Cadmium Stress Response in Catharanthus roseus leaves through Proteomic Approach

S.Pradeep Kumar, P.Arun Mozhi Varman, and B.D. Ranjitha Kumari# Stress Physiology & Medicinal Plant Biotechnology lab,

Department of Plant Science, Bharathidasan University, Tiruchirappalli -620 024. Tamil Nadu, India #corresponding author e-mail: ranjithakumari2004@yahoo.co.in

Abstract—Cadmium has been considered as an extremely significant pollutant due to its high toxicity and great solubility in water. A common consequence of most abiotic and biotic stresses is that they result, at some stage of stress exposure, in an increased production of reactive oxygen species (ROS). It is known that changes in ROS production may activate contrasting responses, an increase or a decrease in ROS scavenging systems, which will guarantee cell survival or induce cell death respectively according to the situation faced by the plant. The aim of the present work was to study the effects of a Cd exposure of C. roseus in a controlled environment using morphological, physiological and proteomic approaches. These findings might provide a framework for future investigations in order to highlight the biological impact of cadmium as well as to study the role of stress-induced proteins in the cadmium response pathways.

Keywords: Antioxidants; cadmium; chlorophyll; MALDITOF-MS; oxidative stress.

I. INTRODUCTION Industrial activity combined with a low conscience of the

consequences of environmental pollution during a long period created a worldwide problem of soil, air and water contamination with various pollutants. Heavy metals are among the most widespread soil contaminants. The effects of certain heavy metals such as Cd, Hg and Pb on cell systems have received attention in recent decades due to the increasing exposure of living organisms to these metals in the environment [1]. Among these, cadmium is generally considered as a non-essential heavy metal and its presence in the environment is essentially due to anthropogenic activities [2]. Cadmium has been considered as an extremely significant pollutant due to its high toxicity and great solubility in water. It can reach high levels in agricultural soils and is easily assimilated by plants [3]. The aim of the present work was to study the effects of a Cd exposure of C. roseus in a controlled environment using morphological, physiological and proteomic approaches. The plant response in young leaves exposed to 25µM Cd during 24 d, 2D gel electrophoresis coupled with mass spectrometry (MS) analysis has been used to address the proteomic changes of total soluble proteome. Differentially expressed proteins between control and treated plants were identified and their possible roles in Cd stress responses are discussed. These

findings provide a framework for future investigations in order to highlight the biological impact of cadmium, as well as to study the role of stress induced proteins in the cadmium response pathways.

II. MATERIALS AND METHODS Plant material, growth conditions and cadmium treatment

C. roseus seeds were germinated and grown under ambient conditions at temperatures ranging from 18 to 24 °C (night and day, respectively). When plants reached the desired size (15-17 cm), they were divided in 2 sets followed by transfer and acclimation to hydroponic culture in a modified 1/4- strength Hoagland’s solution. The first one acted as control, while in the second set, the nutritive solution was enriched with CdCl2 up to a final concentration of 25 μM. Sampling started at day 3, 6, 12 and 24. Leaves from the same foliar stage were used for proteomic analysis (leaf number 5 counted from the apex) and chlorophyll content analysis (leaf number 6). The next foliar stage below (leave numbers 7 and 8 pooled together), as well as roots and the upper tier of stems were used for cadmium content analysis. Immediately upon cutting, leaves for further analysis were frozen in liquid nitrogen.

A. Determination of chlorophyll For the chlorophyll determination leaves were ground with a mortar and pestle according to [4].

B. Cadmium content analysis The harvested plant material (roots and leaves) was

washed in distilled water and dried at 80 °C. The dried samples were digested in a mixture of HNO3–HClO4 (4:1, v/v). The residues were solubilized in 7.0 mM HNO3, and Cd concentrations in the tissue extracts were measured by atomic absorption spectrophotometry (Perkin Elmer-Analyst 300, Norwalk, CT, USA). The results were based on the average of seven replicated determinations.

C. Isolation of total soluble proteins Total soluble proteins were extracted following the

TCA/Acetone method [5].

64

2010 International Conference on Biology, Environment and Chemistry IPCBEE vol.1 (2011) © (2011) IACSIT Press, Singapore

D. Two-dimensional gel electrophoresis and image analysis We performed a 2-DE analysis using 24-cm IPG

strips (pI 3–10, Bio-Rad) in a PROTEAN IEF Cell and PROTEAN plus Dodeca Cell (Bio-Rad) according to the manufacturer’s instructions. The IPG strips were rehydrated at 20 °C for 14 h with 450 µl of rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 65 mM DTT, and 0.2% Ampholine 3.5/10) containing 250 µg of protein.

E. In-gel digestion The gel spots of interest were excised manually and

subjected to in-gel digestion with trypsin (porcine, side chain-protected; Promega, Madison, WI).

F. Mass spectrometry and protein identification All MALDI-TOF mass spectra were collected with an

Ultraflex mass spectrometer and analyzed by the peak list-generating FlexControlTM 2.2 software (Bruker Daltonics, Germany).

III. RESULTS AND DISCUSSION

A. Morphological and physiological changes induced by Cd The results presented here provide information on the

stress response of actively growing C. roseus plants during Cd exposure. The present study was carried out in this respect in order to elucidate molecular mechanisms and, more precisely, proteomic changes in C. roseus plants upon cadmium stress exposure. Proteomic data, as well as morphological are combined with physiological analysis to provide some insights into the mechanism behind cadmium exposure stress. For morphological symptoms, it appears that the most important effects could be observed chlorosis on the youngest foliar stages. Older foliar stages also showed chlorosis symptoms, rather an accelerated senescence combined with an early abscission, whereas in control plants up to 24 days, no similar shedding could be observed. Chlorophyll a and b, as well as their ratio, were decreased from 3 to 12 d on, and slightly started to increase from 18 d on. These significant differences confirmed visual symptoms, which indicated a presence of chlorosis. Cadmium has been

shown to affect the chlorophyll content in maize plants [6].In a study on Canna indica, chlorophyll a and b content, as well as the ratio, were strongly diminished by hydroponic Cd exposure [7]. In a study on rice plants, the effect of cadmium on PSII has been described as directly on the donor side, accompanied by a decrease in efficency of PSII, and a decrease in chlorophyll and carotenoids [8].

B. Catharanthus. roseus leaf proteome in response to Cd stress Leaf samples were taken from C. roseus plants during

control and Cd stress, and total soluble protein extracted using the modified TCA/acetone method and analyzed by2-D PAGE, in combination with modified CBB staining. The average number of detectable spots in this study reached ~600 on each 2-DE gel (Fig. 1)

C. Changes in expression of photosynthetic proteins Exposure of Cd stress led to impair the photosynthetic

function of C. roseus (Fig. 2). Meanwhile 19 differentially expressed identities were found to be associated with the photosynthetic process (Table 2), and their dynamics showed the effects of Cd stress on photosynthesis at the protein level. These proteins are implicated in four functional subgroups.

IV. FIGURES AND TABLES

TABLE I. CD CONTENT IN C. ROSEUS SHOOTS AND ROOTS AFTER CD EXPOSURE AND THE P-VALUE OF THE STUDENT’S T TEST FOR EACH SAMPLING DATE BETWEEN CONTROL AND CD TREATED PLANTS. NDA

INDICATES NOT DETECTED.

Day Shoot (µg/g DW) Root (µg/g DW)

0 nda Nd

3 29.5 ± 1.24 164.3 ± 7.82

6 94. 7± 8.56 287.1 ± 15.57

12 257.1 ± 24.1 345.9 ± 24.31

24 328.9 ± 19.3 365.2 ± 19.48

TABLE II. LIST OF THE DIFFERENTIALLY EXPRESSED PROTEIN SPOTS IN CONTROL AND CADMIUM-TREATED PLANTS OF C ROSEUS IDENTIFIED BY MALDI-TOF-MS. PUTATIVE PROTEIN IDENTIFICATION AND ACCESSION NUMBER OF THE CLOSEST MATCH IN THE DATABASE ARE INDICATED. ASPOT

ABUNDANCE IS EXPRESSED AS THE RATIO OF INTENSITIES OF UP-REGULATED (PLUS VALUE) OR DOWN-REGULATED (MINUS VALUE) PROTEINS BETWEEN STRESS AND CONTROL.

65

Protein NCBI

accession no.

Spot ID Average fold change a C b day 3 day 6 day 12 day24

Photosynthesis Putative chlorophyll a/b-binding protein type III (PSI) precursor

XP464478 ID4524 -1.32 -2.42 -1.28 nsc 65

Chlorophyll a/b-binding protein CP26 precursor

AAX95978 ID4429 -2.84 -3.28 -2.67 -1.84 52

Rubisco large subunit CAG34174 ID4314 ns -0.87 -2.73 -2.04 54 Rubisco large subunit CAG34174 ID4293 -0.81 -1.43 -2.31 -2.98 48 Rubisco large subunit YP052757 ID4226 -0.59 -0.80 -1.54 -1.82 82 Ribulose 1,5-bisphosphate carboxylase AAA62122 ID4246 -1.04 -2.95 -3.61 -2.19 49 Carbonic anhydrases BAA95793 ID4197 -0.91 -1.20 -1.97 ns 39 Fructose-bisphosphate aldolase class-I AAX95073 ID4364 ns -1.33 -2.56 -1.47 53 Glyceraldehyde 3-phosphate dehydrogenase

AAB82133 ID5564 ns 2.83 2.95 1.75 63

Rubisco large subunit AAS46127 ID5738 1.89 ns ns ns 61 Putative 33-kDa oxygen-evolving protein of photosystem II

NP918587 ID5607 ns 1.64 3.82 2.97 54

Thylakoid lumenal 20-kDa-like protein BAD68170 ID5507 ns ns 1.76 2.88 52 Oxygen-evolving complex protein 1 2002393A ID4260 -2.78 -4.08 -3.63 -2.49 59 Probable photosystem II oxygen-evolving complex protein 2 precursor

NP911136 ID4213 -1.55 -2.87 -2.71 -2.01 57

NADP dependent malic enzyme BAB20887 ID5223 ns 1.59 1.69 1.38 45 Photosystem II reaction center psb28 protein

NP567814 ID4491 -1.86 -2.49 -1.75 1.22 53

Glutamate-1-semialdehyde 2,1-aminomutase 2

NP190442 ID4204 ns -1.78 -2.41 -2.52 49

Magnesium protoporphyrin IX methyltransferase

NP194238 ID4331 ns -1.24 -1.48 -1.93 45

Protein biosynthesis 50 S ribosomal protein L12 O22386 ID4532 ns -1.20 -0.89 -0.72 73 Translation elongation factor T XP466527 ID4218 -0.84 -1.86 -2.10 -2.08 54 Putative ribosome recycling factor XP478772 ID5586 ns ns 1.93 2.04 47 Protein folding and assembly HSP70 AAN87001 ID5371 ns 1.37 2.41 1.86 39 Putative dnaK-type molecular chaperone BiP

XP463871 ID5284 2.38 1.87 1.72 ns 49

GroEL protein BAB02911 ID5840 ns 1.42 ns ns 64 mitochondrial HSP60 AAN15422 ID5383 0.94 1.91 1.84 1.19 52 Protein-disulfide isomerase AAX85991 ID5754 1.72 2.18 1.43 0.72 87 Protein degradation 26 S proteasome subunit α-type 2 AAT78811 ID5262 ns 1.62 1.28 ns 47 Oligopeptidase A-like XP468533 ID5281 0.83 1.94 1.44 1.26 35 Cell rescue/defense Catalase 3 NP0010310

73 ID5394 1.52 2.89 2.53 2.53 53

Ascorbate peroxidase XP479627 ID5732 2.08 1.54 1.14 ns 56 NB-ARC domain-containing protein ABA95337 ID5423 ns 0.98 1.82 0.74 44 Putative MLA1 XP480115 ID5500 0.67 1.82 1.28 0.71 48 Putative hydrolase XP462957 ID5554 0.74 1.83 ns ns 48 Harpin-binding protein 1 AAR26485 ID4222 ns -2.62 -2.65 -2.75 64 Hydroxyproline-rich glycoprotein-like

BAD27963 ID4154 ns -2.61 -1.81 ns 81

66

REFERENCES [1] A.Cavallini, L. Natali, M.Durante,and B. Maserti Mercury uptake,

distribution and DNA affinity in durum wheat (Triticum durum Desf.) plants. Science of the Total Environ, 1999, 244: 119–127.

[2] Stohs SJ, Bagchi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18: 321-36.

[3] Lagriffoul A, Mocquot B, Mench M, Vangronsveld J (1998) Cadmium toxicity effects on growth, mineral and chlorophyll contents, and activities of stress related enzymes in young maize plants (Zea mays L.). Plant Soil 200 (2): 241–250

[4] Graan T, Ort DR (1984) Quantitation of the rapid electron donors to P700, functional plastoquinone pool, and the ratio of the photosystems in spinach chloroplasts. J Biol Chem 259: 14003-140010.

[5] Dai S, Chen T, Chong k, Xue Y, Liu S, Wang T (2007) Proteomics identification of differentially expressed proteins associated with

pollen germination and tube growth reveals characteristics of germinated Oryza sativa pollen. Molecular & Cellular Proteomics 6: 207–230.

[6] C. Shuiping, R. Feng, W. Grosse, and W. Zhenbin, Effects of cadmium on chlorophyll content, photochemical efficiency, and photosynthetic intensity of Canna indica Linn. Int J Phytorem 4(3): 239-245.

[7] Guo Y, Marschner H (1995) Uptake, distribution and binding of cadmium and nickel in different plant species. J Plant Nutr 18: 2691–2706.

[8] Pagliano C, Raviolo M, la-Vecchia F, Gabbrielli R, Gonnelli C, Rascio N, Barbato R, La Rocca N (2006) Evidence for PSII donor side damage and photoinhibition induced by cadmium treatment on rice (Oryza sativa L.).J Photochem Photobiol B 84 (1): 70–78.

Fig. 1 Changes in protein abundance in leaves of C. roseus during cadmium stress at 25 µM Cd treatment. Total leaf soluble proteins were separated by

isoelectric focusing followed by SDS–PAGE. Proteins were stained with CBB and analyzed using Delta 2D software. Three replicate gels, each with protein isolated from an independent biological replicates were run that were then computationally combined into a representative standard gel by Delta 2D.

67

Fig. 2. Functional classification and distribution of all 54 identified proteins as listed in Table 2.

68