Arsenate Exposure Affects Amino Acids, Mineral Nutrient Status and Antioxidant

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1 1 Supporting Information Arsenate Exposure Affects Amino ids, Mineral Nutrient Status and Antioxidant in Rice (Oryza sativa L.) Genotypes S. Dwivedi, R.D. Tripathi, *,, P. Tripathi, A. Kumar, R. ve, S. Mishra, R. Singh, D. Sharma, U.N. Rai, D. Chakrabarty, P.K. Trivedi, B. Adhikari, M.K. g, O. P. Dhankher and R. Tuli, Pages=15 Contents: Table S1. Physico-chemical properties and metal composition of soil after supply of different As V concentrations Figure S1. Effect of As V on Fe accumulation in different parts of rice cultivars Figure S2. Effect of As V on P accumulation in different parts of rice cultivars Figure S3. Effect of As V on Zn accumulation in different parts of rice cultivars Figure S4. Effect of As V on S accumulation in different parts of rice cultivars Figure S5. Effect of As V on number of tillers; plant height; panicle length; grain weight and total amino acid content in different rice cultivars. Figure S6. Effect of As V on chlorophyll a; chlorophyll b; total chlorophyll; carotenoid; protein and cysteine in different rice cultivars S1

2 MATERIALS AND METHODS Soil analysis. The soil samples were collected post harvesting of the plants, in polythene bags and brought to the laboratory for analysis. Sulphur (1) and available N content in soil were estimated (2) along with ph and EC of soil by ion meter (Orion, USA), while water holding capacity was measured through hydrometery. The ph and EC of soils were measured using ion meter (Orion, USA) while, water holding capacity was measured through hydrometery. The total organic carbon, bulk density, particle density and available phosphorus were measured by following the protocol of rter and Gregorich (3) Plant harvest and DCB treatment. At maturity, plants were harvested and separated into root, shoot, husk, and grains for metals and metalloids analysis. Roots were washed with deionized water (thrice) and 1 g roots were treated with DCB solution following the protocol of Taylor and Crowder (4) for the removal of adsorbed As onto the plaque Amino id Analysis. The pico tag method (5) was followed for the estimation of amino acids on HPLC system. 2 mg of homogenized seeds were hydrolyzed in 1 ml 6 N HCl for an hour at ºC in an oven. Samples were then filtered for further analysis. 1 µl of samples and standard (2.5 µ moles ml -1 in.1 N HCl) were dried in a vacuum oven at 55 ºC for min at 75 milli torr. This was redried twice by adding 2 µl of redrying solution (Ethanol:Triethylamine:Water, 2:1:2). Then samples were derivatizated by adding 2 µl of derivatization reagent (Ethanol:Triethylamine:Water:Phenyoisothiocynate, 7:1:1:1) and again vacuum dried. These samples were diluted to 1 ml with pico tag sample diluent, filtered (.22 µm syringe filters) and injected (2 µl) into the system. The S2

3 5 51 samples were analyzed on a pico tag column eluted with a gradient of (A) an aqueous buffer and (B) 6% acetonitrile in water Growth, photosynthetic pigments and protein estimation. Plant height and panicle length was measured by meter scale, however number of tillers were counted manually during the harvesting of the rice plants. The grain weight was measured using an electronic balance. Chlorophyll content was estimated by the method of Arnon (6) and that of carotenoid by using the formula given by Duxbury and Yentsch (7). Protein content was estimated following the method of Lowry et al. (8). Cysteine content was estimated following the method of Gaitonde (9) Quality control and assurance. The standard reference materials of metals (E-Merck, Germany) were used for the calibration and quality assurance for each analytical batch. Analytical data quality of metals was ensured with repeated analysis (n=3) of quality control samples, and the results were found within (±2.82) the certified values. Recovery of Fe, Zn, and Se from the plant tissue was found to be more than 96.5%, as determined by spiking samples with a known amount of metal, while for As, rice flour NIST 1568a was used as a reference material with known spiked samples, and recovery of total As were 85.3% (±2.8; n=5) and 89.5% (±3.1; n=5), respectively. The detection limit for Fe and Zn were 1 and.2 mg l 1, respectively, while for As and Se, it was 1 µg l RESULTS Effect of As V on Fe accumulation. A significant genotypic variation among the rice cultivars was observed with respect to Fe (mg kg -1 dw) levels in the roots (ranging from S3

4 to 16114), which increased by irrigation with As V containing water (Fig. S1 A). Maximum accumulation was found in IET-4786 (21151) followed by IR-36 (19774), PNR-519 (19756) and Triguna (15822) at 8 mg l -1 As. The accumulation of Fe in the shoots was much lower and followed a different pattern in comparison to roots (Fig. S1 B). Fe uptake in IR-36 enhanced significantly up to 12 mg l -1 while in other cultivars it increased only up to 8 mg l -1 As supply. The amount of Fe in husk (Fig. S1 C) and grain (Fig. S1 D) also varied significantly. Similar to root and shoot, an initial increase in Fe accumulation at lower dose (up to 8 mg l -1 ) of As followed by inhibition at higher concentration (12 mg l -1 ) was also observed in husk and grain, except IR Effect of As V on P accumulation. A decrease in the level of P (mg kg -1 fw) was observed at higher As dose (12 mg l -1 ). P (mg kg -1 dw) content in roots (Fig. S2 A) was highest in PNR-519 (368, R=.982**) followed by Triguna (2, R=.888*), IET-4786 (285, R=.995**) and IR-36 (275, R=.919*) in control plants. The level of P in roots increased with increasing As supply up to 12 mg l -1 in all cultivars. The level of P in shoots of control plants followed the order Triguna (388)> IET (317)> PNR-519 (8)> IR-36 (4) (Fig. S2 B). The level of shoot P increased upon As supply upto 8 mg l -1, except IET-4786, which showed increasing trend upto 12 mg l -1 As. The P content in husk (Fig. S2 C) and grains (Fig. S2 D) also increased with increase in As supply up to 12 mg l -1 in all the varieties in comparison to control, except the grains of IET-4786, which increased up to 4 mg l -1 As supply Effect of As V on Zn accumulation. Zn accumulation (mg kg -1 dw) in rice roots varied significantly amongst the genotypes. The roots (Fig. S3 A) of PNR-519 (12, R=.951*) S4

5 accumulated maximum amount of Zn, followed by IR-36 (98, R=.991***), IET-4786 (92, R=.968**), and Triguna (81, R=.993***) at 8 mg l -1 As supply. Zn content in shoots (Fig. S3 B) increased only up to 4 mg l -1 As supply in all varieties, except PNR- 519 where it increased up to 12 mg l -1. As supply, up to 8 mg l -1 increased Zn content in husk (Fig. S3 C) of all the cultivars except IR-36 (67) where Zn accumulation was found to increase upto 12 mg l -1. The maximum Zn level in grains (Fig. S3 D) was found in IET-4786 (37, R=.948*), while minimum in IR-36 (29, R=.991**) at 8 mg l -1 As supply Effect of As V on S accumulation. By increasing As concentration in soil, S (mg kg -1 dw) content in roots (Fig. S4 A) increased upto 8 mg l -1 As in Triguna and PNR-519 while upto 12 mg l -1 in variety IR-36 and IET Significant genotypic difference between S accumulation in roots was observed i.e. PNR-519 accumulated approximately 6 fold higher S than IR-36 (19) in control plants. Arsenic exposure caused a significant increase in S accumulation in all plant parts in selected rice cultivars (Fig. S4 A-D). Although, upon higher As supply a decrease in level of S was observed in various plant parts of different cultivars but not lower than control except for roots of Triguna at 12 mg l -1 As supply. Variable trend of S transport and accumulation was observed in different rice cultivars. Triguna and IR-36 showed comparatively good translocation of S in the above ground parts than PNR-519 and IET-4786 in both control and As treated plants. Whereas, As treatment enhanced the level of S in grains maximum being in IET-4786 and minimum in Triguna. The S accumulation in grain (Fig. S4 D) was maximum in IET (121, R=.833 NS ), followed by Triguna (942, R=.923*), PNR-519 (9, R=.977**) and low S was detected in IR-36 (714, R=.94*) at the 8 mg l -1 As. S5

6 Effect of As V on growth and yield of rice plants. The number of tillers (Fig. S5 A) was found to be a varietal character which ranged from 17 to 22 in selected rice cultivars, however, by increasing concentration of As V in soil, the number of tillers increased in all the four cultivars up to 8 mg l -1 As, but decreased significantly at higher As supply (Fig. S5 A). There was no significant change observed in terms of plant height and panicle length up to 4 mg l -1 As but at higher As V doses (8 and 12 mg l -1 ), significant decrease in plant height and panicle length were noticed (Fig. S5 B, C). Similarly, an increase in seed weight (Fig. S5 D) was observed by As supply only up to 4 mg l -1 in all the cultivars in comparison to control. In general, As V toxicity had led to early flowering in all selected rice cultivars leading to early seed maturity. The photosynthetic pigments (chl a, b, and total chl) increased at 4 mg l -1 in all the cultivar followed by a decline up to 12 mg l -1 (Fig. S6 A-C). IET-4786 and PNR-519 seems to be more prone to As toxicity showing 55% and 5% reduction in total chl contents, in comparison to IR-36 and Triguna, which showed a reduction of 26% and 15%, respectively. In contrast, carotenoid (Fig. S6 D) contents of PNR-519 and IET-4786 increased by increasing As concentration up to 12 mg l -1, however, a dose dependent decline was observed in Triguna and IR-36. The leaf protein content in all the rice cultivars was induced significantly in a concentration dependent manner (Fig. S6 E) S6

7 Table S1: Physico-chemical properties and metal composition of control pot soil and after supply of different As V concentrations. All the values are mean of triplicates ±S.D. ANOVA significant at p.1. Different letters indicate significantly different values at a particular treatment (DMRT, p.5). Parameters Control As (4 mg l -1 ) As (8 mg l -1 ) As (12 mg l -1 ) ph 7.6 α ± α ± α ± α ±.44 Electrical conductivity (Ω) γ ± β ± β ± α ±9.21 Total organic carbon (%) 2.21 α ± α ± α ±.4 2. α ±.2 Water holding capacity (%) α ± α ± α ± α ±5.1 Bulk density (g cm -3 ) 1.26 α ± α ± α ± α ±.5 Particle density (g cm -3 ) 1.68 α ± α ± α ± α ±.4 Available nitrogen (%).49 γ ± α ±.3.81 β ±.2.9 β ±.1 Metals (mg kg -1 ) Available phosphorus δ ± α ± β ± γ ±16.66 Fe α ± α ± α ± α ±299.5 Zn 221 α ± α ± α ± α ±21.89 As 5.43 γ ± β ± β ± α ±2.81 S 3.18 α ± α ± α ± α ±.2 Se 6.52 α ± α ± α ± α ± S7

8 A 75 Control mg l-1 B Control mg l-1 Root-Fe (mg kg -1 dw) Shoot-Fe (mg kg -1 dw) 6 45 Cd C 4 D Husk-Fe (mg kg -1 dw) Bd Grain-Fe (mg kg -1 dw) 2 1 Ad Bd Figure S1 (A-D): Effect of As V on Fe accumulation in different parts of rice cultivars. Root Fe (A); Shoot Fe (B); Husk Fe (C); Grain Fe (D). All values are mean of triplicates ±S.D. ANOVA significant at p.1. Different capital letters indicate significantly different values among As treatments in a particular rice cultivar and small letters indicate significantly different values among rice cultivars at a particular treatment (DMRT, p.5). S8

9 1 A 75 Control mg l-1 B Control mg l-1 Root-P (mg kg -1 dw) Shoot-P (mg kg -1 dw) 6 45 Husk-P (mg kg -1 dw) C Grain-P (mg kg -1 dw) D Cd Figure S2 (A-D): Effect of As V on P accumulation in different parts of rice cultivars. Root P (A); Shoot P (B); Husk P (C); Grain P (D). All values are mean of triplicates ±S.D. ANOVA significant at p.1. Different capital letters indicate significantly different values among As treatments in a particular rice cultivar and small letters indicate significantly different values among rice cultivars at a particular treatment (DMRT, p.5) S9

10 182 A 375 Control mg l-1 B Control mg l-1 Root-Zn (mg kg -1 dw) Shoot-Zn (mg kg -1 dw) C 6 D Husk-Zn (mg kg -1 dw) Bd Grain-Zn (mg kg -1 dw) Bd Figure S3 (A-D): Effect of As V on Zn accumulation in different parts of rice cultivars. Root Zn (A); Shoot Zn (B); Husk Zn (C); Grain Zn (D). All values are mean of triplicates ±S.D. ANOVA significant at p.1. Different capital letters indicate significantly different values among As treatments in a particular rice cultivar and small letters indicate significantly different values among rice cultivars at a particular treatment (DMRT, p.5). 19 S1

11 12 A 6 Control mg l-1 B Control mg l-1 Root -S (mg kg -1 dw) 9 6 Ad Cd Shoot-S (mg kg -1 dw) 45 5 C 16 D Husk-S (mg kg -1 dw) Bd Bd Grain-S (mg kg -1 dw) Figure S4 (A-D): Effect of As V on S accumulation in different parts of rice cultivars. Root S (A); Shoot S (B); Husk S (C); Grain S (D). All values are mean of triplicates ±S.D. ANOVA significant at p.1. Different capital letters indicate significantly different values among As treatments in a particular rice cultivar and small letters indicate significantly different values among rice cultivars at a particular treatment (DMRT, p.5). S11

12 4 A 12 control mg l-1 B Control mgl-1 No. of Tillers 2 1 Plant height (cm) C 6 D Panicle length (cm) 2 1 Grain weight (g) 4 2 Amino acid (µ moles g -1 dw) E Ad Ad Bd Cd 198 Triguna IR-36 PNR-519 IET Figure S5 (A-E): Effect of As V on number of tillers (A); plant height (B); panicle length (C); grain weight (D) and total amino acid content (E) in different rice cultivars. All values are mean of triplicates ±S.D. ANOVA significant at p.1. Different capital S12

13 letters indicate significantly different values among As treatments in a particular rice cultivar and small letters indicate significantly different values among rice cultivars at a particular treatment (DMRT, p.5). 25 S13

14 Chlorophyll a (mg g -1 fw) A.4 control mg l-1 B Control mgl-1 Chlorophyll b (mg g -1 fw) Total chlorophyll (mg g -1 fw) C Ad Cd rotenoid (mg g -1 fw) D Cd Protein (mg g -1 fw) E Cysteine (n mol g -1 fw) F Cd Figure S6 (A-F): Effect of As V on chlorophyll a (A); chlorophyll b (B); total chlorophyll (C); carotenoid (D); protein (E) and cysteine (F) in different rice cultivars. All values are mean of triplicates ±S.D. ANOVA significant at p.1. Different capital letters indicate S14

15 significantly different values among As treatments in a particular rice cultivar and small letters indicate significantly different values among rice cultivars at a particular treatment (DMRT, p.5) References (1) APHA. Standard methods for the examination of water and wastewater (19th. St. ed.) Washington, USA, 21. (2) Jackson, M. L. Soil chemical analysis. Printice Hall of India Private Ltd., New Delhi, (3) rter, M.R., Gregorich, E.G. Soil sampling and methods of analysis. Second Edition. CRC Press, Taylor and Francis Group. 27, 264. (4) Taylor, G. J.; Crowder, A. A. Use of DCB technique for extraction of hydrous iron oxides from roots of wetland plants. Amer. J. Bot. 1983, 7 (8), (5) Bidlingmeyer, B. A.; Cohen, S. A.; Tarvin, T. L. Rapid analysis of amino acids using pre-column derivatization. J. Chromatogr. 1984, 336 (1), (6) Arnon, D. I. Copper enzymes in isolated chloroplast: polyphenoloxidases in Beta vulgaris. Plant Physiol. 1949, 24 (1), (7) Duxbury, A. C., Yentsch, C. S. Plankton pigment monograph. J. Mar. Res. 1956, 15, (8) Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. Protein measurement with folin phenol reagent. J. Biol. Chem. 1951, 193, (9) Gaitonde, M. K. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem. J. 1967, 14 (2), S15