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Complexity of nutrient use efficiency in plantsReich, Martin

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Chapter 4

Impact of sulfate salinity onthe uptake and metabolismof sulfur in Chinese cabbage

Martin Reich, Tahereh Aghajanzadeh, C. Elisabeth E. Stuiver,Aleksandra Koralewska and Luit J. De Kok

In: Sulfur Metabolism in Plants - Molecular Physiology and Ecophysiology. De Kok,L. J., Hawkesford, M. J., Rennenberg, H., Saito, K. and Schnug, E. (eds.), Springer:227-238.

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Abstract

Increasing soil salinity is a major threat to crop production in many agriculturalareas throughout the world. Although sodium chloride (NaCl) is one of the mostabundant salts in soils, others viz. sulfate salts may also be present in high concentra-tions in some soil types. Sulfate salts, e.g. Na2SO4, are still widely under-representedamongst salt stress studies and the mechanism of its toxicity is poorly understood.Exposure of Chinese cabbage to Na2SO4 already reduced growth at levels ≥ 20 mM,accompanied by an increase in the total sulfur content of both roots and shoots,which in the shoot for a greater part could be ascribed to an accumulation of sul-fate. Moreover, there was an increase in the total water-soluble non-protein thiolcontent (glutathione) in roots and shoots. Enhanced sulfur metabolite levels (sulfate,glutathione) would down-regulate the expression and activity of the sulfate trans-porters and APS reductase (glutathione). Indeed, Na2SO4 exposure resulted in adown-regulation of the sulfate uptake capacity of the roots at ≥ 5 mM, whereas thetranscript level of the sulfate transporters Sultr1;2 and Sultr4;1 and APS reductasein the roots was reduced at ≥ 20 mM. Apparently in the shoot this regulatory signaltransduction pathway was overruled by the toxic effects of Na2SO4, since in contrastto the roots, the transcript levels of Sultr4;1 and APS reductase were enhanced inthe shoot at ≥ 30 mM and ≥ 5 mM Na2SO4, respectively.

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4.1 Introduction

Salt tolerance of plants and its improvement is one of the most prominent topics incrop research due to both the acuteness of the threats of salinity for agriculture andthe complex physiology that underlies salt tolerance in plants (Flowers, 2004; Paridaand Das, 2005; Peleg et al., 2011). Sulfur metabolism may have significance in thetolerance of plants to salinity. For instance, salt stress may result in an enhanced glu-tathione level, which presumably has adaptive significance in the protection of plantsagainst reactive oxygen species (Noctor et al., 1998; Mittler, 2002; Tausz et al., 2004;Szalai et al., 2009). The production of reactive oxygen species may be increased ifNa+ accumulates in other cell compartments than the vacuole (Zhu et al., 2007).The enhanced glutathione levels appeared to be coupled to increased levels of Na+

in the cytosol, since an enhanced level of glutathione (and cysteine) was absent intransgenic Brassica napus that over-expressed a vacuolar Na+/H+ antiporter uponNaCl exposure (Ruiz and Blumwald, 2002). However, the most important mecha-nisms in plants to avoid Na+ toxicity are the so-called includer/excluder strategieswhere Na+ is actively transported either back to the outside of the cell/plant and/orinto the vacuole in order to prevent cytosolic Na+ accumulation (Blumwald, 2000;Munns and Tester, 2008). These strategies also evolved in halophytes, accompaniedby anatomical adaptations such as succulence (increased cell size by salt accumula-tion in vacuoles) or specialized organs for salt exclusion via the leaves (salt glands).Another crucial factor is the cellular K+/Na+ ratio, which needs to be kept high inorder to prevent an inhibition of enzymes regulated by K+ (Maathuis and Amtmann,1999; Tester and Davenport, 2003; Chen et al., 2005; Zhu, 2007; Cuin et al., 2008).In addition to NaCl plants also may have to deal with Na2SO4 salinity (Garcıa andHernandez, 1996) and many areas are even dominated by sulfate salts (Chang et al.,1983; Keller et al., 1985). Although salt stress is usually mainly attributed to Na+

toxicity many studies showed that the accompanying anion might change the severityof the toxicity (e.g. Renault et al. (2001). In many species it had been shown thatsulfate salinity might be more toxic than chloride salinity (Eaton, 1942; Paek et al.,1988; Bilski et al., 1988; Datta et al., 1995; Renault et al., 2001). The physiologicalbasis of the toxic effects of sulfate salinity has still to be resolved. In this study theimpact of Na2SO4 salinity on the uptake, distribution and assimilation of sulfatewas studied in Chinese cabbage.

4.2 Material and Methods

Plant culture and harvest

Chinese cabbage (Brassica pekinensis (Lour.) Rupr. cv. Kasumi F1 (Nickerson-Zwaan, Made, The Netherlands) was germinated in vermiculite. Ten day-old seedlingswere pre-grown on a 10% Hoagland nutrient solution for 2 days and subsequentlygrown on a 25% Hoagland nutrient solution (pH 5.9; for composition see Koralewskaet al., 2007) at Na2SO4 concentrations of 0.5, 5, 10, 20, 30 and 40 mM in 30 litercontainers (10 sets per container, three plants per set) which were placed in a cli-mate controlled room for 11 days. Day and night temperatures were 21 and 17◦C (±1◦C), respectively, relative humidity was 60-70 % and the photoperiod was 14 h at a

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photon flux rate of 230 ± 20 µmol m−2 s−1 (within the 400-700 nm range) at plantheight, supplied by Philips HPI-T (400 W) lamps. After 11 days of exposure plantswere harvested and shoot and root fresh weight were determined. For determinationof the dry matter content, fresh plant tissue was dried at 80◦C for 24 h. For anionanalysis, roots were rinsed in ice-cold de-mineralized water (for 3 x 20 s) to removesulfate from the free space. Shoots and roots were separated, weighed, frozen inliquid N2 and stored at -20◦C until further analysis.

Extraction and determination of anions, water-soluble non-protein thiols, sulfur and nitrogen content

Anions were extracted from frozen plant material and determined refractometricallyafter separation by HPLC (Shahbaz et al., 2010). Water-soluble non-protein thiolswere extracted from freshly harvested plant tissue (Shahbaz et al., 2010) and thetotal water-soluble non-protein content was determined colorimetrically accordingto De Kok et al. (1988). For determination of the total sulfur and nitrogen contents,oven-dried plant material was pulverized by a Retsch Mixer-Mill (type MM2; Haan,Germany). Total sulfur content was determined with the barium sulfate precipita-tion method (Koralewska et al., 2008) and total nitrogen content was determinedaccording to a modified Kjeldahl method (Barneix et al., 1988).

Sulfate uptake capacity

Sulfate uptake capacity was determined as described by (Koralewska et al., 2007).Three sets of plants (three plants per set) per treatment were transferred to 25% Hoagland solution labeled with 35S-sulfate (2 MBq l-1) and incubated for 30min at 30◦C, at 0.5 mM Na2SO4. Subsequently, plants were removed and rootsrinsed in ice-cold non-labeled nutrient solution for 3 x 20 s. Roots and shootswere separated and digested in 1 N HCl at room temperature for 7 days. Theextracts were filtered through one layer of Miracloth and 100 µl of the filtrate wasmixed with 1 ml Emulsifier Scintillator Plus (Perkin Elmer, Boston, MA, USA).Radioactivity was measured with a liquid scintillation counter (TRI-CARB 2000CA Liquid Scintillation Analyzer, Perkin Elmer, Waltham, MA, USA).

Gene expression of sulfate transporters

Total RNA from roots and shoots was isolated by a method based on Verwoerd et al.(1989), which involved an additional phenol-chloroform-isoamyl alcohol extractionof the aqueous phase after the first centrifugation, or by using TRI REAGENTTM

(SIGMA), a mixture of guanidine thiocyanate and phenol in a mono-phase solu-tion. The final air-dried pellet was dissolved in an appropriate volume of diethylpyrocarbonate-treated water. The quality of the RNA preparations was checked byelectrophoresis of a 2 µg aliquot on a 1% (w/v) Tris-acetate/agarose gel. The con-centration was calculated from the absorbance at 260 nm in water. Determination ofthe expression of sulfate transporter was carried out according to Church and Gilbert(1984), with pre-hybridization and hybridization at 65 and 60◦C, respectively. Tenµg of total RNA per slot was separated on a 1.2% (w/v) agarose/formaldehyde

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Figure 4.1: The impact of Na2SO4 salin-ity on growth and shoot to root ratioof Chinese cabbage. Data represent themean of two experiments with 15 to 19measurements with three plants in each±SD (* = p < 0.01; unpaired Student’st-test)

gel and blotted onto a positively charged nylon membrane (Hybond-N+). Sequencediversity, especially in the 3’ non-coding region, allowed the use of partial cDNA frag-ments for gene-specific hybridization to the respective Brassica sulfate transportermRNA. The cDNA fragments were labeled with 32P-dCTP and used as hybridiza-tion probes. After hybridization with probes for sulfate transporters, membraneswere washed at 65◦C twice with 2xSSC, 0.1% SDS for 5 and 30 min, once with1xSSC, 0.1% SDS and twice with 0.1xSSC, 0.1% SDS for at least 30 min each, andexposed to Kodak BioMax MS film or to Cyclone MultiPurpose Phosphor Screen(Perkin Elmer, UK).

Statistical Analysis

Statistical analysis was performed with an unpaired Student’s t-test.

4.3 Results and Discussion

Although in natural ecosystems Brassicaceae species occur in dry and saline habitatsand even in extreme sulfur-enriched gypsum-bearing soils (Ernst, 1990; Dixon, 2007),some of the modern cultivated hybrids and cultivars appear to be very sensitive tosulfate salinity. The results of the present study showed that the shoot growth ofChinese cabbage was already inhibited at 20 mM Na2SO4 (Fig. 4.1). The shootgrowth was slightly more susceptible to salt stress than root growth and the latterwas only significantly reduced at 40 mM Na2SO4, resulting in a slight decrease inthe shoot to root ratio at ≥ 30 mM Na2SO4. Dry matter content of shoots wasenhanced at Na2SO4 concentrations ≥ 30 mM, up to two-fold at 40 mM, whereas

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Figure 4.2: The impact of Na2SO4 salin-ity on dry matter content of shoots androots of Chinese cabbage. Data representthe mean of two experiments with threemeasurements with three plants in each±SD (* = p < 0.01; unpaired Student’st-test)

that of roots was only slightly enhanced at 40 mM Na2SO4 (Fig. 4.2). Exposure ofplants to Na2SO4 salinity resulted in an increase in the total sulfur content of bothroots and shoots (Fig. 4.3), which in the shoot for a greater part could be ascribed toan accumulation of sulfate (Fig. 4.4). The sulfate content increased gradually withthe Na2SO4 concentration, but it was strongly enhanced at 40 mM Na2SO4 and itscontent in roots and shoots was increased 1.5-fold and four-fold, respectively. Ap-parently, the regulatory control of the uptake of sulfate by the roots was overruled atNa2SO4 concentrations exceeding 30 mM. In both shoots and roots, total nitrogenand nitrate decreased with the Na2SO4 concentration, indicating that the uptakeand assimilation of nitrate was negatively affected by sulfate salinity (Figs. 3 and4). Similar to previous observations there was apparently no direct linkage betweenthe uptake and assimilation of sulfate and nitrate (Stulen and De Kok, 2012). Simi-lar to observations with NaCl (Ruiz and Blumwald, 2002), Na2SO4 salinity resultedin an increase in the total water-soluble non-protein thiol content (presumably GSH)in roots and shoots at concentrations ≥ 20 mM (Fig. 4.5). Evidently this increaseremained relatively low and only at toxic Na2SO4 concentrations, and at sulfate lev-els in shoots four-fold higher than that of the control, a substantial increase of thiolcontent (two-fold) occurred. From the present data, the increased thiol/glutathionelevel appears to be a consequence of the excessive sulfate accumulation and not anadaptive protective response against salinity. Sulfate uptake plays a major role inthe control of plant sulfur homeostasis (Vauclare et al., 2002). The uptake and dis-tribution of sulfate is mediated by distinct sulfate transporters, which activity maybe controlled at a transcriptional, translational and/or post-translational level, andis regulated by the plant sulfur requirement for growth (Hawkesford and De Kok,2006; De Kok et al., 2011). Sulfate salinity had a substantial effect on the expres-

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Figure 4.3: The impact ofNa2SO4 salinity on total sulfur(S) and nitrogen (N) content ofshoots and roots of Chinese cab-bage. Data represent the mean ofthree measurements with 12 plantsin each ± SD (*/x = p < 0.01; un-paired Student’s t-test)

sion and activity of the sulfate transporters and the expression of APS reductase ofChinese cabbage (APR; Fig. 4.6). However, there were considerable differences inresponse of the different sulfate transporters and APS reductase between roots andshoots of Chinese cabbage. The Group 1 transporters are responsible for the primaryuptake of sulfate by the root and in sulfate-sufficient Brassica species only Sultr1;2is expressed (Hawkesford and De Kok, 2006; Koralewska et al., 2007, 2009; De Koket al., 2011). Also upon Na2SO4 salinity Sultr1;2 was the sole Group 1 sulfate trans-porter expressed in roots and the transcript levels of Sultr1;1 were negligible (Fig.4.6). Na2SO4 salinity resulted in a decreased expression of Sultr1;1 in the roots at≥ 30 mM, whereas the sulfate uptake capacity was already decreased ≥ 5 mM. Thelatter further decreased with the Na2SO4 concentration and was reduced more than2.5-fold at 40 mM (Fig. 4.6). Apparently, Na2SO4 salinity affected the regulation ofthe sulfate transporters in the roots already at lower concentrations at translationaland/or post translational than at transcriptional level. The Group 4 transportersare involved in the vacuolar efflux of sulfate (Hawkesford, 2003; Kataoka et al., 2004;Hawkesford and De Kok, 2006; De Kok et al., 2011) and there was also a decreasein the transcript level of Sultr4;1 in roots at 40 mM Na2SO4, whereas Sultr4;2 washardly expressed at all. The latter was in agreement with previous observations thatSultr1;1 and Sultr4;2 were only expressed in sulfate-deprived Brassica tissue (Ko-ralewska et al., 2009; De Kok et al., 2012; Shahbaz et al., 2014). The transcript levelof APR, the key regulating enzyme in the sulfate reduction pathway (Hawkesfordand De Kok, 2006; De Kok et al., 2011) was also reduced in the roots at ≥ 30 mM(Fig. 4.6). Sultr1;1 was hardly and Sultr4;2 was only slightly expressed in bothroots and shoots. Sulfate salinity only resulted in an increased transcript level ofSultr4;1 and Sultr4;2 at 40 mM Na2SO4, whereas that of APR was increased at ≥5 mM Na2SO4 (Fig. 4.6). Of the Group 1 and 4 transporters, only Sulftr4;2 wassubstantially expressed in the shoot of Chinese cabbage (Fig. 4.5). Contrary to the

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Figure 4.4: The impact of Na2SO4 salin-ity on sulfate and nitrate content of shootand roots of Chinese cabbage. Data repre-sent the mean of three measurements withthree plants in each ±SD (*/x = p < 0.01;unpaired Student’s t-test)

observations in the root, its transcript levels increased at ≥ 30 mM Na2SO4, togetherwith that of APS reductase (Fig. 4.6). The latter needs to be further investigated.It has been suggested that sulfate itself, or reduced sulfur compounds, may have arole in the regulation of expression and activity of the sulfate transporters and APSreductase (Hawkesford and De Kok, 2006; De Kok et al., 2011). For instance, hightissue levels of these compounds would down-regulate the expression and activityof the sulfate transporters (sulfate, glutathione) and APS reductase (glutathione).Indeed, in the roots this relationship between the content of these sulfur compoundsand the expression and activity of the sulfate transporters and expression of APSreductase does exist although in the shoot the toxic effects of Na2SO4 salinity ap-parently overruled this regulatory signal transduction pathway. Sulfate salinity wasdescribed as having a greater inhibitory effect on growth than chloride salinity inwheat (Datta et al., 1995), sugar beet and tomato (Eaton, 1942), wild potato (Bilskiet al., 1988) and on germination in barley (Huang and Redmann, 1995), alfalfa (Red-man 1974) and wheat (Hampson and Simpson, 1990). Comparative studies withinBrassica species are still very scarce. Additionally, the results of a study by Paeket al. (1988) on calli of Brassica campestris revealed that Na2SO4 had a strongernegative impact on biomass. The authors of the study also noted that sulfate accu-mulated much less under Na2SO4 than chloride under NaCl salinity (also found inChapter 5 of this thesis). This unequal uptake of Na+ and its anion under sulfatesalinity could explain the increased inhibitory effect on growth (also concluded byMeiri et al. (1971); Navarro et al. (2003)). Another observation from older studiesis that excess sulfate inhibits calcium uptake (Hayward and Wadleigh, 1949) butthis also holds true for NaCl and the application of additional calcium usually leadsto an amelioration of salt stress (Cramer, 2002; Kaya et al., 2002; Shabala et al.,2006). However, Johansen and Loneragan (1975) observed that Na2SO4 reduced75% of K+ uptake compared to the absence of Na+ while the same concentration of

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Sulfate salinity and sulfur metabolism Chapter 4

Figure 4.5: The im-pact of Na2SO4 salinityon total water-soluble non-protein thiols of shoot androots of Chinese cabbage.Data represent the mean ofthree measurements withthree plants in each ±SD(x = p < 0.01; unpairedStudent’s t-test)

NaCl reduced it only by 50%. A link to cation homeostasis therefore seems likely.Interestingly, Na+ accumulated mainly in shoots when jack pines were exposed toNaCl whereas it mainly accumulated in the roots under Na2SO4 salinity (Apostolet al., 2002). This is another hint that the translocation to the shoot and its controlmight be a crucial process under salt stress. Besides promoting the toxicity of Na+,sulfate could also have direct toxic effects (Visscher et al., 2010). Chapter 5 of thesisattempts to test these proposed mechanisms of sulfate toxicity by using differentsalts and additional calcium. Salt tolerance is known to be a physiological complexand trait, which challenges attempts at improvement. As Brassica is a diverse genuswith high agricultural importance, many efforts have been made to identify and de-velop salt tolerant cultivars. Salt tolerance in Brassica napus, for example, could beincreased tremendously in transgenic plants with an enhanced Na+ accumulation inthe vacuole (Zhang et al., 2001). In another transgenic approach, plants of Bras-sica juncea with an introduced bacterial pathway for the synthesis of glycine-betaineshowed increased germination rates and seedling growth (Prasad et al., 2000) butthe occurrence of sulfate salinity and its increased toxicity compared with chloridesalinity also suggests correlations of salt tolerance with sulfate uptake, assimilationand whole plant distribution. More research needs to be carried out on the toxicityof excessive sulfate and its possible role in exacerbating Na+ toxicity.

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Figure 4.6: The impact ofNa2SO4 salinity on sulfate up-take capacity and gene expres-sion of sulfate transporters (Sultr)and APS reductase (APR; North-ern blot analysis) of shoot androots of Chinese cabbage. EqualRNA loading was determined byethidium bromide staining of gels(shown in the bottom panels).Data on sulfate uptake capacityrepresent the mean of three mea-surements with three plants ineach ± SD (* = p < 0.01; unpairedStudent’s t-test)

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