The potential of zinc accumulation in Gynura pseudochina (L.) DC.: study in a tissue culture system Presented Mr. Natthawoot Panitlertumpai

Transcription

1 The potential of zinc accumulation in Gynura pseudochina (L.) DC.: study in a tissue culture system Presented Mr. Natthawoot Panitlertumpai p_natthawoot@yahoo.com (advisor) woranan_n@yahoo.com

2 Phytoremediation Remediation Soil Contaminated Soil

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4 Phaenark et al., 2009 (International Journal of Phytoremdiation, vol.11, pp ) Mining area Extractable concentration Zn mg/kg Cd mg/kg

5 Table. Summary of elemental levels in background soil, metalliferous (or contaminated) soil, critical load in soil above which biodiversity and ecosystem function are adversely af- fected, and Commission des Communautés Européennes (EU) and Environmental Protection Agency (USA) permissible limits. a b Lakin 1972, c Reeves and Baker 2000 d ca e f g Bannick et al. 2002; Sand ~50% of loam/silt levels; clay ~1.5X loam/silt levels, h i H2O, j Soil, k Wendy Ann Peer et al. 2005

6 Gynura pseudochina (L.) DC. Panitlertumpai et al., 2003 (the 29 th Congress on Science and Technology of Thailand 2003, Khon Kaen, Thailand)

7 Gynura pseudochina (L.) DC. Thlaspi arvense g Zn/kg-ash 4 g Cd/kg /kg-ash ~ g Zn/kg-ash

8 Gynura pseudochina (L.) DC. Panitlertumpai et al, 2009 (Minerals Engineering ; accepted td in revise process).

9 Suitable conditions for practical application of hyperaccumulation The mechanism of accumulation and level of zinc tolerance

10 SDS-PAGE X-ray absorption spectroscopy

11 Tissue culture system

12 Objective Study the mechanism of zinc accumulation and tolerance in protein of Gynura pseudochina (L.) DC. by tissue culture system. 1. The protein were studied by extraction and separation with SDS-PAGE 2. The from of zinc on crude protein was studied by X-ray absorption spectroscopy

13 Methods

14 Plant culture and metal treatment Non-contaminated shoot tips (3 cm) Sectioned apical bud Cultured on 20 ml ½ MS Cutting nodal sterilized following with Gambrog (1995) tissue lengths 1cm medium for 2 months segment 0.5 cm Culture on 20ml MS plus 2 mg l -1 of IAA and IBA for 4 weeks Culture on 20ml ½ MS for 4 weeks Exposed 2 ml of sterilize zinc solution 0, 100, 250, 500, 750 and 1,000 mg l -1 for 14 days Exposed 2 ml of sterilize zinc 20

15 Zinc accumulation and tolerance After 14 days Washed with deionized water and blotted dried Determined yellowish colour or chlorosis of leave Determined weight and height and separated plant Filtered No g Analysis zinc by FAAS (Shimaszu AA-680) Digestion with the modified method of Miller(1998) by HNO 3 and HClO 4 Sample dry 80 o C for 24 hr, determine dry weight and mashed sample

16 Protein analysis 100 mg of sample Collect crude protein and keep at C for analysis Ground with phenol saturated buffer followed Seangdee et al., (2003) Wash with acetone Centrifuge at 13,000 rpm Precipitate 5 volume of 100 mm ammonium acetate, incubate 20 O C for 30 min Freeze dried Press a pellet e protein Pack in Mylar film Dissolve protein of leaves in 1X Protein sample buffer Load μl of total protein SDS-PAGE 30 ma 90 min Proton Factory BL-12C, Japan X-ray absorption spectroscopy Stained and estimate molecular weight by standard

17 Results

18 Effect of zinc concentration on growth Dry wei ight (g) Height Dry weight Concentration (mg l -1 ) Fig. Dry weight and height of G. pseudochina (L.) DC. after 14 days treated with various concentration of zinc Heigh ht (cm)

19 Dry weight (g) Height Dry weight Concentration (mg l -1 ) Height (cm) Fig. Dry weight and height of G. pseudochina (L.) DC. after 14 Fig. Dry weight and height of G. pseudochina (L.) DC. after 14 days treated with various concentration of zinc.

20 Table. Toxicity of zinc concentration that result in yellowish color or chlorosis of leaves Zinc concentration Total number Number of % LD 50 (mg l -1 ) of plant appearance symptom

21 Zinc accumulation and translocation factor Table. Zinc accumulation (averages±sd; n=8) in G. pseudochina (L.) DC. exposed to difference concentration of zinc and translocation factors (n=4). Zinc addition (mg l -1 ) Zinc accumulation in plant (mg g -1 dry weight) Root Stem Leave Shoot Translocation Factor (TF) ±0.14 a 0.29±0.14 a 0.16±0.04 a 0.45±0.11 a 0.67±0.06 a ±0.37 ab 0.62± ab 0.57± ab 1.19±0.37 b 0.67± a ±0.63 b 0.94±0.17 b 0.91±0.20 bc 1.85±0.26 b 0.72±0.08 a ±1.66 c 2.07± c 1.28±0.31 c 3.35±0.29 c 0.74± a ±0.95 d 3.02±0.68 d 1.83±0.26 d 4.85±0.75 d 0.80±0.06 a 1, ±0.75 d 3.98±0.86 e 2.25±0.57 e 6.23±1.05 e 0.98± b Panitlertumpai et al, 2009 (Minerals Engineering ; accepted in revise process).

22 Effect of zinc on protein patterns M C Fig. SDS-PAGE of proteins from control (C) and Zn treatment of 100, 250, 500 and 1000 mg l -1 of zinc solutions and protein marker (M)

23 XANES spectrum of zinc on protein Nomalize ed intensity (a.u.) ZnSO 4 7H 2 O (a) ZnCl 2 (b) Zn(NO 3 ) 2 (c) ZnO (d) ZnS (e) Protein-Zn (f) Energy (ev) Zinc in protein was possibly ZnS (80.95% fitting) and ZnO (19.05% fitting)

24 EXAFS spectrum of zinc on protein Atom = Element N= oscillation yielded coordination number R= inter atomic distance Zn-S coordinate shell were Experiment data 2.31 (N), 2.29 A o (R) and Rule data (σ 2 ) Difference from the residual of the immediately preceding fitting

25 - G. pseudochina (L.) DC. had ability to tolerance and growth in high zinc concentration - Zinc content in plant was depended on the zinc concentration - G. pseudochina (L.) DC. had a potential for a hyperaccumulator with zinc high concentration over short periods - Zinc might induce protein molecular mass 26.4 kda for zinc stress - The first of sulfur ligand bond zinc on the protein

26 Junior National Mahasarakham Photon Factory, science talent and university KEK project technology development agent

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