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Research Article

Influence of and on antioxidants, and growth in Indian mustard

Humayra Bashir1, Mohamed M. Ibrahim2,3, Rita Bagheri1, Javed Ahmad1, Ibrahim A. Arif2,3, M. Affan Baig1 and M. Irfan Qureshi1* 1 Proteomics and Bioinformatics Lab, Department of Biotechnology, Jamia Millia Islamia, New Delhi 110025, India 2 Department of Botany and Microbiology, Science College, King Saud University, PO Box 2455, Riyadh, Saudi Arabia 3 Department of Botany and Microbiology, Faculty of Science, Alexandria University, PO Box 21511, Alexandria, Egypt Received: 13 August 2014; Accepted: 16 December 2014; Published: 12 January 2015 Associate Editor: Wen-Hao Zhang Citation: Bashir H, Ibrahim MM, Bagheri R, Ahmad J, Arif IA, Baig MA, Qureshi MI. 2015. Influence of sulfur and cadmium on antioxidants, phytochelatins and growth in Indian mustard. AoB 7: plv001; doi:10.1093/aobpla/plv001

Abstract. Soils in many parts of the world are contaminated with heavy metals, leading to multiple, deleterious effects on plants and threats to world food production efficiency. Cadmium (Cd) is one such metal, being toxic at rela- tively low concentrations as it is readily absorbed and translocated in plants. Sulfur-rich compounds are critical to the impact of Cd toxicity, enabling plants to increase their cellular defence and/or sequester Cd into mediated by phytochelatins (PCs). The influence of sulfur on Cd-induced stress was studied in the hyperaccumulator Indian 2− 2− mustard ( juncea) using two sulfur concentrations (+S, 300 mMSO4 and S-deficient 2S, 30 mMSO4 ) with and without the addition of Cd (100 mM CdCl2) at two different time intervals (7 and 14 days after treatment). Com- pared with control plants (+S/2Cd), levels of oxidative stress were higher in S-deficient (2S/2Cd) plants, and greatest in S-deficient Cd-treated (2S/+Cd) plants. However, additional S (+S/+Cd) helped plants cope with oxidative stress. Superoxide dismutase emerged as a key player against Cd stress under both 2S and +S conditions. The activity of ascorbate , reductase and catalase declined in Cd-treated and S-deficient plants, but was up-regulated in the presence of sulfur. Sulfur deficiency mediated a decrease in ascorbate and glutathione (GSH) con- tent but changes in ascorbate (reduced : oxidized) and GSH (reduced : oxidized) ratios were alleviated by sulfur. Our data clearly indicate that a sulfur pool is needed for synthesis of GSH, non- and PCs and is also important for growth. Sulfur-based defence mechanisms and the cellular antioxidant pathway, which are critical for tolerance and growth, collapsed as a result of a decline in the sulfur pool.

Keywords: Antioxidants; cadmium; growth; oxidative stress; phytochelatins; sulfur.

sulfur on agricultural land. Based on crop demand, Introduction -use efficiency and current inputs, the worldwide In recent years, sulfur deficiency has become a major sulfur-deficit has been estimated to be 10.4 million problem for agricultural productivity, reducing both crop tonnes annually (Haneklaus et al. 2007). Increased food quality and yields. Legitimate approaches to reducing production will further raise sulfur requirements, elevat- emissions of sulfur into the atmosphere have resulted ing this sulfur-deficit to 12.5 million tonnes by 2015 in a concomitant decrease in atmospheric deposition of (Randazzo 2009).

* Corresponding author’s e-mail address: [email protected]; [email protected]

Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

AoB PLANTS www.aobplants.oxfordjournals.org & The Authors 2015 1 Bashir et al. — Synergistic effects of S-deficiency and Cd on mustard

As an essential macronutrient required for the proper reported that Cd induces the production of com- growth and development of plants, sulfur plays a critical pounds and transcripts of the S-assimilation pathway. It role in a number of cellular processes, such as in protein is noteworthy that a PC-deficient Arabidopsis thaliana disulfide bridges (Saito 2000), mediating electron trans- mutant, cad1, exhibits Cd, As and Zn hypersensitivity port in iron–sulfur (Fe–S) clusters, the redox cycle, detoxi- (Cobbett and Goldsbrough 2002; Tennstedt et al. 2009). fication of heavy metals and xenobiotics, vitamin Cadmium activates the S-assimilation pathway respon- co-factors (Hell and Hillebrand 2001) and the sible for the synthesis of (Cys), a precursor of of secondary products (Hell 1997; Saito 2000). Sulfur is thus GSH . Glutathione (an NPT) acts as an import- a major plant nutrient (Takahashi et al. 1997; Leustek et al. ant antioxidant in mitigating Cd-induced stress (Meuwly 2000; Saito 2000; Lee and Korban 2002) that contributes to and Rauser 1992). It also plays an important role in PC an increase in crop yield, directly adding nutritional value synthesis, which has a proven role in Cd detoxification and improving the efficiency of use of other essential plant (Park et al. 2012). nutrients (Salvagiotti et al.2009), particularly nitrogen and Plants are also provided with an efficient mechanism phosphorus. Research on plant adaptations to S-related for protection against ROS and peroxidation reactions. stresses has shifted from an emphasis on excessive inputs The majority of ROS-scavenging pathways in plants and acidification to how deficiencies impact crop produc- involve superoxide dismutase (SOD), which is found in tion. Sulfur deficiency not only impacts crop quality and almost all cellular compartments, the water– yield, but it also raises the demand for adequate fertiliza- in and the ascorbic acid (AsA)–GSH cycle in tion to resist biotic and abiotic stresses. Exposure of plants chloroplasts, cytosol, mitochondria, and peroxi- to excessive toxic metals like cadmium (Cd) may affect the somes, in which ascorbate peroxidase (APX) and GR play

uptake and metabolism of S and negatively impact the crucial roles. Catalase (CAT) removes H2O2 in peroxisomes yield and plant resistance to abiotic stresses (Gill and (Mittler 2002). Tuteja 2011). A varying degree of antioxidative response and toler- Cadmium is a non-essential heavy metal that is readily ance is exhibited by different plant species. Several studies absorbed and rapidly translocated in plants, which makes have demonstrated that most of the main S-responsive it highly bio-available and thus toxic, even at relatively low genes involved in assimilation are induced by Cd, concentrations. Cadmium exerts its phytotoxicity by inter- suggesting the existence of a general adaptive response fering with several basic events of plant growth, develop- to an increase in cellular demand for reduced S (Nocito ment and (Qadir et al.2004). Cadmium induces et al.2006). Hence the availability of S to plants is crucial. oxidative stress leading to the overproduction of harmful Brassica juncea (L.) Czern. (Indian Mustard) belongs to (ROS) (Zhang et al. 2010). These the family and is an important oilseed crop ROS may cause damage to cell membranes, , that is known to tolerate considerable amounts of Cd DNA replication and repair. A major effect of this metal, (Qadir et al. 2004). In this study, we investigated the observed in most plants studied to date, is the inhibition effects of Cd, S-deficiency and their combination on of photosynthesis by altering chlorophyll synthesis (Pietrini S-assisted defence against oxidative stress, and changes et al.2003; Maksymiec and Krupa 2006) and the light- in cellular antioxidant activity, the contents of NPTs, PCs harvesting Chl a/Chl b protein complex II (Qureshi et al. and photosynthetic pigments, and growth parameters 2010), and interfering with RuBisCO activation (Prasad in B. juncea. 1995). Therefore, Cd-mediated reductions in photosyn- thetic activity lead to declines in crop yield (Ouzounidou Methods et al. 1997). Cadmium also interferes with processes such as carbohydrate and nitrogen metabolism (Sanita` di Toppi Experimental design and Gabbrielli 1999), catalysis (Van Assche and Seeds of mustard (B. juncea cv. Pusa Jaikisan), procured Clijsters 1990) and water balance (Perfus-Barbeoch et al. from the Indian Agriculture Research Institute (IARI), 2002). were treated with 1 % (v/v) sodium hypochlorite solution Sulfur plays a critical role in allowing plants to protect for 10 min followed by thorough washing in deionized themselves against heavy metal toxicity, especially Cd water. The seeds were germinated on moist soil (Soilritew; toxicity (Gill and Tuteja 2011). Numerous studies have 1 kg per pot). For S-, conditions were as described shown that S is involved in the biosynthesis of heavy in Abdallah et al. (2010). One set of plant cultures, consid- metal detoxification agents (Anjum et al. 2012), such as ered as the control, received Hoagland nutrient solution 2− non-protein thiols (NPTs) including phytochelatins (PCs), (Hoagland and Arnon 1950) with 300 mMSO4 (herein glutathione (GSH) (Noctor et al. 2011) and Cd -sulfide referred to as ‘S-sufficient’) and was designated as (+S). crystallites (Robinson et al. 1993). Harada et al. (2002) A second set received the same Hoagland nutrient

2 AoB PLANTS www.aobplants.oxfordjournals.org & The Authors 2015 Bashir et al. — Synergistic effects of S-deficiency and Cd on mustard

solution with an S concentration 10 times lower Ascorbate peroxidase assay 2− (30 mMSO4 , herein referred to as ‘S-deficient’) than The APX extraction and assay were performed as de- that of the control, and was designated as (2S). After scribed by Qureshi et al. (2007). Frozen (0.3 g) was 10 days of growth, both sets were divided into two further homogenized in 3 mL cold extraction buffer (0.5 M phos- sets. Cadmium (100 mM CdCl2) prepared in corresponding phate buffer containing 1 % (w/v) polyvenylpyrrolidone + Hoagland nutrient media was supplied to one set of S (PVP), 1 % (v/v) Triton-X 100, 100 mM EDTA, pH 7.8) in and 2S daily according to the water-holding capacity an ice bath. The crude extract was centrifuged at (WHC) of the soil. The plants were grown in a growth 6708 × g for 15 min at 4 8C. The supernatant was used chamber: 16/8 h light/dark period, photon flux density to measure APX activity. All reagents were prepared 22 21 of 150 + 10 mmol photons m s , 25/20 8C(day/ fresh before the assay. The assay reaction mixture con- night) temperature and 75 % relative humidity. There tained 0.1 M potassium phosphate buffer (pH 7.4), were four replicates of each treatment: (T ¼ +S/2Cd), 1 0.5 mM ascorbate, 0.3 % (v/v) H2O2 and 100 mLenzyme + + + (T2 ¼ 2S/2Cd), (T3 ¼ 2S/ Cd), (T4 ¼ S/ Cd). extract in a total volume of 1 mL. The assay was allowed were harvested from the plants after 7 and 14 days of to equilibrate at 25 8C for 1 min before the addition of Cd treatment and immediately used for biochemical , which initiated the reaction. Ascor- parameter, leaf area and also fresh and dry weight bate peroxidase assay activity was determined by moni- (DW) analysis. toring the rate of ascorbate oxidation as indicated by a reduction in the absorbance at 290 nm for 3 min at Thiobarbituric acid reactive substances (TBARS) 25 8C. A control reaction was prepared by replacing the The magnitude of oxidative stress was measured by esti- ascorbate with reaction buffer. A unit of APX is defined mating the content of TBARS following the method of as the amount required to oxidizie 1 mmol of ascorbate 21 Heath and Packer (1968). Fresh tissue was ground to a min at 25 8C (290 nm extinction coefficient of 21 21 powder using liquid nitrogen in a pre-chilled mortar and 2.8 mmol cm ). pestle. The powder was mixed into a paste in 1 % (w/v) trichloroacetic acid (TCA; 10 mL g21 fresh weight, FW). assay The extract was centrifuged at 9660 × gfor5min. To prepare the crude enzyme extract, 0.5 g leaf material A 1.0-mL aliquot of supernatant was placed in a separate was ground to a powder in liquid nitrogen using a pre- tube, to which 4.0 mL of 0.5 % (w/v) thiobarbituric acid chilled mortar and pestle. The powder was homogenized (TBA) was added. The mixture was heated at 99 8Cfor in 2 mL of cold 0.1 M potassium phosphate buffer (pH 7.2) 30 min. It was then quickly cooled in an ice bath and cen- in an ice bath. The crude extract was centrifuged at trifuged at 2817 × g for 5 min to clarify the reaction mix- 5433 × gfor15minat48C. The supernatant was col- ture. The absorbance of the supernatant at 532 nm was lected and used for assay. The glutathione reductase measured and corrected for unspecific turbidity by sub- (GR) assay was modified from Anderson (1985).The tracting the value at 600 nm. 1-mL assay contained 0.02 mM oxidized glutathione (GSSG) and 0.2 mM NADPH in a buffer (0.1 M potassium Superoxide dismutase assay phosphate buffer, pH 7.2). The assay was initiated with the addition of 0.2 mL of enzyme extract and activity The SOD assay was performed using the method of was monitored by a decrease in absorbance at 340 nm Dhindsa et al. (1981), based on the ability of SOD to inhibit for 3 min at 25 8C. A unit of activity is the amount of photochemical reduction of nitroblue tetrazolium (NBT). enzyme that catalyses the reduction of 1 mmol of GSSG The assay mixture, consisting of 1.5 mL reaction buffer min21 at 25 8C. (containing 0.1 M sodium phosphate buffer, pH 7.5, 1 % w/v PVP), 13 mM of L-, 0.1 mL of enzyme ex- Catalase assay tract with equal amounts of 1 M Na2CO3, 2.25 mM NBT so- lution, 3 mM EDTA and 60 mM riboflavin and 1.0 mL of Catalase (CAT) activity was determined using the method double-distilled water (DDW), was incubated under a of Aebi (1984). Fresh leaf material (0.5 g), ground in 5 mL 15-W fluorescent lamp at 28 8C. The absorbance of the of extraction buffer (0.5 M Na phosphate, pH 7.3, 3 mM irradiated reaction mixtures at 560 nm was compared EDTA, 1 % w/v PVP, 1 % v/v Triton X-100) was centrifuged with the non-irradiated mixture and per cent inhibition at 13 148 × g for 20 min at 4 8C. Catalase activity in the of colour was plotted as a function of the volume of supernatant was determined by monitoring the dis- enzyme extract corresponding to 50 % reduction of appearance of H2O2, according to the decrease in absorb- NTB, which was considered as one unit of enzyme activity ance at 240 nm. The reaction was run in a final volume of and expressed as mg21 protein min21. 2 mL of reaction buffer (0.5 M Na phosphate, pH 7.3)

AoB PLANTS www.aobplants.oxfordjournals.org & The Authors 2015 3 Bashir et al. — Synergistic effects of S-deficiency and Cd on mustard

containing 0.1 mL of 3 mM EDTA, 0.1 mL of enzyme ex- the addition of GSH-containing extract, the absorbance

tract and 0.1 mL of 3 mM H2O2 for 5 min. Catalase activity at 412 nm was monitored. The reaction blank was pre- was calculated by using a coefficient of absorbance of pared by replacing the plant extract with 5 % (w/v) 0.036 mM21 cm21. One unit of enzyme determines the 5-sulfosalicylic acid. To the same tube 0.2 units per assay

amount necessary to decompose 1 mmol of H2O2 per min. GR from yeast and 50 mL of 0.4 % (w/v) NADPH were added and the reaction was allowed to run for 30 min at 25 8C, Ascorbate content after which the absorbance at 412 nm was measured. Ascorbate (AsA) was estimated using a modification of The concentration of GSH was determined through com- Law et al.’s method (1983). Fresh leaf material (0.1 g) parison with a standard curve of the reaction rate as a was ground to a powder in a mortar and pestle using liquid function of the concentration of GSSG and was expressed nitrogen. The powder was homogenized in 2 mL of extrac- as nmol g21 FW. tion buffer and was centrifuged at 6708 × g for 10 min. To 400 mL of supernatant, 10 % (w/v) TCA (200 mL) was Non-protein thiols added. The mixture was vortex-mixed and cooled in ice Non-protein thiols (NPTs) were estimated with the method for 5 min and 10 mL of 5 M NaOH was added. The super- described by Howe and Merchant (1992), using Ellman’s natant mixture was centrifuged at 822 × gfor5min. reagent. A calibration curve was prepared using GSH The supernatant fraction was divided into two separate (Sigma, MO, USA) to estimate NPTs in samples. The amount 21 centrifuge tubes (200 mL each) to measure total (AsA + of NPT was expressed as nmol g FW. DHA) and reduced (AsA) ascorbate. To estimate total as- Phytochelatins corbate, 100 mL of dithiothreitol (DTT) and 200 mL of reac- tion buffer (150 mM potassium phosphate buffer) were content (PCs) in the tissue was calculated added and the assay mixture was thoroughly mixed by subtracting the amount of GSH from the amount of 21 and incubated for 15 min at 25 8C; 100 mL of 0.5 % (w/v) total NPTs, and expressed as nmolg FW. N-ethylmaleimide was then added. The contents of the − PCs (nmol g 1 FW)=NPT − total GSH. other tube were mixed with 200 mL of reaction buffer (150 mM potassium phosphate buffer) and 200 mLof double-distilled water. Both samples were vortex-mixed Chlorophyll estimation and growth parameters and incubated at room temperature for 30 s. To each Chlorophyll content was estimated using the method of tube was then added 400 mL of 10 % (w/v) TCA, 400 mL Hiscox and Israelstam (1979). Fresh leaves were col- of 44 % (v/v) H3PO4,444mLof4%(w/v)bipyridyland lected, washed with deionized water and kept in vials. 200 mLof3%(w/v)FeCl3. After vortex-mixing, samples Ten millilitres of DMSO were added to the vials, which were incubated at 37 8C for 60 min and the absorbance were then kept in an oven at 65 8C for 1 h. Absorbance was recorded at 525 nm on a UV–VIS spectrophotometer was recorded at 480, 645, 520 and 663 nm on a Beckman (Model DU 640, Beckman, USA). A calibration curve was DU 640B spectrophotometer. The chlorophyll concentra- prepared from different concentrations of AsA. The tion was calculated with the help of formulae given by 21 amount was expressed in nmol g FW. Arnon (1949) and expressed as mg g21 FW. Plants were uprooted carefully from the soil, washed Glutathione content with double-distilled water to remove soil and kept be- Glutathione content was determined using the method of tween moist filter paper to avoid desiccation. The leaf Anderson (1985), which measures total GSH [reduced area was measured with the help of a portable leaf area glutathione (GSH) and oxidized glutathione (GSSG)] using meter (Model LI 3000A, LI-COR, USA) and expressed in a GR-catalyzed reaction. Glutathione was extracted by cm2 per plant. To determine the leaf dry matter, the homogenizing 0.5 g of frozen fresh leaves in 2 mL of 5 % plants were dissected at the stem and petiole junction (w/v) 5-sulfosalicylic acid to reduce the oxidation of GSH. and the leaves were dried in an oven at 65 8C for 2 days. The powder was homogenized in 2 mL of cold 0.5 M potas- The dried samples were then weighed to determine the sium phosphate buffer (pH 7.8) in an ice bath. The crude plant DW. extract was centrifuged at 5433 × gfor15minat48C and used for subsequent assay. Statistical analysis The 1.0-mL assay for oxidized GSH contained 40 mLof All the estimates of sample variability are given in terms 0.15 % (w/v) 5-5′-dithio-bis(2-nitrobenzoic acid) (DTNB) of the standard error (SE). A Student’s t-test was used to and 0.5 mL plant extract in 0.1 M potassium phosphate identify statistical differences between pairs of means at buffer (pH 7.8). The assay mixture (minus GR and leaf ex- a confidence level of ≥95 % for each set of data using tract) was allowed to equilibrate at 30 8C for 5 min. After ANOVA. The data are means + SE from 10 samples in

4 AoB PLANTS www.aobplants.oxfordjournals.org & The Authors 2015 Bashir et al. — Synergistic effects of S-deficiency and Cd on mustard

four replicates (n ¼ 4, P ≤ 0.05, significant at 5 % level, and CAT (10 and 28 %) as noted at 7 DAT and 14 DAT, re- P ≤ 0.01, significant at 1 % level). spectively. However, SOD activity was up-regulated in 2S/+Cd plants both at 7 DAT (32 %) and 14 DAT (21 %). Results The activity of SOD further increased to 136 % (7 DAT) and 153 % (14 DAT) under Cd stress when in the presence of Thiobarbituric acid reactive substances sufficient S (+S/+Cd). To assess S-deficiency and Cd-induced oxidative cell dam- Cadmium treatment of S-deprived plants proved the age, the content of TBARS was determined. Under most deleterious, leading to a decline in the activity of S-deficiency, a 32 % increase in TBARS was observed APX (47 and 45 %), GR (55 and 54 %) and CAT (36 and over the control; a further 84 % increase occurred follow- 30 %) at 7 DAT and 14 DAT, respectively. However, ing the application of Cd to these plants. However, the S-sufficient plants showed significant resistance to Cd presence of S (+S/+Cd) lowered the oxidative threat by stress (+S/+Cd). The activity of APX (13 and 24 %), GR 33 % at 7 DATand 32 % at 14 DATduring Cd treatment (26 and 46 %) and CAT (16 and 53 %) was increased at (Fig. 1). 7 DAT and 14 DAT, respectively.

Response of enzymatic and non-enzymatic antioxidants Non-enzymatic antioxidants To study the stress response in plants exposed to Cd and Significant changes in the content of non-enzymatic anti- the protection offered in the presence of S, changes in the oxidants including ascorbates (ascorbate, AsA; dehy- + activities of enzymatic and non-enzymatic antioxidants droascorbate, DHA; total ascorbate, AsA DHA) and involved in the AsA-GSH cycle were analysed in the leaves their ratios (AsA/DHA) were seen under S-deficiency and of mustard. Cd stress. Sulfur-deprived (2S/2Cd) plants showed a 23 % (7 DAT) and 47 % (14 DAT) increase in DHA content. Antioxidative Cadmium stress during S-deprivation (2S/+Cd) further The activity of antioxidative enzymes viz. SOD (Fig. 2A), increased the DHA content by 40 % at 7 DAT and 58 % + + APX (Fig. 2B), GR (Fig. 2C) and CAT (Fig. 2D) was deter- at 14 DAT. Sulfur-sufficient but Cd-treated ( S/ Cd) mined over an experimental period of 7 DAT and 14 DAT plants showed a 33 % (7 DAT) and 60 % (14 DAT) increase of Cd exposure in both S-sufficient and S-deprived plants. in the DHA content. Sulfur deficiency significantly suppressed the activities of In contrast to DHA, the AsA content suffered a decline SOD (42 and 38 %), APX (35 and 28 %), GR (30 and 50 %) under every treatment and at both time points. AsA declined by 37 and 22 % (2S/2Cd), 42 and 57 % (2S/+Cd) and 33 and 22 % (+S/+Cd) at 7 DAT and 14 DAT, respectively. Similarly, the total ascorbate (AsA + DHA) content suffered a decline, but of a lesser magni- tude, in all treatments and at all time points. The total as- corbate content declined by 24 and 10 % (2S/2Cd), 24 and 38 % (2S/+Cd) and 19 and 8 % (+S/+Cd) at 7 DAT and 14 DAT, respectively. The AsA/DHA ratio showed sig- nificant alterations; compared with the control (+S/2Cd) value of 3.66 (7 DAT) and 4.95 (14 DAT), the AsA/DHA ratio dropped to 1.87 and 2.63, 1.50 and 1.33 and 1.83 and 2.41 under 2S/2Cd, 2S/+Cd and +S/+Cd, respectively (Table 1). The GSH contents (reduced form, GSH; oxidized form, GSSG; total GSH, GSH + GSSG) and their ratios (GSH/ GSSG) also showed significant alterations. Sulfur-deprived (2S/2Cd) plants showed a decrease of 23 % (7 DAT) and 12 % (14 DAT) in the GSSG content. Cadmium stress dur- + Figure 1. Effect of S-deficiency and Cd stress on the magnitude of ing Sulfur deficiency (2S/ Cd) further decreased the oxidative stress in mustard leaf. The values are mean and standard GSSG content to 12 % at 7 DATand 23 % at 14 DAT. Sulfur- error of the mean (mean + SE) of 10 samples and four replicates sufficient but Cd-treated (+S/+Cd) plants, however, (n ¼ 4, *P ≤ 0.05; **P ≤ 0.01; NS, non-significant). +S ¼ 300 mM showed a 160 % (7 DAT) and 124 % (14 DAT) increase in 2− ¼ 2− ¼ + ¼ SO4 , 2S 30 mMSO4 , 2Cd No CdCl2, Cd 100 mMCdCl2. the GSSG content.

AoB PLANTS www.aobplants.oxfordjournals.org & The Authors 2015 5 Bashir et al. — Synergistic effects of S-deficiency and Cd on mustard

Figure 2. Effect of S-deficiency and Cd stress on the activity of antioxidant enzymes. (A) Superoxide dismutase, (B) APX, (C) GR and (D) catalase (CAT) in mustard leaf. The values are mean and standard error of the mean (mean + SE) of 10 samples and four replicates (n ¼ 4, *P ≤ 0.05; 2− 2− **P ≤ 0.01; NS, non-significant). +S ¼ 300 mMSO4 , 2S ¼ 30 mMSO4 , 2Cd ¼ No CdCl2, +Cd ¼ 100 mM CdCl2.

A similar trend was exhibited by the GSH content in all However, S-sufficient but Cd-treated (+S/+Cd) plants treatments and at both time points. Glutathione content showed a 32% (7 DAT) and 79% (14 DAT) increase in declined by 28 and 18 % (2S/2Cd), 26 and 34 % the NPT content over the control (Table 2). (2S/+Cd) at 7 DAT and 14 DAT, respectively. A significant Similarly, the PC content (PCs ¼ NPTs 2 total glutathi- increase of 110 % (7 DAT) and 81 % (14 DAT) in the GSH one) declined under each treatment, except +S/+Cd, at content over the control was observed in S-sufficient both time points. The PC content declined by 80 and but Cd-treated (+S/+Cd) plants. However, the total GSH 92 % (2S/2Cd), 59 and 60 % (2S/+Cd) at 7 DAT and 14 content (GSH + GSSG) declined in all treatments, except DAT, respectively. However, the presence of sufficient S +S/+Cd, and at both time points. The total GSH content during Cd stress (+S/+Cd) helped the plant to almost declined by 16 and 17 % (2S/2Cd), 27 and 32 % maintain (22 % at 7 DAT) or increase (+75 % at 14 (2S/+Cd) at 7 DAT and 14 DAT, respectively. However, DAT) the PC content over the control (Table 2). thepresenceofsufficientSduringCdstress(+S/+Cd) helped the plant to accumulate 119 % (7 DAT) and 89 % Chlorophyll and growth (14 DAT) more total GSH over the control. The GSH/GSSG Sulfur deficiency resulted in a significant decrease in ratio showed significant alterations; compared with the chlorophyll content; Chl a decreased by 21 % (7 DAT) control (+S/2Cd) value of 4.36 (7 DAT) and 4.50 (14 and 23 % (14 DAT). Moreover, application of Cd to DAT), the GSH/GSSG ratio dropped to 4.06 and 4.23, 3.66 S-deprived (2S/+Cd) plants resulted in a drastic decrease and 3.81 and 3.52 and 3.63 under 2S/2Cd, 2S/+Cd of 53 % at 7 DATand 55 % at 14 DAT. However, plants with and +S/+Cd, respectively (Table 2). sufficient S were able to resist damage with a decline of 32 and 38 % at 7 DAT and 14 DAT, respectively. The Chl NPTs and PCs b content decreased by 25 % (7 DAT) and 16 % (14 DAT) The NPT content declined by 65 and 69 % (2S/2Cd), 49 in 2S/2Cd, 39 % (7 DAT) and 39 % (14 DAT) in 2S/+Cd and 51 % (2S/+Cd) at 7 DAT and 14 DAT, respectively. and 16 % (7 DAT) and 26 % (14 DAT) in Cd-treated plants

6 AoB PLANTS www.aobplants.oxfordjournals.org & The Authors 2015 Bashir et al. — Synergistic effects of S-deficiency and Cd on mustard

4; + + ¼ supplied with a sufficient amount of S ( S/ Cd). Total Chl NS ¼ NS Cd

9.15 13.1 +

n (a b) content decreased by 22 % (7 DAT) and 21 % (14 5.33 + Cd + + , 22 %) 8%) 60 %)** 2 + DAT) in 2S/2Cd, 49 % (7 DAT) and 50 % (14 DAT) in 1 + 2 2 ( ( ( S/ 2S/+Cd and, 28 % (7 DAT) and 35 % (14 DAT) in 1 98.4 237.2 335.6

No CdCl Cd-treated plants supplied with a sufficient amount of S

¼ (+S/+Cd). In fact, compared with the control, the ratio

Cd of Chl a to Chl b tended to decrease in plants treated 4.9 9.2 2 5.10 , Cd + + 2

57 %)** 38 %)* with Cd particularly under Sulfur deficiency (Table 3). 58 %)** 2 + 1 4 + 2 2 ( ( (  S/ Leaf area was also reduced by 28 % (7 DAT) and 31 % 2 97.4 129.4 226.8 MSO (14 DAT) in S-deprived plants and the reduction was more m

30 pronounced (48 % (7 DAT) and 54 % (14 DAT)) when NS NS ¼ ...... S-deficient plants were exposed to Cd (Fig. 3A). The pres- S 12.7 SE) of 10 samples and four replicates ( 4.8 2 11.7 Cd +

, ence of S in Cd-stressed plants, however, had a lesser ...... 22 %) 10 %) 47 %)** + + 2 2 + 2 2 + 2

4 effect of 235 % (7 DAT) and 236 % (14 DAT). Sulfur ( ( ( S/

2 deficiency led to a reduction of leaf DW by 27 % (7 DAT) MSO

m and 40 % (14 DAT). Upon exposure to Cd, S-deficient DHA and AsA/DHA ratio) under different combinations of

300 plants further decreased DW, by 40 and 50 % at 7 DAT +

¼ and 14 DAT, respectively. However, the DW of leaves S 15.3 (00) 238.4 12.3 (00) 329 +

2.6 (00) 90.6 in S-sufficient plants treated with Cd decreased by 21 % Cd + + + 2

Cd). (7 DAT) and 38 % (14 DAT) (Fig. 3B). S/ 2 1 304.9 61.6 14 366.5 S/ + Discussion NS ...... 7.9 12.1 Cadmium stress as well as S deprivation leads to oxidative 4.17 Cd + + 33 %)* 19 %) 33 %)*

+ stress in plants (Pilon et al. 2006; Bashir et al. 2013) due to 1 2 2 + ( ( ( S/ peroxidation of biomolecules including lipids. In this 1 164.6 254.5 89.9 study, an increase in the magnitude of oxidative stress was observed in response to S-deficiency and Cd stress. Cadmium induced more oxidative stress, particularly in 7.2 10.2 4.18 Cd + + Sulfur deficient conditions. This indicated the formation 42 %)** 24 %)* 40 %)** + 1 2 2 + ( ( ( S/ of more ROS in leaves, not only under S-deficiency but FW. The values are mean and standard error of the mean (mean 2 142.3 236.9 94.6

1 also under Cd stress, with the most formed under dual 2 stress (2S/+Cd). However, the presence of S reduced

NS the magnitude of oxidative stress (Fig. 1), indicating 7.5 10.6 3.85

Cd that Cd becomes more deleterious under Sulfur defi- + + 37 %)** 24 %)* 23 %) + 2 + 2 2 ciency. This could be because antioxidant systems in ( ( ( S/

2 plants might suffer a limitation of S for the synthesis of antioxidant enzymes and other /proteins, and ROS quenching molecules such as GSH. Thus, it is evident that Cd severely impairs the plant’s ability to counter- 12.2 (00) 155.3 13.4 (00) 238.2

3.2 (00) 82.9 attack ROS during S-deficiency. Hu et al. (2015) also Cd + + + 2 found that a source of S (SO2) helped wheat to resist S/ 1 7 ...... Controloxidative stress. Control

Antioxidative enzymes Cadmium influences the ascorbate-GSH antioxidant sys- FW) 313.9

1 tem (Markovska et al. 2009) and metabolism of essential 2 elements (Dong et al. 2006), including non-enzymatic FW) 67.3 FW) 246.6 0.01; NS, non-significant). Figures in parentheses are per cent variations with respect to control ( 1 1

≤ a 2

. (GSH; AsA; -tocopherol and carotenoids) and enzymatic 2 2 P (SOD,APX,GR,CAT,etc.)antioxidants.Inthepresent Changes in different attributes of ascorbates (reduced ascorbate, AsA; oxidized ascorbate, DHA; total ascorbate, AsA

DHA (nmol g study, SOD, APX, GR and CAT activities were studied. M CdCl + 0.05; ** m The activity of all these enzymes was lower under ≤ P AsA (nmol g Treatment/parameter Days after treatment (DAT) AsA AsA/DHA 3.66 1.87 1.50 1.83 4.95 2.63 1.33 2.41 DHA (nmol g Sulfur (S) and Cadmium (Cd) in mustard leaf. Units are expressed in nmol g Table 1...... * 100 S-deficiency, which may be attributed to the limitation

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Table 2. Changes in different attributes of thiols (2SH) including reduced GSH, oxidized glutathione (GSSG), NPTs and PCs, and soluble protein content under different combinations of Sulfur (S) and Cd in mustard leaf. Values for GSH, GSH and GSH + GSSG are expressed in nmol g21 FW, NPT and PCs in nmol SH mg21 protein and soluble protein in mg g21 FW. The values are the mean and standard error of the mean (mean + SE) of 10 samples and four replicates (n ¼ 4; *P ≤ 0.05; **P ≤ 0.01; NS, non-significant). Numbers in parentheses are per cent variations with 22 22 respect to control (+S/2Cd). +S ¼ 300 mMSO4 , 2S ¼ 30 mMSO4 , 2Cd ¼ No CdCl2, +Cd ¼ 100 mM CdCl2.

Treatment/ Days after treatment (DAT) parameter ...... 7 14 ...... 1S/2Cd 2S/2Cd 2S/1Cd 1S/1Cd 1S/2Cd 2S/2Cd 2S/1Cd 1S/1Cd Control Control ...... GSSG 23.2 + 1.8 (00) 17.9 + 1.2 20.5 + 1.4 60.4 + 2.8 27.8 + 1.8 (00) 24.3 + 1.9 21.5 + 1.8 62.4 + 4.2 (223 %)* (212 %)NS (+160 %)** (212 %)NS (223 %)NS (+124 %)** GSH 101.2 + 6.1 (00) 72.8 + 6.2 75.1 + 7.1 212.7 + 7.2 125.2 + 3.9 (00) 102.8 + 4.8 82.1 + 3.4 226.7 + 9.8 (228 %) (226 %) (+110 %)** (218 %)NS (234 %)* (+81 %)** GSH + GSSG 124.4 + 6.7 (00) 90.7 + 4.9 95.6 + 5.4 273.1 + 9.6 153 + 4.5 (00) 127.1 + 5.2 103.6 + 5.2 289.1 + 12.1 (216 %)NS (227 %)* (+119 %)** (217 %)NS (232 %)* (+89 %)** GSH/GSSG 4.36 4.06 3.66 3.52 4.50 4.23 3.81 3.63 NPTs 445.3 + 31.3 (00) 155.8 + 10.1 227.8 + 16.2 586.1 + 36.4 495.2 + 40.2 (00) 154.7 + 7.4 241.6 + 15.3 887.5 + 100.2 (265 %)** (249 %)* (+32 %)* (269 %)** (251 %)* (+79 %)** PCs 320.9 + 18.4 (00) 65.1 + 4.6 132.2 + 9.3 313 + 22.6 342.2 + 24.4 (00) 27.6 + 2.6 138 + 8.4 598.4 + 44.7 (280 %)** (259 %)** (22%)NS (292 %)** (260 %)** (+75 %)** & h uhr 2015 Authors The Bashir et al. — Synergistic effects of S-deficiency and Cd on mustard

in the amount of S-containing amino acids and hence NS lower synthesis of enzymes. Under dual (2S/+Cd) stress 0.06 0.15 0.04 Cd 38 %)* 35 %)* 26 %) + + + the activity of APX, GR and CAT further decreased as a re- 1 2 2 2 ( ( ( S/ sult of S deficiency. Interestingly, Cd increased the activity 1 1.56 2.24 0.68 of SOD despite limited S availability, which perhaps is due to the channelling of infrastructural S towards the synthe- sis of more SOD, to counter ROS. A similar observation of lesser degree was made in Arabidopsis by Bashir et al. 0.05 0.12 0.05 Cd 55 %)** 50 %)** 39 %)* + + +

1 (2013). Unlike SOD, APX and GR activities did not increase 2 2 2 ( ( ( S/ under dual (2S/+Cd) stress but decreased further, indi- 2 1.14 1.70 0.56 cating that Cd-tolerant mustard prefers the strengthen- ing of SOD rather than the ascorbate-GSH antioxidant

NS system. These results also clearly show that due to the 0.14 0.16 0.05 lower activity of the ascorbate-GSH antioxidant system Cd 23 %)* 21 %)* 16 %) + + + 2 accumulation of H2O2 caused high damage to chloro- ...... 2 2 2 ( ( ( S/

2 phylls. The maximum decrease in APX, GR and CAT activ- ities occurred under Cd stress during S-deficiency, which ...... could be attributed to the binding of Cd metal with the thiol groups of these enzymes (Romero-Puertas et al. 2007). SOD showed a different response perhaps due to 0.14 (00) 1.93 0.21 (00) 2.70 0.06 (00) 0.77

Cd Cd entrapment by peptides, GSH/GSH oligomers or anti- + + + 2 oxidant enzymes, helping up-regulate SOD activity. Sulfur S/ 1 2.52 3.44 14 Control 0.92 deficiency significantly suppressed APX and GR activities...... The decline in APX and GR activities may be due to GSH depletion and a subsequent reduction in the NS ascorbate-GSH cycle (Gomes-Junior et al.2006)as 0.04 0.07 0.08

Cd showninFig.3B and C. The recovery of CAT activity at 16 %) 32 %)* 28 %)* . + + + 1 2 2 2 2 0.01; NS, non-significant). Numbers in parentheses are per cent variations with respect to control ( ( ( S/ 14 DAT in S-deficient plants might be due to the presence ≤ 1 0.71 1.62 2.33

P of comparatively more Fe (metal ligand of CAT) in the ab- M CdCl

m sence of S and that the defence system acts better 0.05; ** 100 against oxidative stress and/or compensates for the de- ≤ ¼ crease in other antioxidant enzymes such as APX and P 0.03 0.05 0.10 Cd Cd 4; * 39 %)* 53 %)** 49 %)** + GR but at non- locations in the cell. The scen- + + + 1 , 2 2 2 ¼ 2 ( ( ( S/ ario was totally different in S-sufficient plants exposed n 2 0.52 1.12 1.64 toCd.Theactivitiesofallfourenzymes(SOD,APX,GR

No CdCl and CAT) were up-regulated; the maximum SOD was ¼ 136–153 %. NS NS Cd 2 , 0.03 0.09 0.14 Cd FW) and chlorophyll a/b ratio in mustard leaf treated with different combinations of Sulfur (S) and Cd. The values are mean and standard 2 Ascorbate, glutathione and sulfur-rich compounds 25 %)* 21 %) 22 %) 2 + + + 1 2 4 2 2 2 2 ( ( ( S/ The ascorbate-GSH cycle appears to be of great import- 2 MSO ance in controlling the cellular redox status, especially m

30 upon exposure to Cd. Both ascorbate and GSH are essen-

¼ tial for scavenging ROS (Anjum et al.2008) and are im- S 2

, portant in controlling metal . The ratio of 2 2 .04 (00) 0.64 0.10 (00) 1.87 0.17 (00) 2.51 4

SE) of 10 samples and four replicates ( AsA in the reduced form (AsA) to that in the oxidized Cd + + + + 2 state (DHA) is considered an important indicator of the S/ MSO m 1 0.85 Days after treatment (DAT) 7 Control 2.38 ...... 3.23 redox status of the cell and the degree of oxidative stress

300 experienced. Data (Table 1) show that there was an over-

¼ all increase in DHA and a decrease in AsA and total ascor- S

+ bate content, indicating inhibition of ascorbate synthesis. Changes in chlorophyll contents (mg g b ratio 2.80 2.92 2.15 2.28 2.74 2.51 2.03 2.29 Cd). b However, the Asa/DHA ratio varied; from 4.95 (control) it + / 2 b a a a droppedto2.63(2S/2Cd), 1.33 (2S/+Cd) and 2.41 S/ Chl Chl Chl Chl Treatment/ parameter + + + error of the mean (mean Table 3...... ( ( S/ Cd) at 14 DAT. The AsA/DHA ratios can be attributed

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Figure 3. Effect of S-deficiency and Cd stress on (A) leaf area (cm2 leaf21) and (B) dry weight (mg plant21) of mustard leaf. The values are mean and standard error of the mean (mean + SE) of 10 samples and four replicates (n ¼ 4; *P ≤ 0.05; **P ≤ 0.01; NS, non-significant). +S ¼ 300 mM 2− 2− SO4 , 2S ¼ 30 mMSO4 , 2Cd ¼ No CdCl2, +Cd ¼ 100 mM CdCl2.

to APX activity, which helps in AsA regeneration and in- free Cd concentration in the and helps sup- creases the AsA/DHA ratio. press activation of the stress-related responses in plant Glutathione is the main S-storage compound and an metabolism (Metwally et al. 2005). It has been shown important antioxidant in plants. The content and redox that S-nutrition status is associated with plant response + level of GSH was measured to determine the effect of to Cd, at least at the plasma membrane H ATPase level S-deficiency on the amount of GSH. In S-deficient plants, (Astolfi et al. 2005), with a concomitant uptake of sulfate the GSH content decreased compared with the control. at a higher rate (Nocito et al. 2002). Interestingly, the This decrease was more pronounced when S-deficient levels of NPTs and PCs fell far below those of control mustard was exposed to Cd. However, when S was sup- plants, indicating that the cell utilizes reserves of S-rich plied to these plants, there was a marked increase in compounds that are found in relatively high concentra- GSH levels (Table 2). Thus, S plays a key role in limiting tions in Indian mustard, perhaps making it Cd-tolerant the cellular damage and inducing defence mechanisms and a hyperaccumulator. Adaptation of sulfate uptake against the ROS in response to Cd treatment through and assimilation is assumed to be a crucial determinant the accumulation of GSH. The combination of Cd stress for plant survival in a wide range of adverse environmen- with S-deficiency provided novel data in the present tal conditions since different S-containing compounds study, but otherwise the results were consistent with are involved in plant responses to both biotic and abiotic the results obtained by Smeets et al. (2005), who reported stresses (May et al. 1998; Rausch and Wachter 2005). that heavy metals such as Cd bind to GSH, forming Cysteine provided to plants through sulfate assimilation metal-thiolate compounds. This suggests that GSH pathways acts as a source of reduced S for the biosyn- might be involved in the synthesis of PCs, which could de- thesis of a number of S-containing compounds, including toxify Cd ions. It also plays an indirect role in protecting GSH. Cysteine synthesis has previously been shown to be membranes by maintaining a-tocopherol and zeaxanthin improved with the addition of S sources, and a Cys defi- in the reduced form. Glutathione levels and GSH/GSSG ra- ciency limits the synthesis of GSH (Nikiforova et al. 2006; tios are often indicative of the stress faced by the plant Chekmeneva et al. 2011). Therefore, GSH synthesis in (Tausz et al. 2004). The response of GSH and redox state plant cells as observed in the present study relies on has varied in different studies. In this study, S-deficiency, and is regulated by the plant’s S supply. A sufficient Cd stress under S-deficiency and Cd stress during suffi- amount of S helped plants not only to maintain, but cient S decreased the GSH/GSSG ratio, from 4.50 in also to accumulate GSH, NPTs and PCs by 90, 79 and the control to 4.23 (2S/2Cd), 3.81 (2S/+Cd) and 3.63 57 %, respectively, over the control at 14 DAT. The level (+S/+Cd) at 14 DAT, showing a ratio-maintaining cap- of different types of PCs was shown to increase with

ability of plant only in the presence of sufficient S. The 100 mMCdCl2 in mustard (D’Allessandro et al.2013) data suggest a direct correlation between GR activity using a proteomic approach, strongly supporting the pro- and GSH/GSSG ratio. tective role of PCs against Cd. Phytochelatin binds to toxic The addition of sufficient S tended to recover GSH levels metal and then the metal–PC complex is sequestered in and maintain the redox status of the cells during short- the and perhaps induces specific transporters and long-term exposure to Cd. Under Cd stress, the for- that mediate Cd tolerance to Arabidopsis (Park et al.2012). mation of Cd–GSH and Cd–PC complexes reduces the However, the efficiency of PC-based Cd-detoxification is

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subject to the availability of other nutrients such as iron 31 % in leaf area and 40 % in DW) or Cd stress (up to (Astolfi et al. 2011). 36 % in leaf area and 38 % in DW), indicating that the ef- fect of Cd treatment and Sulfur deficiency is synergistic. Chlorophyll and growth Moreover, leaf area was found to be more sensitive in re- Cadmium may directly inhibit the process of photosyn- sponse to Cd stress under S-deficiency. Ouzounidou et al. thesis by its interaction with enzymes (Siedlecka and (1997) suggested that the inhibitory action of heavy me- Krupa 1999). Indirectly, Cd may inhibit the synthesis of tals on -shoot and leaf growth seems principally to be photosynthetic pigments or cause their degradation; due to chromosomal aberrations and abnormal cell divi- S-deficiency further worsens the scenario (Bashir et al. sions. It may also be correlated with Cd-induced inhib- 2013). The effects of Cd on N and S assimilation have ition of photosynthetic processes and other enzymes been studied in several plants. The current results showed involved in leaf expansion. Decreased plant growth that both Cd and S-deficiency significantly reduced the caused by heavy metals is a collective consequence of in- chlorophyll content and the Chl a to Chl b ratio in a hyper- hibition of photosynthesis, translocation of photosyn- accumulator plant, Indian mustard. Both S and Cd, alone thetic products and cell division (Dra˛z˙kiewicz and or in combination, cause leaf (Bashir et al. 2013). Baszyn´ski 2005). Several studies have suggested that Cd-induced leaf 2+ chlorosis might be due to impairment of the Mg inser- Conclusions tion into protoporphyrinogen (Gillet et al. 2006) or direct Chl destruction as a consequence of Mg ion substitution In conclusion, we confirmed that S-deficiency increases in both Chl a and b (Ku¨pper et al. 1998). Our results the susceptibility of plants to Cd-generated oxidative showed clear and very rapid inhibition of Chl a, rather damage and modulates the AsA-GSH cycle. We propose than Chl b, mainly in response to Cd stress, with or with- that S helps the accumulation of GSH and other S-rich out S-deficiency. As an early response to S-deficiency, the compounds to detoxify metals. In turn, SOD expression Chl a/Chl b ratio increased slightly (2S/2Cd at 7 DAT) but is up-regulated to reduce the concentration of superoxide in the remaining treatments there was a significant de- radicals. This study demonstrates that SOD is not the first crease in the Chl a/Chl b ratio. Cadmium may also impair line of defence against metal stress and that S-rich com- the S uptake, leading to leaf chlorosis and a disrupted pig- pounds play a prime role. Further, S-deprived plants lack ment ratio. It has also been proposed that a reduction in S-defence and efficient antioxidative mechanisms, mak- chlorophyll might be caused by direct interference by Cd ing Cd more dangerous. Under S-deficiency, S-containing with enzymes of the Chl biosynthesis pathways or by Cd defence metabolites (GSH, PCs, etc.) as well as enzyme interference with the correct assembly of the pigment– co-factors (Fe-S clusters) decline. The clear decrease in protein complexes of the photosystems (Baryla et al. Fe-S clusters, under S-deficiency, is crucial for the main- 2001; Qureshi et al.2010). It is also possible that the tenance of photosynthesis-related molecules and activ- Chl decrease may be due to strong oxidation of the photo- ities, but the presence of Cd under such conditions chemical apparatus and a reduction in chloroplast dens- might exacerbate oxidative stress, with a highly adverse ity and size. Sulfur deficiency in combination with Cd effect on growth. further alleviated chlorophyll inhibition. However, in the presence of sufficient S, we observed lower destruction Sources of Funding of chlorophyll. S is required to repair Fe-S-containing The authors extend their appreciation to the Deanship of protein complexes, including the incorporation of Fe-S Scientific Research at King Saud University for funding clusters into apoproteins and the stabilization of biomole- this work through research group no. RGP-VPP RG- cules with sulfolipids. It has also been proposed that Cd 1435-042. may influence the biosynthesis of chlorophyll by affecting protochlorophyllide reductase, which, however, contains oxygen-tolerant Fe-S clusters (Nomata et al. 2008), indi- Contributions by the Authors cating the role of S in protection. The research design and preparation of the manuscript When the overall impact of S-deficiency and Cd stress are credited to M.I.Q., H.B. and M.M.I. H.B., R.B., J.A. and on leaf area and leaf biomass was analysed, S showed a M.A.B.contributedtodatacollectioninreplicates. positive role under Cd stress. Sulfur deficiency and Cd M.I.Q., H.B. and I.A.A. analysed the data. caused a significant decrease in leaf area and DW, at least with sufficient S-supply. In the current study, growth inhibition was more severe in 2S/+Cd (up to 54 % in leaf Conflicts of Interest Statement area and 50 % in DW) plants than in S-deficiency (up to None declared.

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