown in plastic pots of 28 cm diameter containing 12.5 kg well washed sand,but after germination, seedlings were thinned to three of almost uniform sizein each pot. The experiment was arranged in a completely randomized de-sign with four replicates. All pots were irrigated uniformly with full strengthHoagland’s nutrient solution for 18 days after which time NaCl treatments(0 or 150 mM) in Hoagland’s nutrient solution were begun. Salt solutionwas applied in aliquots of 50 mM every day. Each pot received two liters ofappropriate treatment solution after every week. This volume was sufficientto remove previously present salts. Moisture content of the sand was main-tained daily by adding 200 ml distilled water to each pot. Different levels ofKH2PO4 [(NS (no spray), WS (spray of water + 0.1% Tween-20 solution), 5+ 4, 10 + 8, 15 + 12, and 20 + 16 mg g−1 K + P in 0.1% Tween-20 solution)]were applied foliarly two times to 18-day old non-stressed and salt stressedsunflower plants. First foliar application of KH2PO4 solution was done oneweek after the commencement of salt treatment. The second foliar applica-tion was done one week after the first application. Each KH2PO4 treatmentor blank solution was prepared in 0.1% Tween-20 solution and its pH wasmaintained at 6.5 to ensure the maximum penetration of salt into the leaftissue and to avoid the leaf injury.

Twenty two days after the initiation of salt treatment, the data for gasexchange characteristics, chlorophyll fluorescence, leaf chlorophyll content,and water relation parameters were recorded.

Gas Exchange Characteristics

An open system LCA-4 ADC portable infrared gas analyzer (Analytical De-velopment Company, Hoddeson, England) was used for the measurements

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1044 M. S. Akram and M. Ashraf

of net carbon dioxide (CO2) assimilation rate (A), transpiration rate (E),stomatal conductance (gs), and sub-stomatal CO2 concentration (Ci). Thesemeasurements were made on a fully expanded youngest leaf from 10 AM to 2PM with the following conditions/adjustments of the leaf chamber: leaf sur-face area 11.35 cm2, leaf chamber volume gas flow rate (v) 396 mL min−1,ambient pressure (P) 99.95 kPa, ambient CO2 concentration (Cref) 342.1µmol mol−1, molar flow of air per unit leaf area (Us) 221.1 mol m−2 s−1,temperature of leaf chamber (Tch) varied from 39.2 to 43.9◦C, leaf cham-ber molar gas flow rate (U) 251 µmol s−1, PAR (Q leaf) at leaf surface wasmaximum up to 918 µmol m−2. Water use efficiency (WUE) was calculatedas CO2 assimilation rate/transpiration.

Chlorophyll Fluorescence

The chlorophyll fluorescence was measured with a Plant Efficiency An-alyzer (PEA, Handsatech Instruments Ltd., King’s Lynn, UK) followingStrasser et al. (1995). The transients were induced by red light of 3000 µmolm−2 s−1 provided by an array of six light emitting diodes (peak 650 nm),which focused on the leaf surface to give homogenous illumination over ex-posed area of sample surface. All the samples were exposed to darkness for30 min prior to fluorescence measurements. Maximal quantum yield of PSIIwas calculated as Fv/Fm (Fm = maximum fluorescence with all PSII reactioncenters open; Fv = variable fluorescence).

Water Relation Parameters

Leaf water potential measurements were made with a Scholander typepressure chamber (Arimad-3000, 2 Tokyo, Japan) early in the morning be-tween 6.00 to 8.00 a.m. on a fully expanded youngest leaf excised from eachplant. The same leaf that was used for water potential measurements wasfrozen into 2 cm3 propylene tubes at −20◦C in an ultra-low freezer for twoweeks, after which time the leaf tissue was thawed and extracted by mechan-ically crushing the material. After centrifugation (8000 g) for four min. thesap was used directly for osmotic potential determination in a vapor pressureosmometer (model 5520, Wescor, Logan, UT, USA). The leaf turgor poten-tial was calculated as the difference between osmotic potential and waterpotential values following Nobel (1991) and Taiz and Zeiger (1998).

Photosynthetic Pigments

Chlorophylls a and b were determined following Arnon (1949). Freshleaves (0.2 g) were cut and extracted overnight with 80% acetone at −4◦C.The extract was centrifuged at 10,000 g for 5 min. Absorbance of the super-natant was read at 645 and 663 nm using a spectrophotometer (model 220,

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Dihydrogen Phosphate and Salt Stress 1045

Hitachi, Tokyo, Japan). The concentrations of chlorophylls a and b wereworked out by using the appropriate formulae.

Relative Membrane Permeability

Electrolyte leakage was used to determine relative membrane permeabil-ity (RMP) of the leaf cells following Yang et al. (1996). A number of 1-cm2

discs were cut from a fully expanded young leaf from each plant. These discs(0.5 g) were then placed into test tubes containing 20 ml deionized distilledwater. After vortexing the samples for 3 sec, initial electrical conductivity(EC0) of each sample was measured. The samples were then incubated at4◦C for 24 h and electrical conductivity (EC1) was measured again. The sam-ples were then autoclaved at 120◦C for 15 min, cooled to room temperatureand electrical conductivity (EC2) measured for the third time. The relativepermeability of cell membrane was calculated using the following equation:

Relative permeability (%) = (EC1 − EC0)/(EC2 − EC0) × 100

Growth and Yield

After all the physiological measurements, one plant from each pot washarvested and used for measuring biomass and mineral nutrients of plants,while the remaining two plants were used for the estimation of yield and yieldcomponents. Plant roots were carefully removed from the sand and washedin cold lithium nitrate (LiNO3) solution isotonic with the correspondingtreatment in which plants had been growing. One mM of calcium nitrate[Ca(NO3)2. 4H2O] solution was added to LiNO3 solution to maintain mem-brane integrity. Fresh weights of all shoot and root samples were recorded,and thereafter all these samples were oven-dried at 65◦C for one week anddry weights measured. At maturity, heads of sunflower were removed fromplants and data for achene yield and yield components were recorded.

Inorganic Elements

For determining the concentration of N, P, K+, Ca2+, Mg2+, and Na+ inplant tissues, leaves and roots were washed with double distilled water beforedrying to remove the deposition of foliar applied potassium and phospho-rous as KH2PO4. Then the dried ground leaf and root material (0.1 g)was digested with sulfuric acid and hydrogen peroxide (Merck, Darmstadt,Germany). Cations such as Na+, K+, and Ca2+ in the digests were deter-mined with a flame photometer (model PFP-7, Jenway, Stone, UK). Magne-sium (Mg2+) was determined with an atomic absorption spectrophotometer(Perkin Elmer Analyst 300, Wellesley, MA, USA). Nitrogen was estimated by

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1046 M. S. Akram and M. Ashraf

micro-Kjeldhal’s method (Bremner, 1965). Phosphorus (P) was determinedspectrophotometrically following Jackson (1962). For the determination ofCl− contents, leaf and root samples (100 mg) were ground and extractedin hot distilled water at 80◦C. Cl− content was determined with a chlorideanalyzer (Model 926, Sherwood Scientific Ltd., Cambridge, UK).

Statistical Analysis

A completely randomized design (CRD) with four replicates was usedfor setting up the experiment. The COSTAT computer package (CoHortSoftware, Berkeley, CA, USA) was used for working out two way analyses ofvariance of all variables.

RESULTS

Salt stress (150 mM of NaCl) of the growth medium caused a markedreduction in shoot fresh and dry weights of sunflower plants. However,exogenously applied varying levels of potassium and phosphorus in the formof KH2PO4 improved the shoot biomass of both salt-stressed and non-stressedplants of sunflower. Under non-saline conditions post-hoc analysis of datashowed that shoot fresh weight was improved at all K + P levels, while shootdry weight only at 20 + 16 mg g−1 K + P level applied as a foliar spray(Table 1). In salt-stressed plants, application of 15 + 12 mg g−1 K + P as

TABLE 1 Post-hoc analysis of data when different levels of K + P were applied foliarly to sunflowerplants grown under non-saline conditions

Source of Shoot fresh Shoot dryvariation df weight weight Leaf P Root P Leaf K+ Root K+

No spray vs water sprayK + P 1 9.57ns 0.032ns 0.002ns 0.0001ns 1.53ns 0.281nsError 6 97.363 11.62 0.205 0.61 16.86 9.156

No spray vs 5/4 mg g−1 (K/P)K + P 1 746.3∗ 28.69ns 0.031ns 0.002ns 6.125ns 2.0nsError 6 98.95 13.95 0.204 0.572 16.39 6.25

No spray vs 10/8 mg g−1 (K/P)K + P 1 1066.9∗ 40.59ns 0.039ns 0.075ns 52.53ns 1.531nsError 6 88.53 14.35 0.268 0.958 20.53 5.489

No spray vs 15/12 mg g−1 (K/P)K + P 1 1259.2∗ 29.92ns 0.066ns 0.007ns 16.53ns 7.031nsError 6 144.8 10.45 0.206 0.687 33.74 12.48

No spray vs 20/16 mg g−1 (K/P)K + P 1 1580.6∗ 110.2∗ 0.037ns 0.0177ns 57.78ns 3.781nsError 6 198.6 12.19 0.295 0.567 19.4 4.739

ns = non-significant, ∗ = significant at 0.05 level, K + P = (potassium + phosphorus).

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Dihydrogen Phosphate and Salt Stress 1047

FIGURE 1 Fresh and dry weights of shoots and roots, yield components, and photosynthetic pigmentsof sunflower (Helianthus annuus L.) when varying levels of K + P as KH2PO4 were applied foliarly to18-day old plants subjected to normal or saline conditions (Mean ± S.E values).

KH2PO4 caused a maximum increase in shoot biomass (shoot fresh and dryweights) (Figures 1a and 1b).

Growth medium salinity significantly reduced the root fresh and dryweights of sunflower plants. However, exogenous application of different

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1048 M. S. Akram and M. Ashraf

levels of K and P from KH2PO4 did not alter root fresh or dry weight of bothnon-salinized and salinized plants (Figures 1c and 1d).

Yield and yield components such as achene yield and 100-achene weightwere significantly reduced due to salt stress, but foliar-applied different levelsof K and P as KH2PO4 markedly improved all these yield attributes of bothnon-stressed and salt stressed plants. Highest values of 100-achene weight,achene yield, and number of achenes per plant were recorded in both saltstressed and non-stressed plants at 5 + 4 mg g−1 K + P applied foliarly(Figures 1e, 1f, and 1g).

Salt stress caused a significant reduction in chlorophyll a and b con-tent, but not in chlorophyll a/b ratio of the sunflower plants. Chlorophylla and b contents and chlorophyll a/b ratio in both salt-stressed and non-stressed plants remained unchanged due to exogenously applied KH2PO4

(Figures 1h-j).All gas exchange parameters like net CO2 assimilation rate (A), transpi-

ration rate (E), stomatal conductance (gs), and sub-stomatal CO2 concen-tration (Ci) were significantly reduced due to salt treatment. Varying levelsof foliarly applied potassium (K) + phosphorus (P) caused a significant im-provement in all these gas exchange attributes. Net CO2 assimilation rate(A) was found to be maximum in non-salinized and salinized plants at 10 +8 and 5 + 4 mg g−1 K + P levels, respectively (Figure 2a). Transpiration ratein the non-stressed plants increased consistently with increase in exogenouslevel of KH2PO4, whereas in the salt-stressed plants a maximum increasewas observed at 10 + 8 mg g−1 K + P. In contrast, a slight improvement ings was observed in both salt-stressed and non-stressed plants due to exoge-nous application of KH2PO4 (Figures 2b and 2c). Sub-stomatal CO2 (Ci) inboth salt-stressed and non-stressed plants was enhanced due to exogenousapplication of varying levels of KH2PO4 (Figure 2d). Furthermore, wateruse efficiency (A/E) was also enhanced in both salinized and non-salinizedplants due to exogenously applied KH2PO4. Overall, foliar spray of 10 +8 mg g−1 K + P level was found to be most effective in improving WUE inthe non-stressed plants of sunflower, whereas in the salt-stressed plants therewas an inconsistent pattern of increase or decrease in WUE with increase insupplementary KH2PO4 (Figure 2e).

Root zone salt regime significantly reduced the quantum yield (F v/F m)of photo-system II (PSII). However, application of different levels of KH2PO4

as a foliar spray had a non-significant effect on the photochemical efficiencyof photosystem II (Figure 2f).

Although different water relation parameters (water potential, osmoticpotential and turgor potential) in the sunflower plants were significantly re-duced due to salt stress, no significant effect of exogenous KH2PO4 was ob-served on either parameter (Figures 2g–i). Salt stress of the rooting mediumsignificantly (P ≤ 0.001) decreased the relative water contents (RWC) ofsunflower plants, however, varying levels of KH2PO4 applied foliarly had

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Dihydrogen Phosphate and Salt Stress 1049

FIGURE 2 Gas exchange characteristics, quantum yield (Fv/Fm) of photosystem II, and leaf water poten-tial, osmotic potential, turgor potential, and relative water content of sunflower (Helianthus annuus L.)when varying levels of K + P as KH2PO4 were applied foliarly to 18-day old plants subjected to normal orsaline conditions (Mean ± S.E values).

no significant effect on this attribute (Figure 2j). Although statistically non-significant RWC of the salt stressed plants was consistently improved withincrease in exogenous KH2PO4. Although a marked (P ≤ 0.001) increase inrelative leaf cell membrane permeability was observed due to growth medium

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1050 M. S. Akram and M. Ashraf

salt stress, various levels of KH2PO4 applied foliarly had no significant effecton the salt-stressed or non-stressed plants of sunflower.

As a general phenomenon known in many mesophytes, salt stress signif-icantly caused an increase in leaf or root Na+, Cl−, and a decrease in K+,Ca2+, Mg2+, N and P contents. However, two way analysis of variance andpost-hoc analysis show that effect of external KH2PO4 was non-significant onleaf or root Na+, K+, P, Cl−, Ca2+, Mg2+ and N in the stressed or non-stressedsunflower plants (Table 1; Figures 3a–j and Figures 4a–f).

DISCUSSION

Overcoming salt-induced deficiencies of essential nutrients by foliar ap-plication of N, K, P and Ca is considered as a very viable approach to counter-act the adverse effects of salt stress on plants (Kaya et al., 2001a, 2003; Ikeda etal., 2004; Delgado and Sanchez-Raya, 2007). With this in mind, in the presentstudy it was examined whether exogenously applied varying levels of supple-mentary potassium and phosphorus as KH2PO4 could mitigate the adverseeffects of salt stress on sunflower plants. However, foliar-applied potassium(K) + phosphorus (P) in the form of KH2PO4 improved the growth and yieldof sunflower plants under both non-saline and saline conditions. These re-sults can be related to the earlier findings of Kaya et al. (2001b) in whichit was found that foliar-applied KH2PO4 improved the growth of spinach(Spinacia oleracea) plants under both non-saline and saline conditions. Simi-larly, foliar-applied potassium fertilizers have been beneficial for improvinggrowth and fruit yield of citrus and other fruit crops (Embleton and Jones,1972; Diver et al., 1985).

Of all the major essential nutrients, K has a vital role in plant metabolism(Marschner, 2005). However, supplementary K is known to promote growthand counteract the salt-induced growth inhibition in different crops species,e.g., strawberry (Kaya et al., 2001a), spinach (Kaya et al., 2001b), cucum-ber and pepper (Kaya et al., 2003), and rice (Ikeda et al., 2004). Further-more, Din et al. (2001) found that foliar spray with K2SO4 counteractedsalt-induced inhibition in growth and yield of rice plants.

Like K, exogenous application of phosphorus is also effective in promot-ing growth and yield of salt-stressed plants of different crops, e.g., tomato(Bernstein et al., 1974; Cerda and Bingham, 1978), and certain cereal andpasture plants (Manchanda et al., 1982; Gibson, 1988). However, in thepresent study a significant improvement in growth and yield was observedby the application of KH2PO4 in sunflower plants, but it is not possible todifferentiate whether the improved growth was due to only K or both K andP. Thus, a further study needs to be carried out to differentiate how far K orP could contribute to growth improvement in sunflower plants.

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Dihydrogen Phosphate and Salt Stress 1051

FIGURE 3 Na+, K+, P, Cl− and Ca2+ concentrations of leaf and root of sunflower (Helianthus annuusL.) when varying levels of K + P as KH2PO4 were applied foliarly to 18-day old plants subjected to normalor saline conditions (Mean ± S.E values).

In the present study, photosynthetic rate was increased with increasein the level of exogenously applied KH2PO4. Thus, KH2PO4-induced im-provement in growth and yield of sunflower could be related to enhancedphotosynthetic efficiency (A). Such a positive relationship between growthand photosynthesis has already been found in different crops subjected to

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1052 M. S. Akram and M. Ashraf

FIGURE 4 Mg2+ and N concentrations and K+/Na+ ratios in the leaves, roots and relative permeabilityof sunflower (Helianthus annuus L.) when varying levels of K + P as KH2PO4 were applied foliarly to18-day old plants subjected to normal or saline conditions (Mean ± S.E values).

salt stress e.g., sugar beet (Terry and Ulrich, 1973), cotton (Longstreth andNobel, 1979; Bednarz et al., 1998), and almond (Basile et al., 2003). Increasesin photosynthetic rate due to foliar application of K + P levels might havebeen due to up-regulation of stomatal or non-stomatal factors (Brugnoliand Bjorkman, 1992; Athar and Ashraf, 2005; Dubey, 2005). While workingwith cotton, Bednarz et al. (1998) concluded that under mild K deficiency

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Dihydrogen Phosphate and Salt Stress 1053

stomatal limitations are the major factors affecting the photosynthetic rate,whereas metabolic limitations become a dominant limiting factor under se-vere K deficiency. Likewise, Basile et al. (2003) reported that reduced Kavailability in soil caused a reduction in leaf K and affected the photosyn-thetic capacity via biochemical limitations. Thus, the results from the presentstudy suggests that the increase in photosynthetic rate due to exogenous ap-plication of K + P may have been due to some metabolic factors includingphotosynthetic pigments, concentration and activity of rubisco, supply ofATP and NADPH to photosynthetic carbon reduction (PCR) cycle and useof assimilation products (Lawlor and Cornic, 2002; Athar and Ashraf, 2005).

In the present study, exogenously applied K + P caused an increase inwater use efficiency of both salt stressed and non-stressed plants. Other waterrelation parameters such as leaf water potential and relative water contentwere also increased in the salt stressed sunflower plants due to exogenousapplication of K + P. This could have been due to the vital role of K+ asa major osmoticum in the vacuole, for maintaining high tissue water con-tent under stressful environment (Marschner, 1995). In view of some recentreports, stomatal regulation largely depends upon the distribution of K inepidermal cells, guard cells and leaf apoplast (Shabala et al., 2002). Theregulation of voltage-dependent K+-selective inward (KIR) and outward rec-tifying channels (KOR) present at plasma membrane of guard cells, play animportant role in K+ transport, which in turn results in stomatal regulation(Pilot et al., 2001; Schroeder et al., 2001; Shabala, 2003). In addition tospecific K+-selective channels, guard cells also possess a wide range of non-selective cation channels (NSCC). These channels are likely to be involvedin release of solutes during osmoregulation (Demidchik et al., 2002). Thereare some reports which show that gene expression of all these channels ischanged due to salt stress (Golldack et al., 2003; Pilot et al., 2003), as hasbeen earlier observed in the leaf epidermis of Arabidopsis (Dennison et al.,2001) and ice plant (Su et al., 2002).

Generally, plant water status is maintained by regulating stomatal con-ductance and transpiration rate. In the present study, salt stress reducedboth stomatal conductance and transpiration rate. However, varying levelsof KH2PO4 applied foliarly caused an increase in transpiration rate in the saltstressed plants, but stomatal conductance remained unaffected. From theseresults, it is obvious that changes in plant water status in the salt stressedplants may have been due to some factors other than stomatal regulation.Recently, gene ERECTA (a putative leucine rich repeat receptor-like kinase)has been identified in Arabidopsis, which is responsible for directly regulatingplant transpiration efficiency (Masle et al., 2005). In view of the roles of Kin activation of various enzymes and channels, a possible activation of suchtype of kinase (ERECTA) by K cannot be ruled out. It has been reported thathigh transpiration rate increases salt stress susceptibility in plants (An et al.,2001; Li et al., 2001), but in the present study, such an increase in Na+ or Cl−

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accumulation in the leaves of salt stressed plants supplied with increasinglevel of KH2PO4 has not been observed, particularly in those plants showinghigh rates of transpiration. These results are in parallel to those of An et al.(2001) who have shown that Na+ translocation to shoot was independent ofthe transpiration rate in a salt tolerant cultivar of soybean.

Salt stress causes perturbance in ion homeostasis, which results in os-motic stress and ion toxicity, both of which can generate reactive oxygenspecies (ROS). The ROS can trigger phytotoxic reactions such as lipid per-oxidation of membranes resulting into increased cell membrane permeabil-ity and ion leakage (Mittler, 2002; Sairam et al., 2002; Kukreja et al., 2005).While evaluating salinity tolerance of different wheat cultivars, Sairam etal. (2002) and Farooq and Azam (2006) reported that cell membrane per-meability could be used as one of the potential indicators of salinity tol-erance. In the present study, salt stress increased cell membrane perme-ability and ion leakage in the sunflower plants, but foliar spray with dif-ferent doses of potassium dihydrogen phosphate did not affect cell mem-brane permeability. These results are not in agreement with the earlierfindings of Kaya et al. (2001b) in which it was observed that foliarly applied5 mM KH2PO4 reduced the ion leakage in spinach grown under salineconditions.

Salt-induced reduction in leaf or root K+ in the sunflower plants wasnot ameliorated by varying levels of leaf-applied KH2PO4. These results arein contrast with Satti and Al-Yahyai (1995) who showed that application ofadditional P and K in nutrient solution corrected P and K deficiencies intomato. Similarly, working with tomatoes, Chapagain (2001) and Chapagainand Wiesman (2004) found that foliar spray of 2% KH2PO4 or 1% KH2PO4

increased leaf K+, and fruit yield and quality of greenhouse tomatoes. Theresults for tissue ion contents show that salt stress increased the accumulationof Na+ and Cl− but decreased that of N, P, K+, Ca2+ and Mg2+ in thesalt stressed sunflower plants. However, leaf-applied varying levels of K + Pshowed non-significant effect on leaf or root K+ and P contents in the saltstressed plants.

Overall, exogenously applied KH2PO4 improved growth and acheneyield of salt-stressed sunflower plants. The improved growth of salt-stressedsunflower plants due to exogenous application of KH2PO4 was found to beassociated with enhanced photosynthetic capacity, water use efficiency andrelative water content.

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