REGULAR ARTICLE

Improving NaCl resistance of red-osier dogwood: roleof CaCl2 and CaSO4

Sylvie Renault & Maha Affifi

Received: 20 February 2008 /Accepted: 23 July 2008 / Published online: 14 August 2008# Springer Science + Business Media B.V. 2008

Abstract The influence of Ca2+ salts on the resistanceof red-osier dogwood (Cornus sericea) seedlings tosalinity was investigated. Red-osier dogwood seed-lings were exposed to 5 and 10 mM of CaCl2 orCaSO4 in the presence or absence of 50 mM NaCl for40 days in a controlled environment. Seedlingsexposed to CaCl2 and CaSO4 recovered from NaCl-induced transpiration reduction after 20 days at aconcentration of 10 mM and after 30 days at aconcentration of 5 mM; while in absence of additionalCa2+, the seedlings recovered only after 40 days.Addition of 10 mM Ca2+ to NaCl treatment alsolimited the accumulation of proline in leaf tissues andcaused an increase in leaf and lateral shoot K+ content.These results suggest that 10 mM Ca2+ could alleviate,at least in part, the osmotic effect of NaCl on red-osierdogwood via control of stomatal closure. On the otherhand, ion analysis showed that Ca2+ addition was ableto reduce the NaCl-induced Na+ concentration only instem tissues suggesting that Ca2+ had only a limitedeffect on the ionic stress. The present study alsoshowed an unexpected NaCl-induced increase in Ca2+

content of leaves, lateral shoots and stems that was notobserved in our previous hydroponics experiments andseems to be more characteristic of plants growing onsandy soils.

Keywords Cornus sericea . Resistance . Salinity .

NaCl . Ca2+ salts

Introduction

Calcium plays an important role in plant survival ofsalinity stress by increasing the resistance to the stress(Greenway and Munns 1980; Hasegawa et al. 2000).Studies have shown that Ca2+ can reduce the negativeeffect of salinity on plants and alleviate the growthinhibition mainly by mitigating the ionic effect ofsalinity rather than the osmotic effect (for review seeRengel 1992). Supplemental Ca2+ can maintainmembrane integrity in both roots and shoots bylimiting Ca2+ displacement from the membrane byNa+ ions (Cramer et al. 1985; Läuchli 1990). Sodiumuptake can also be reduced and K+ uptake increasedby the presence of Ca2+ in the soil solution (Cramer etal. 1985, 1987). Furthermore, Na+ binding to the cellwall can be limited by addition of Ca2+ (Stassart et al.1981; Kurth et al. 1986). However, the responses varydepending not only on plant species but also on thesalts (NaCl or Na2SO4) and the source of Ca2+ ions(CaCl2 or CaSO4) (Volkmar et al. 1998; Caines andShennan 1999).

Plant Soil (2009) 315:123–133DOI 10.1007/s11104-008-9737-7

Responsible Editor: John McPherson Cheeseman.

S. Renault (*) :M. AffifiDepartment of Biological Sciences,University of Manitoba,Z320 Duff Roblin Building,Winnipeg, MB R3T 2N2, Canadae-mail: renaults@cc.umanitoba.ca

Most of the research on Ca2+ and salinity interac-tions has focused on agricultural crop species. Red-osier dogwood (Cornus sericea) is a woody plantwidely distributed across North America. It is a cold-and flood-tolerant species that can also grow on drysites. This species is an excellent soil stabilizer that haspotential for early stages of reclamation of some salt-affected areas in Western Canada as a result of oil sandmining (Renault et al 2001). Our earlier studies(Renault et al. 2001; Renault 2005) have shown thatNa+ was preferentially stored in the root tissues of red-osier dogwood exposed to NaCl and Na2SO4 suggest-ing that the seedlings could control the transport ofNa+ from root to shoot thus limiting shoot injury. Wefound that the negative effects of 50 mM Na2SO4 onshoot height and stem cell wall dry weight of red-osierdogwood could be alleviated by the presence of 5 mMCaCl2 (Renault 2005). However, our results haveshown that 50 mM Na2SO4 caused a more severereduction in root and shoot dry biomass of red-osierdogwood than equimolar Na+ concentration from NaCl(Renault et al. 2001). Therefore, it cannot be speculat-ed that the interaction of NaCl and various Ca2+ saltswill be similar to a Na2SO4 and Ca2+ interaction.

The objective of this study was to determine theeffects of CaCl2 and CaSO4 (5 and 10 mM) on thegrowth and ion content of the woody species red-osierdogwood exposed to 50 mM NaCl. It has beensuggested that additional Ca2+ alleviates more theeffects of ionic stress than osmotic stress. To validatethis hypothesis, we measured transpiration rates andproline content of red-osier dogwood leaves of plantexposed to NaCl in presence and absence of Ca2+.

Material and methods

Plant material and treatments

Red-osier dogwood (Cornus sericea L syn. Cornusstolonifera Michx) seeds were collected in northernAlberta, Canada (57°01.67′ N, 111°30.60′ W) inproximity to oil sands mining operations north ofFort McMurray. After 6 weeks of stratification at 4°C,seeds were sown in a mixture of peat moss and sand(3:1, v/v). Plants were fertilized monthly prior to thetreatment with N/P/K (20:20:20) fertilizer. The exper-iment was conducted in late summer in a greenhouseunder the following conditions: day/night temperature

25:18°C, 16 h photoperiod supplemented by 400 Whigh-pressure sodium lamps (P.L. Light Systems,Beamsville, Ontario, Canada).

Three-month-old seedlings were transplanted into6-inch pots containing a mixture of peat moss and sand(pH=6) and placed in trays to prevent leaching. Eachpot was treated twice (over a 1-week period) with500 ml water containing one of the following solu-tions: (1) distilled water (control), (2) 50 mMNaCl, (3)5 mM CaCl2, (4) 50 mM NaCl + 5 mM CaCl2, (5)10 mM CaCl2, (6) 50 mM NaCl + 10 mM CaCl2, (7)5 mM CaSO4, (8) 50 mM NaCl + 5 mM CaSO4, (9)10 mM CaSO4 or (10) 50 mM NaCl + 10 mMCaSO4. The NaCl concentration selected for thisexperiment was similar to the concentration of saltsfound in some of the oil sands mine wastes in Alberta.The calcium concentration of the peat moss/sandmixture was around 2 mM. Water was regularlyadded to the trays to keep soil moisture relativelyconstant and prevent salt leaching; water was alsoadded from time to time to the top of the pots toprevent formation of a salt crust. There were threeplants per pot in four replicated experiments placed intrays for a total of 120 seedlings.

Shoot heights were determined at the end of theexposure period (40 days). Seedlings were then har-vested, washed twice with distilled water and dividedinto roots, stems and leaves to determine fresh biomass.Leaves showing any damage (necrotic tips and necrotictissues) were separated from other leaves and kept forelemental analysis. Samples were freeze-dried to deter-mine the dry weight per plant and tissue water content.Freeze-dried tissues were then ground to analyze Na+,Ca2+, K+ and Cl− tissue content and proline.

Transpiration measurement

Transpiration and stomatal conductance were measuredafter 10, 20, 30 and 40 days of treatment. Measurementswere performed using one of the mature leaves from thethird pair from the top of the seedling. All measurementswere conducted in the greenhouse between 10:00 A.M.

and 2:00 P.M. using a steady state porometer (LI-1600,LI-COR, Inc., Lincoln, NE, USA).

Ion analysis

Levels of Na+, K+ and Ca2+ in freeze-dried groundtissues (1.5 g) were determined using an inductively

124 Plant Soil (2009) 315:123–133

coupled plasma optical emission spectrometer (Liberty200; Varian, Walnut Creek, CA, USA) after digestionfor 1 h with concentrated nitric acid at 200°C(Thompson and Walsh 1983). Three sub-samples of0.25 g (one for each of the three seedlings per replicate)were combined to provide one sample per replicate.Chloride concentration was determined using a Cl−

selective electrode (Accumet, Fisher Scientific). Drytissues (50 mg) from a powder mix of three plants fromeach pot were extracted with 10 ml of 0.5 M HNO3

(Rieger and Litvin 1998). After 30 min, 200 µL of ISA(Ionic Standard Adjuster, 5 M NaNO3) was added tothe extracts and the reading was recorded after 1 min.

Proline determination

Proline was extracted and determined as described byBates et al. (1973) with slight modifications. Threemilliliters of 3% sulfosalicylic acid were added to 0.1 gof dry ground leaf tissues and incubated in a water bathat 100°C for 30 min. After cooling, samples werecentrifuged at 4,900×g for 5 min at 25°C. Onemilliliter of the obtained supernatant was added to2 ml mixture reagent (60% acetic acid and 1%ninhydrin) and boiled for 1 h. After cooling, 3 ml oftoluene were added with subsequent separation of thetoluene phase. The absorbance was recorded at 520 nmwith a spectrophotometer (Pharmacia Biotech, ultro-spec 2,000 UV/Visible spectrophotometer, England)using toluene as a blank. The proline concentrationwas calculated against a standard curve of L-proline.

Data analysis

A one-way ANOVA was used to analyze all data(version 15; SPSS Inc., Chicago, IL, USA). Orthogonalcontrasts were used to determine: (1) the effect of NaClon plants with and without Ca2+ exposure, (2) tocompare the effects of different Ca2+ concentrations (5versus 10 mM) and (3) to compare the effects ofdifferent Ca2+ salts (CaCl2 versus CaSO4; α=0.05).

Results

Transpiration, water content and proline content

Our results showed that after ten days of treatment, NaClreduced (P<0.001) the transpiration rates of red-osier

dogwood in absence and presence of Ca2+ (Fig. 1). Tendays later, on the 20th day of treatment, the plantstreated with NaCl and 10 mM Ca2+ started to shownon significant changes in transpiration rates comparedto their respective controls; while the plants exposed toNaCl and 5 mM Ca2+ still exhibited a decrease (P<0.001) in transpiration rates with no differencesbetween CaSO4 and CaCl2. Thirty days after thebeginning of the treatment, plants exposed to NaCland all Ca2+ treatments did not exhibit any changes intranspiration rates when exposed to NaCl whereas inabsence of Ca2+, NaCl still reduced the transpirationrates (P=0.06). It was only at the end of the treatmentexposure (40 days) that NaCl did not significantlyaffect transpiration rates in any of the treatments.Stomatal conductance of the leaves (data not shown)showed the same results as the transpiration.

The presence of 50 mM NaCl caused an increase inboth root and stem water content (P<0.025) comparedto the untreated plants both in absence and presence ofCa2+ (Table 1). Sodium chloride also increased leafwater content (P<0.01) of red-osier dogwood seed-lings but to a larger extent than in the other tissues (upto 10% of control in absence of Ca2+). In presence ofadditional Ca2+, NaCl still increased leaf water content(P<0.02) compared to control plants but to a lowerextent (6% to 8% for 5 mM Ca2+ and 4% to 5% for10 mM Ca2+). In the lateral shoots, the water contentwas also increased (P<0.02) by NaCl in all treatments.Overall, we did not find any significant differencesbetween the effects of the two Ca2+ salts on watercontent of red osier dogwood tissues.

The decrease in transpiration was coupled with anincrease (P=0.023) in proline concentration in plantleaves in absence of Ca2+ salts. However, in thepresence of Ca2+ (with the exception of 5 mM CaCl2),NaCl did not significantly change the proline contentof the leaves (Fig. 2).

Growth

Although red-osier dogwood seedlings survived the40 days of salt treatments, their growth was affectedby NaCl. Root dry weight of red-osier dogwoodexposed to NaCl was reduced by 39% (P<0.01) whileshoot (including stem, leaf and lateral shoot) dryweight was not affected (Table 2) resulting in asignificant reduction (P<0.01) in root to shoot ratio(35%). In root tissues, the NaCl-induced decrease

Plant Soil (2009) 315:123–133 125

(P<0.01) in biomass was still recorded in presence ofCa2+. However, a difference was observed betweenthe two Ca2+ salts at low concentration; CaCl2 limitedthe decrease to 33% whereas CaSO4 limited it to16%. Sodium chloride did not affect stem, leaf andlateral shoot dry weight as well as the shoot height inabsence of Ca2+; however, addition of Ca2+ to NaCl

treatment led to a decrease (P<0.03) in both stem andleaf dry weights and also shoot height. The reductionin stem biomass (P=0.01) was more pronounced inpresence of CaCl2; whereas CaSO4 produced a greaterreduction in shoot height.

Sodium chloride did not affect red-osier dogwoodleaf number after forty days of treatment, whereas the

Table 1 Water content of root, stem, leaf and lateral shoots of red-osier dogwood seedlings exposed to 50 mM NaCl in presence orabsence of additional Ca2+ for 40 days

Treatments Water content (% fresh weight)

Root Stem Leaf Lateral shoot

Control 80.4±0.3 63.0±0.7 60.9±0.8 65.5±0.8NaCl 81.9±0.5 65.1±0.6 67.2±1.0 68.7±1.0Control + 5 mM CaCl2 81.3±0.6 63.4±0.5 62.9±0.7 64.8±1.0NaCl + 5 mM CaCl2 83.3±0.5 65.9±0.6 68.3±0.5 71.3±0.5Control + 10 mM CaCl2 81.8±0.6 62.8±0.6 63.2±1.0 67.4±0.9NaCl + 10 mM CaCl2 83.9±0.5 66.7±0.7 65.8±2.1 73.2±0.9Control + 5 mM CaSO4 81.0±0.5 63.9±0.4 63.7±0.7 67.0±0.7NaCl + 5 mM CaSO4 82.4±0.4 65.2±0.4 67.4±0.5 69.8±0.7Control + 10 mM CaSO4 81.0±0.5 64.0±0.4 64.1±0.7 67.1±0.9NaCl + 10 mM CaSO4 83.3±0.7 65.5±0.4 67.6±0.4 71.2±0.8

Values represent the mean±SE (n=12).

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b Control

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NaCl

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Fig. 1 Transpiration of red-osier dogwood seedlingsexposed to 50 mM NaCl inpresence or absence ofCaCl2 (a) and CaSO4 (b).Values represent means+SE(n=12)

126 Plant Soil (2009) 315:123–133

addition of 10 mM CaCl2 caused a decrease (P=0.016)in the leaf number exposed to NaCl compared with therespective controls, mainly due to leaf abscission(Table 3). The number of lateral shoots and nodeswas not modified by any of the salt treatments. After40 days of treatment, NaCl increased the number ofnecrotic leaves (P<0.01) in all treatments.

Ion content

Damaged leaves of red-osier dogwood treated withNaCl were the tissues that had the highest Cl−

concentration (over 15 mg g−1 dry weight) both withand without Ca2+ addition. Roots, leaves and lateralshoots had relatively similar amounts of Cl− (6–12 mg g−1 dry weight) in their tissues when exposedto NaCl (with and without Ca2+ addition), whilelower amounts were found in stem tissues (less than3.1 mg g−1 dry weight) (Table 4). Plants exposed toNaCl and 10 mM CaCl2 had more Cl− in their tissuesthan the other plants.

In red-osier dogwood tissues Na+ concentrationwas lower than Cl− concentration (Table 5). Thehighest amount of Na+ was found in roots followed

Table 2 Growth parameters (shoot height, root/shoot ratio, root, stem, leaf and lateral shoot dry biomass per plant) of red-osierdogwood seedlings exposed to 50 mM NaCl in presence or absence of additional Ca2+ for 40 days

Treatments Shoot heighta (cm) Root/shoot ratio Dry weight (g)

Roots Stems Leaves Lateral shoots

Control 17.2±2.0 0.63±0.04 1.76±0.16 0.95±0.10 1.48±0.08 0.34±0.10NaCl 14.2±1.0 0.41±0.02 1.08±0.09 0.88±0.05 1.44±0.12 0.31±0.06Control + 5 mM CaCl2 18.4±1.5 0.56±0.03 1.61±0.10 1.02±0.06 1.50±0.10 0.39±0.08NaCl + 5 mM CaCl2 13.7±1.6 0.47±0.03 1.08±0.12 0.76±0.06 1.10±0.12 0.44±0.09Control + 10 mM CaCl2 16.0±1.6 0.59±0.06 1.38±0.11 0.93±0.08 1.36±0.13 0.23±0.04NaCl + 10 mM CaCl2 12.5±1.4 0.49±0.05 0.87±0.11 0.67±0.08 1.06±0.18 0.22±0.07Control + 5 mM CaSO4 19.2±1.7 0.54±0.03 1.43±0.11 0.95±0.09 1.49±0.13 0.33±0.07NaCl + 5 mM CaSO4 12.7±1.6 0.51±0.04 1.20±0.11 0.79±0.07 1.29±0.16 0.35±0.06Control + 10 mM CaSO4 20.8±1.6 0.57±0.02 1.60±0.10 1.09±0.07 1.56±0.09 0.20±0.04NaCl + 10 mM CaSO4 14.0±1.8 0.43±0.02 1.10±0.15 0.91±0.11 1.24±0.18 0.38±0.09

Values represent the mean±SE (n=12).a Shoot height is expressed as the height difference between the beginning and the end of the treatments.

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Fig. 2 Proline content ofred-osier dogwood leavesexposed to 50 mM NaCl inpresence or absence ofCaCl2 (a) and CaSO4 (b).Values represent means+SE(n=12)

Plant Soil (2009) 315:123–133 127

by stems and lateral shoots while non-damaged leavesdid not contain significant amount of Na+ (Table 5).The damaged leaves had also relatively low concen-tration of Na+ (0.20 to 0.45 mg g−1 dry weight).Addition of Ca2+ reduced Na+ concentration only inthe stem (P=0.03).

The root tissues did not show any change in K+

concentration both in the absence and presence ofCa2+ when exposed to NaCl (Table 6). In stems, thesmall increase (P<0.045) in K+ that occurred inplants exposed to NaCl, was alleviated when Ca2+

salts were added with the exception of 10 mMCaCl2. In non-necrotic leaves and lateral shoots, anincrease in K+ (P<0.02) was recorded in NaCltreatment only in presence of 10 mM Ca2+ whileno change occurred in other NaCl treatments.

Sodium chloride increased (P<0.02) the amount ofCa2+ in leaves (100%), lateral shoots (73%) and stems(34%) of red-osier dogwood plants whereas nochange occurred in roots (Table 7). In presence ofadditional Ca2+, the NaCl-induced change in Ca2+

concentration did not occur in stems and it was stillobserved (P<0.08) in leaves and lateral shoots but toa lower extent (35–60% and 24–37% for 5 and10 mM respectively).

Discussion

The presence of 50 mM NaCl decreased transpirationrate and stomatal conductance of red-osier dogwoodseedlings within the first 10 days of the treatment.

Table 3 Number of leaves, lateral shoots, necrotic leaves and nodes of red-osier dogwood seedlings exposed to 50 mM NaCl inpresence or absence of additional Ca2+ for 40 days

Treatments Leaves Lateral shoots Necrotic leaves Nodes(Number per plant)

Control 19.7±1.1 15.1±1.9 0.7±0.5 13.4±0.4NaCl 18.7±1.3 17.6±1.8 6.4±1.1 13.5±0.4Control + 5 mM CaCl2 20.2±1.0 16.9±1.8 0.2±0 13.7±0.6NaCl + 5 mM CaCl2 16.5±1.3 16.7±1.1 5.3±0.9 13.5±0.4Control + 10 mM CaCl2 18.2±1.3 14.7±1.8 3.6±0.9 13.4±0.4NaCl + 10 mM CaCl2 14.2±1.6 14.6±1.5 5.3±1.2 14.2±0.2Control + 5 mM CaSO4 20.6±0.8 17.0±1.6 1.4±0.8 13.7±0.5NaCl + 5 mM CaSO4 19.4±1.5 17.5±1.3 6.3±0.9 13.9±0.3Control + 10 mM CaSO4 20.5±1.2 13.7±1.7 1.1±0.7 14.2±0.4NaCl + 10 mM CaSO4 18.4±1.6 16.0±1.7 6.7±1.3 13.5±0.5

Values present the mean±SE (n=12).

Table 4 Chloride content of red-osier dogwood tissues (root, stem, leaf and lateral shoot) exposed to 50 mM NaCl in presence orabsence of additional Ca2+ for 40 days

Treatments Root Stem Non necrotic leaf Lateral shoot(Milligram chloride per gram dry mass)

Control 1.13±0.11 0.11±0.03 0.71±0.06 0.41±0.11NaCl 9.31±0.18 2.40±0.37 7.70±1.53 6.78±0.92Control + 5 mM CaCl2 4.90±0.26 0.55±0.08 2.43±0.20 1.74±0.16NaCl + 5 mM CaCl2 9.22±1.20 1.79±0.23 6.84±0.93 7.34±0.47Control + 10 mM CaCl2 5.33±0.37 0.78±0.05 4.35±0.74 4.17±1.15NaCl + 10 mM CaCl2 12.01±1.11 3.04±0.29 10.42±2.84 12.08±2.47Control + 5 mM CaSO4 1.69±0.16 0.12±0.03 0.58±0.15 0.39±0.08NaCl + 5 mM CaSO4 8.75±0.49 2.19±0.31 7.22±0.22 8.26±0.88Control + 10 mM CaSO4 1.35±0.13 0.14±0.02 0.66±0.08 0.36±0.03NaCl + 10 mM CaSO4 9.39±1.12 2.02±0.22 6.15±0.74 8.32±1.32

Values represent the means±SE (n=4).

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Seedlings were able to recover after 40 days, having thesame level of transpiration rate and stomatal conduc-tance compared to the controls. The decrease intranspiration in response to osmotic stress has beenassociated in many plants with the transfer of ahormonal signal, ABA (abscisic acid), from root toshoot. This change in ABA leads to a reduction instomatal opening thus limiting water loss from the plant(Nilsen and Orcutt 1996). Under stressful conditions,following the initial increase in ABA, plants canreduce their ABA content to the normal level (Nilsenand Orcutt 1996). Cummings et al. (1971) showed thatthe response of stomata to ABA was reversible oncethe hormone supply was removed. In some halophytes,such as Aster tripolium, the reduction in transpirationhas been associated with an accumulation of Na+ ions

in the apoplast surrounding the guard cells, inhibitingstomatal opening (Perera et al. 1994).

We found that by adding Ca2+ to the NaCl treat-ments, red-osier dogwood recovered from reducedtranspiration and stomatal conductance after 20 dayswith 10 mM Ca2+ and after 30 days with 5 mM Ca2+

compared to 40 days in the controls. These resultssuggest that the stomata were able to re-open morerapidly when Ca2+ was added to the growth medium.This finding is consistent with a study conducted onAster tripolium (Perera et al. 1995) in which increasingCa2+ reduced the NaCl-induced decrease in stomatalconductance. In the present study, the addition of Ca2+

could have limited the effect of ABA on stomatalclosure. One of the models of signal transductionpathways suggests that ABA inhibits ABI1 and ABI2,

Table 6 Potassium content of red-osier dogwood tissues (root, stem, leaf and lateral shoot) exposed to 50 mM NaCl in presence orabsence of additional Ca2+ for 40 days

Treatments Root Stem Non necrotic leaf Lateral shoot(Milligram potassium per gram dry biomass)

Control 4.69±0.20 3.34±0.16 4.93±0.53 4.91±0.73NaCl 4.40±0.41 4.07±0.25 4.88±0.61 5.75±0.45Control + 5 mM CaCl2 4.88±0.15 4.01±0.24 5.22±0.31 4.79±0.07NaCl + 5 mM CaCl2 4.43±0.30 4.17±0.35 5.39±0.26 6.23±0.33Control + 10 mM CaCl2 4.88±0.32 3.71±0.30 4.40±0.11 5.01±0.67NaCl + 10 mM CaCl2 4.83±0.16 4.57±0.40 5.58±0.44 6.86±0.75Control + 5 mM CaSO4 4.29±0.39 3.61±0.27 5.25±0.54 6.04±0.16NaCl + 5 mM CaSO4 4.46±0.37 3.64±0.24 4.71±0.69 4.91±0.41Control + 10 mM CaSO4 4.75±0.31 3.68±0.16 4.25±0.45 5.14±0.56NaCl + 10 mM CaSO4 3.95±0.54 3.55±0.38 5.17±0.61 5.64±0.81

Values represent the means±SE (n=4).

Table 5 Sodium content of red-osier dogwood tissues (root, stem, leaf and lateral shoot) exposed to 50 mM NaCl in presence orabsence of additional Ca2+ for 40 days

Treatments Root Stem Non necrotic leaf Lateral shoot(Milligram sodium per gram dry biomass)

Control 0.80±0.01 0.06±0.02 0.027±0.007 0.055±0.014NaCl 5.07±0.30 1.61±0.14 0.132±0.037 0.702±0.046Control + 5 mM CaCl2 0.73±0.06 0.04±0.01 0.023±0.002 0.031±0.002NaCl + 5 Mm CaCl2 5.25±0.91 1.18±0.18 0.126±0.088 0.463±0.171Control + 10 mM CaCl2 0.79±0.03 0.05±0.01 0.034±0.013 0.040±0.006NaCl + 10 mM CaCl2 5.31±0.15 1.22±0.19 0.100±0.031 0.719±0.044Control + 5 mM CaSO4 1.02±0.07 0.06±0.02 0.021±0.010 0.043±0.001NaCl + 5 mM CaSO4 4.78±0.34 1.21±0.05 0.075±0.021 0.429±0.084Control + 10 mM CaSO4 0.91±0.07 0.04±0.01 0.015±0.003 0.053±0.011NaCl + 10 mM CaSO4 4.88±0.43 1.18±0.26 0.128±0.070 0.551±0.214

Values represent the means±SE (n=4).

Plant Soil (2009) 315:123–133 129

two Ca2+ dependent protein phosphatases, whichregulate stomatal opening by playing a role of negativeregulators of ABA response (Leung et al. 1994; Wu etal. 2003; Nilson and Assmann 2007). Similarly, in red-osier dogwood, the addition of Ca2+ could havestimulated the activity of the Ca2+ modulated phos-phatases (ABI1 and ABI2) thus decreasing the ABAresponsiveness of the stomata.

To survive the salt-induced osmotic stress, someplants exhibit osmotic adjustment involving theproduction of osmolytes such as proline (Hasegawaet al. 2000). Our results showed that after 40 days oftreatment, proline levels were higher in red-osierdogwood exposed to NaCl in the absence of Ca2+

(with the exception of 5 mM CaCl2), while in thepresence of 10 mM CaCl2 and both concentrations ofCaSO4 the accumulation of salinity-induced prolinewas limited compared to their respective controls.These results suggest that in the presence of Ca2+ theplants are exposed to a lower level of osmotic stress.Similar results have been found in NaCl stressedpeanut plants (Arachis hypogaea) exposed to supple-mental Ca2+ (Girija et al. 2002) while accumulation ofproline occurred in other species such as Sorghumbicolor (Colmer et al. 1996). In peanut plants, theaddition of CaCl2 lowered the proline concentrationby increasing the level of proline oxidase (an enzymethat converts proline to glutamate) and decreasing γ-glutamyl kinase activities which plays an importantrole in proline synthesis (Girija et al. 2002). Inaddition to osmotic adjustment, proline plays manyroles in stressed plants. For instance, it can act asprotein and membrane protectants, a sink storage of

carbon and nitrogen and a scavenger of free radicals(Girija et al. 2002). Furthermore, proline has beenshown to inhibit to various degrees the stomatalopening in Commelina communis (Klein and Itai1989), Commelina benghalensis (Raghavendra andBhaskar Reddy 1987) and Vicia faba (Rai 2002). Thisinhibition has been associated in Commelina bengha-lensis with a decrease in malate synthesis and areduction in the hydrolysis of starch into sugars(Raghavendra and Bhaskar Reddy 1987). The highestconcentrations of proline in red-osier dogwood seed-lings exposed to NaCl, without Ca2+ addition, couldhave thus played a role in the slower transpirationrecovery of these plants.

The early decrease in stomatal conductance andtranspiration observed in presence of NaCl could havecontributed to the increase in plant water content. Thiswater increase may have helped the plants to reducethe Na+ and Cl− concentration in tissues by dilutingthese ions. Plants treated with both NaCl and Ca2+

showed similar trends. However, in the leaves that re-opened their stomata more rapidly, the increase inwater content was limited. The further increase in thewater content of the lateral shoots exposed to CaCl2could have play a role in ion dilution in the newtissues as the lateral shoots were produced only afterthe beginning of the treatments.

With the addition of NaCl, the root biomass of red-osier dogwood growing in peat/sand substrate wassignificantly reduced, while there was no significantchange in shoot biomass, resulting in a decrease inroot/shoot ratio. Previous studies using hydroponicshave shown that the root/shoot ratio of red-osier

Table 7 Calcium content of red-osier dogwood tissues (root, stem, leaf and lateral shoot) exposed to 50 mM NaCl in presence orabsence of additional Ca2+ for 40 days

Treatments Root Stem Non-necrotic leaf Lateral shoot(Milligram calcium per gram dry biomass)

Control 3.37±0.10 5.18±0.21 9.76±0.51 8.95±0.21NaCl 3.41±0.10 6.92±0.26 19.51±0.90 15.51±1.73Control + 5 mM CaCl2 3.43±0.12 6.29±0.29 12.53±1.04 11.25±0.71NaCl + 5 mM CaCl2 3.52±0.05 6.80±0.58 16.88±1.26 16.03±1.29Control + 10 mM CaCl2 3.38±0.04 6.29±0.34 15.63±1.95 13.10±0.73NaCl + 10 mM CaCl2 3.52±0.10 7.10±0.81 19.43±0.89 17.92±0.53Control + 5 mM CaSO4 3.61±0.07 7.47±0.79 11.72±1.12 11.28±1.03NaCl + 5 mM CaSO4 3.55±0.11 6.92±0.26 18.66±0.05 15.17±0.68Control + 10 mM CaSO4 3.57±0.16 7.01±0.37 15.44±2.01 13.40±2.34NaCl + 10 mM CaSO4 3.64±0.15 6.06±0.37 19.47±3.33 17.89±2.88

Values represent the means±SE (n=4).

130 Plant Soil (2009) 315:123–133

dogwood was not affected by 50 mM NaCl as bothroot and shoot biomass were equally reduced by thesalt (Renault et al. 2001). This difference in plantresponses can be attributed to the availability of ions.In a peat/sand mixture, the binding of ions to soilparticles and the presence of microorganisms couldhave contributed to the depletion of ions in therhizospere, while in hydroponics the ions were moreavailable for uptake and are thus more likely to have agreater ionic effect on shoots.

We found that in the presence of Ca2+, the root/shootratio was still reduced but to a lower extent than in theabsence of Ca2. Calcium chloride limited the decrease inroot/shoot ratio mainly by decreasing both stem and leafbiomass. Woody plant fruit crops have been reported tobe relatively sensitive to chloride salts (Maas 1993). Inour experiment, leaf abscission occurred in the presenceof CaCl2 and contributed to the reduction in biomass. InCitrus, leaf abscission has been correlated with highlevels of Cl− in leaf tissues, which would triggersynthesis of ethylene (Gómez-Cadenas et al. 1998).Although leaf abscission could have reduced theconcentration of Cl− and Na+ in the plants, the decreasein total leaf area limited the amount of tissues availablefor photosynthesis thus reducing the amount of carbo-hydrates necessary for growth. Calcium sulfate was alsoable to limit the decrease in root/shoot ratio of red-osierdogwood, mainly by limiting the reduction in rootbiomass (at least at low concentration). However, theplants produced were significantly shorter than the salt-treated plants not exposed to CaSO4. Previous results(Renault et al. 2001) have also shown that 50 mMNa2SO4 reduced plant biomass and shoot height of red-osier dogwood to a greater degree than NaCl inequimolar Na+ concentrations although the osmoticpotential of the 50 mM Na2SO4 solution (−0.43 MPa)was higher than for the NaCl solution (−0.56 MPa)suggesting specific anion effects. Although sulfur isrequired as a macronutrient for plant growth, a highconcentration of SO2�

4 can lead to the formation ofsulfoxide that can inhibit cell division (Strogonov 1974).Furthermore, SO2�

4 and Cl− could affect differentiallynitrogen metabolism: pigeonpea (Cajanus cajan) treatedwith Na2SO4 had a lower nitrogen and protein contentas well as a lower nitrate reductase activity than plantstreated with NaCl (Joshi 1987).

Under saline conditions, some plants limit sodiumuptake and transport in shoot tissues (Husain et al.2004). This work and previous work (Renault et al.

2001; Renault 2005) suggests that red-osier dogwoodfollows this mechanism to limit the toxic effect ofNa+ in shoot tissue. Even in necrotic leaves, Na+

concentration was 10 times lower compared to theconcentration found in the roots (Table 5). AlthoughCa2+ has been shown to have an inhibitory role in Na+

uptake in many plants (Yokoi et al. 2002), in red-osierdogwood exposed to NaCl only a small reduction inNa+ concentration was observed in stem tissues.Chloride concentration was higher than sodiumconcentration, especially in the leaves and lateralshoots where it could have contributed to theobserved leaf necrosis. Foliar injury in woody plantssuch as plum trees and citrus species (Hoffman et al.1989; Maas 1993) has been attributed to highconcentration of Cl−.

Potassium, one element that plays a key role inhomeostasis (Niu et al. 1995), was not affected byNaCl treatment in absence of Ca2+, with the exceptionof the stem tissue where a small increase occurred. Incontrast, in the presence of 10 mM Ca2+ we found anincrease in both leaves and lateral shoots K+

concentration in the NaCl treatment. This increase inK+ could have played a role in the early re-opening ofthe stomata through regulation of K+ channels by Ca2+.Extracellular calcium has been shown to regulate K+

efflux channels in Arabidopsis, limiting K+ loss fromcells (Shabala et al. 2006).

There was an increase in Ca2+ content by two-foldin leaf tissues and lateral shoots. The stem tissues alsohad a smaller but significant increase in Ca2+ in plantsexposed to NaCl while no change occurred in theroots. These increases were unexpected as NaCl hasbeen shown to reduce the endogenous Ca2+ concen-tration in plant tissues as a result of the competitionbetween Ca2+ and Na+ (Rout and Shaw 2001).However, an increase in leaf Ca2+ concentration hasbeen previously found in woody species Prosopiscineraria (Ramoliya et al. 2006) and Asparagusofficinalis grown in sandy soil exposed to salinity(Warncke et al. 2002). It has been suggested that Ca2+

could be redistributed from roots to leaves duringsalinity stress (Ramoliya et al. 2006). In red-osierdogwood, there was no change in Ca2+ content in roottissues to support this hypothesis. It seems morelikely that the increase level of NaCl in the soil hadaffected the amount of Ca2+ available for plantuptake. Warncke et al. (2002) have shown that NaClapplication to a sandy/sandy loam soil resulted in an

Plant Soil (2009) 315:123–133 131

increase in extractable Ca2+. We did not see anyfurther increase in the amount of Ca2+ in the red-osierdogwood tissues when CaSO4 or CaCl2 were added.

Overall, our results showed that red-osier dogwoodplants survived salt stress, at least up to 50 mM, byadjusting not only to the ionic stress but also to theosmotic stress. To limit the ionic stress, the plants wereable to control the transport of Na+ from the roots toshoots thus confirming the results of our earlier studiesdone in hydroponics (Renault et al. 2001). In thispresent study NaCl treatments were applied to red-osier dogwood seedlings growing in soil, not inhydroponics, reflecting a scenario closer to fieldconditions. It also appears that NaCl can induce anincrease in Ca2+ content of leaves, lateral shoots andstems that was not observed in the previous hydro-ponics experiments and seems to be more characteris-tic of plants growing on sandy soils. The adjustment tothe osmotic stress involved a reduction in transpirationrates to limit water loss and an accumulation ofosmolytes (proline).

In red-osier dogwood, Ca2+ seemed to be moreinvolved in helping the plants to survive the osmoticstress, through regulation of transpiration rates, than theionic stress although there was a trend of decreased Na+

concentration in the stems. Our results also indicate thata concentration of 10 mM Ca2+ was more efficient than5 mM Ca2+ to limit the osmotic stress with nosignificant difference observed between CaSO4 andCaCl2. In addition, CaSO4 was the most efficient forlimiting the reduction in root growth at low concentra-tion. It is important to mention that the differencesobserved between the Ca2+ salts could be related totheir chemistry, CaCl2 having a higher solubility and adifferent dissociation constant than CaSO4 in water. Inaddition, the beneficial effects of Ca2+ from CaCl2could have been masked by the negative effect of Cl−

on the plants. To complete this study and to determinethe beneficial effects of treating salt affected areas withCa2+ salts prior to planting red-osier dogwood seed-lings, field experiments should be undertaken.

Acknowledgment Research funds for the project wereprovided to S.R. by the Natural Sciences and EngineeringResearch Council of Canada. Seeds were provided by MartinFung (Syncrude Canada Ltd.). We would like to thank ScottGreen and Karen Kivinen for technical assistance. Thanks toDrs. J. Franklin, D. Weihrauch, M. Sumner and the anonymousreviewers for critical reviews of the manuscript and forproviding helpful comments.

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