Microsoft Word - 18-Bio_36923_pago 713 Original Article Biosci. J., Uberlândia, v. 33, n. 3, p. 713-720, May/June. 2017 PROTEOMIC ANALYSIS OF SAFFRON (Crocus sativus L.) GROWN UNDER CONDITIONS OF CADMIUM TOXICITY ANÁLISE PROTEÔMICA DO AÇAFRÃO (Crocus sativus L.) CULTIVADO SOB CONDIÇÕES DE TOXICIDADE POR CÁDMIO Junfeng RAO 1* ; Weide LV 1 ; Jumei YANG 2 1. Hangzhou Vocational and Technical College, Hangzhou 310018, China; 2. Panan People’s Hospital, Jinhua 322300, China. raojunfenghz@163.com; 13588867756@139.com ABSTRACT: Cd is a highly detrimental global environmental pollutant. Plants have evolved complex defense mechanisms as an adaptation to against Cd toxicity. In this study, a pot experiment was performed to evaluate the protein profile of saffron in response to Cd stress. Fifteen proteins were found to be up-regulated in the leaves of plants under Cd stress and were primarily related to metabolism, signal transduction, stress and defense response and energy. Eleven proteins were found to be down-regulated following Cd treatment, including ribulose bisphosphate carboxylase/oxygenase (Rubisco), ferredoxin-NADP reductase, a 70 kDa heat shock-related protein and three protein synthesis-associated proteins. The results provide valuable insights regarding the molecular mechanism of saffron in response to Cd toxicity and the possible utilization of genetic resources in developing Cd tolerant/low-accumulation saffron. KEYWORDS: Heavy metal. Proteome. Response. Photosynthesis. Saffron INTRODUCTION Cadmium (Cd) pollution has become a serious environmental issue as a consequence of human activities (KULIK et al., 2012; CAO et al. 2014a). Since the occurrence of “Itai-Itai disease” during the 1950-60s in Japan, Cd pollution has generated worldwide concern (CAO et al., 2013). Moderate Cd pollution in the soil could result in considerable Cd accumulation in cereals and subsequently pose a significant threat to human health via the food chain (URAGUCHI and FUJIWARA 2012). In plants, having no biological function and high toxicity, Cd can lead to various problems such as the inhibition of photosynthesis and transpiration, alteration of ion homeostasis, and poor quality products (VESELOV et al., 2003; DENG et al., 2014). Cd affects many important physiological and biochemical processes in plants such as photosynthesis and nutrient homeostasis, which in turn inhibits normal plant growth and development (CLIJSTERS; VANASSCHE, 1985; CAO et al., 2014b). To defend against the detrimental effects of Cd, plants have evolved many detoxification mechanisms, including Cd chelation and vacuolar compartmentalization (HALL, 2002). For example, OsHMA3 (a P-type ATPase 3) has been found to play a key role in xylem Cd transport by mediating vacuolar compartmentalization in the root cells of rice (URAGUCHI; FUJIWARA, 2012). OsDEP, encoding a Cys-rich G protein γ subunit, has also been found to play an important role in Cd tolerance (KUNIHIRO et al., 2013). However, the regulatory mechanism of Cd tolerance in saffron is still unknown. Saffron (Crocus sativus), a perennial herb belonging to the Iridaceae, is heavily used in traditional medicine because of its anticancer, antioxidative, antidepressant, anticonvulsant and learning- and memory-enhancing properties (MACCARONE et al., 2008; SHAMSA et al., 2009; ASADI et al., 2014). There is only one study which reports the effect of Cd on saffron and they only investigated the heavy metal concentration in saffron (ABOU-ARAB; ABOU DONIA 2000). However, the molecular mechanisms underlying the response to Cd toxicity in saffron remain unclear. Comparative proteomic analysis provides a powerful tool to identify the proteins expressed under biotic and abiotic stress (XU et al., 2010). No study has reported the effect of Cd on protein expression in saffron. Thus, the present study was carried out to identify the protein profiles of saffron under Cd stress. These results will be useful for understanding the effect of Cd on saffron at the proteomic level, and may identify key proteins or genes for the genetic enhancement of tolerance to Cd toxicity in plants. MATERIAL AND METHODS Experimental design A pot experiment was conducted in the greenhouse of Hangzhou Vocational & Technical College, Hangzhou, China. A single saffron genotype (Crocus sativus L.) was used. Two kg of Received: 14/10/16 Accepted: 20/02/17 714 Proteomic analysis analysys… RAO, J.; LV, W.;YANG, J. Biosci. J., Uberlândia, v. 33, n. 3, p. 713-720, May/June. 2017 commercial soil was weighed and placed in each pot (5 L, 20 cm × 22 cm × 4 cm). The soil was then artificially contaminated by adding a Cd solution to create a Cd treatment with 20 mg kg-1 Cd; the control contained no added Cd. Following addition of the Cd solution, the soil was allowed to equilibrate for a period of 30 d in the greenhouse. The saffron bulbs were placed on a shaded shelf for germination. After 60 days, uniform healthy plants were selected and transplanted into the pots. Four plants were planted per pot. The experiment used a completely randomized design with 4 replicates. Plant samples were collected after 30 days of treatment for protein analysis. The fresh leaves were immediately frozen in liquid nitrogen and stored at - 80 ºC until analysis. Protein analysis Total leaf protein was extracted according to the protocol of Carpentier et al. (2005). The protein content was measured with a standard Bradford assay using bovine serum albumin as the standard (Bio-Rad, Hercules, CA, USA). Protein spots were separated using two-dimensional gel electrophoresis (2-DE) and then visualized by silver staining of the analytical gels (BAH et al., 2010; DAI et al., 2013). The gels were scanned and calibrated with a PowerLook1100 scanner (UMAX), and protein spots were then analyzed using GE HealthCare Software (Amersham Biosciences). Only those spots with significant and reproducible changes (P ≤ 0.05) were considered to be differentially expressed proteins. The selected protein spots were excised from the stained gels and digested with a trypsin solution using a Spot Handling Workstation (Amersham Biosciences). Trypsin digestion and peptide extraction were conducted according to the protocol of Bah et al. (2010). The digested peptide masses were analyzed using a MALDI-TOF–TOF mass spectrometer (ABI4700 System, USA). The corresponding parameters were based on BAH et al. (2010). Peptide mass fingerprint data were matched to the NCBInr database using the Profound program under 50 ppm mass tolerance. Data were processed using Data Explorer software and proteins were unambiguously identified by searching against a comprehensive non-redundant sequence database using the MASCOT software search engine (http://www.matrixscience.com). Proteins with change of > 1.5-fold or < -1.5-fold and a P value <0.05 were considered differentially expressed. Statistical analysis Statistical analyses were performed using Data Processing System (DPS) statistical software. RESULTS The average number of leaf protein spots on the 2-DE gels for the Cd treatment and the control was 1594 and 1588, respectively. Of these, 94 and 64 protein spots were observed to be significantly up-regulated (>1.5-fold change) and down-regulated (<-1.5-fold change), respectively, after Cd treatment compared with the control. Fifteen (A1-A15) and 11 (B1-B11) protein spots were successfully identified by MALDI-TOF-TOF-MS (Tables 1 and 2, Figs 1 and 2). The functions of the fifteen proteins that were significantly up-regulated in the Cd treatment were categorized as metabolism (46.7%), signal transduction (26.7%), stress and defense response (6.7%), energy (6.7%) and unknown function (13.3%) (Table 1). Seven proteins were involved in metabolism: 5-methyltetrahydropteroyltriglutamate- homocysteine methyltransferase (A1), glyceraldehyde-3-phosphate dehydrogenase (A2), glutamine synthetase (A3), tubulin beta chain (A4), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (A5), alpha-1,4-glucan- protein synthase (A6) and 3 beta-hydroxysteroid- dehydrogenase/decarboxylase isoform 1 (A7). The other up-regulated proteins were as follows: signal transduction (c.f. A8 and A9, S-adenosylmethionine synthetase 1; A11, 1-aminocyclopropane-1- carboxylate oxidase), stress and defense (A12, pathogenesis-related protein 10), energy (A13, ATP synthase subunit alpha) and unknown function (A14 and A15). Table 1. Proteins whose expression were significantly up-regulated under Cd stress Spot ID Protein name Accession number MW (Da) pI FC Score Cov % MP PF A1 5- methyltetrahydropteroyltriglutamate- -homocysteine methyltransferase (Catharanthus roseus) METE_CATRO 85089 6.10 106 126 11 7 M A2 Glyceraldehyde-3-phosphate dehydrogenase, (Oryza sativa) G3PC_ORYSI 36561 6.61 106 132 10 4 M 715 Proteomic analysis analysys… RAO, J.; LV, W.;YANG, J. Biosci. J., Uberlândia, v. 33, n. 3, p. 713-720, May/June. 2017 A3 Glutamine synthetase (Musa acuminata) M0S022_MUSAM 39428 6.03 1.80 94 16 4 M A4 Tubulin beta chain (Cicer arietinum) TBB_CICAR 51171 4.75 2.90 65 25 7 M A5 2,3-bisphosphoglycerate- independent phosphoglycerate mutase (Cryophytum crystallinum) PMGI_MESCR 61316 5.39 3.12 141 13 7 M A6 Alpha-1,4-glucan-protein synthase (Pisum sativum) UPTG_PEA 42059 5.73 1.50 110 17 6 M A7 3beta-hydroxysteroid- dehydrogenase/decarboxylase isoform 1 (Arabidopsis thaliana) HSDD1_ARATH 48493 8.89 3.86 29 2 1 M A8 S-adenosylmethionine synthetase 1 (Populus trichocarpa) METK1_POPTR 43642 5.68 106 245 20 7 ST A9 S-adenosylmethionine synthetase 1 (Populus trichocarpa) METK1_POPTR 43642 5.68 4.05 236 20 7 ST A10 Auxin-responsive protein IAA18 (Arabidopsis thaliana) IAA18_ARATH 29936 9.26 2.89 61 13 6 ST A11 1-aminocyclopropane-1-carboxylate oxidase (Persea americana) ACCO_PERAE 36453 5.08 106 42 4 1 ST A12 Pathogenesis-related protein 10 (Crocus sativus) E5D8G2_CROSA 17502 6.08 22.58 93 37 5 SD A13 ATP synthase subunit alpha, chloroplastic (Eucalyptus globulus) ATPA_EUCGG 55535 5.15 1.52 319 24 12 E A14 Uncharacterized protein (Triticum aestivum) W5A1P2_WHEAT 192765 6.74 1.80 72 12 16 U A15 Uncharacterized protein (Musa acuminata) M0TI68_MUSAM 32323 5.23 1.85 51 4 1 U Cov, coverage; E, energy; FC, fold change; M, metabolism; MP, matched peptides; PF, Putative function; U, unknown; SD, stress and defense response; ST, signal transduction. Figure 1. Representative 2-DE maps of saffron leaf proteins isolated from Cd and control treatment. Total proteins were extracted and separated by 2-DE. In IEF, 100 mg proteins were loaded onto pH 4–7 IPG strips (24 cm, linear). SDS-PAGE was performed with 12.5% gels. The spots were visualized by silver staining. Differentially accumulated protein spots are indicated by green sashes. Fifteen higher expressed spots (A) and eleven suppressed (B) spots are shown in the maps. 716 Proteomic analysis analysys… RAO, J.; LV, W.;YANG, J. Biosci. J., Uberlândia, v. 33, n. 3, p. 713-720, May/June. 2017 Table 2. Proteins whose expression were significantly down-regulated under Cd stress Spot ID Protein name Accession number MW (Da) pI FC Score Cov % MP PF B1 Ribulose bisphosphate carboxylase/oxygenase activase, (Oryza sativa) RCA_ORYSJ 51764 5.43 -2.85 253 16 8 M B2 Ribulose bisphosphate carboxylase large chain (Isophysis tasmanica) RBL_ISOTA 52248 5.87 -1.58 386 22 12 M B3 Stromal 70 kDa heat shock-related protein, chloroplastic (Pisum sativum) HSP7S_PEA 75583 5.22 -2.97 170 17 9 SD B4 Eukaryotic initiation factor 4A (Triticum aestivum) IF4A_WHEAT 47183 5.31 -2.11 59 14 7 PS B5 30S ribosomal protein 2, chloroplastic (Spinacia oleracea) RRP2_SPIOL 28390 8.42 -2.61 91 10 5 PS B6 Chaperonin (Zea mays) B4F848_MAIZE 25796 8.49 -2.68 76 33 6 PS B7 Ferredoxin-NADP reductase, leaf isozyme, (Pisum sativum) FENR1_PEA 40454 8.56 -7.00 118 16 7 T B8 Ferritin-1, chloroplastic (Glycine max) FRI1_SOYBN 28204 5.73 -1.50 104 9 5 T B9 Knotted1 (Spinifex sericeus) A3RCP6_9POAL 9309 4.98 -1.50 78 92 7 Tr B10 Uncharacterized protein (Hordeum vulgare) M0UGX1_HORVD 91892 6.51 -3.20 76 22 15 U B11 Uncharacterized protein (Brachypodium distachyon) I1HXX4_BRADI 39825 4.50 -2.68 72 8 5 U Cov, coverage; FC, fold change; M, metabolism; MP, matched peptides; PF, Putative function; PS, protein synthesis; U, unknown; SD, stress and defense response; T, transport; Tr, transcription. Figure 2. The spot view of individual identified spots, being up-regulated (A) and down-regulated (B) after Cd treatment. A A A A A A A A A A1 A A A1 A A B1 B B B4 B B B7 B B B1 B Cd contr Cd contr Cd contr Cd contr 717 Proteomic analysis analysys… RAO, J.; LV, W.;YANG, J. Biosci. J., Uberlândia, v. 33, n. 3, p. 713-720, May/June. 2017 The 11 suppressed proteins (Cd vs. control) belonged to 6 functional categories (Table 2; Fig. 2). Two were found to play key roles in photosynthesis: ribulose bisphosphate carboxylase/oxygenase activase (B1) and ribulose bisphosphate carboxylase large chain (B2). There were 3 protein synthesis- related proteins: eukaryotic initiation factor 4A (B4), 30S ribosomal protein 2 (B5) and chaperonin (B6). One stress and defense response protein, stromal 70 kDa heat shock-related protein (B3), and 1 transcription-related protein, Knotted1 (B9), were significantly down-regulated by -2.97-fold and - 1.50-fold, respectively, after Cd treatment. Two transport-related proteins, ferredoxin-NADP reductase (B7) and ferritin-1 (B8), were also identified. In addition, 2 uncharacterized proteins (B10 and B11) were also clearly down-regulated. DISCUSSION Cd is believed to induce damage even at very low concentrations, not only affecting many important physiological processes but also reducing the yield and quality of plants (CAO et al., 2013, 2014). Saffron is a traditional precious herb. Our previous study found that Cd significantly suppressed saffron growth and considerable Cd was accumulated (data unpublished). In this study, 15 and 11 protein spots were up-regulated and down- regulated, respectively, in response to Cd stress (Fig. 2). Among the 15 proteins with elevated expression, 4 are involved in signal transduction: S- adenosylmethionine synthetase 1 (SAMS, A8 & A9), 1-aminocyclopropane-1-carboxylate oxidase (ACCO, A11) and auxin-responsive protein IAA18 (A10). It has been found that the ethylene (ET) signaling pathway plays a fundamental role in the response to abiotic stress, including Cd stress (FUJITA et al. 2006). SAMS has long been considered to have a house-keeping function and plays a crucial role in ET synthesis (DAI et al. 2013). In addition, ACCO catalyzes the terminal step of ET synthesis (HERBETTE et al. 2006; CAO et al. 2014). Hence, up-regulation of SAMS and ACCO in response to Cd stress could contribute to ET synthesis, which subsequently activates additional Cd-responsive genes. Auxin-responsive protein IAA18 was also significantly up-regulated (Table 1). Therefore, it is necessary to study the relationship between the ET and auxin signaling pathways in response to Cd toxicity. Seven of the proteins identified as up- regulated were involved in metabolism (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, A2) plays a key role in glycolysis. VESCOVI et al. (2013) found that the cytosolic GAPDH isoenzyme GAPC1 may play a role in signaling oxidative stress or protection in Arabidopsis roots under Cd stress. Glutamine synthetase (GS, A3) catalyzes the formation of glutamate, which is an important component of reduced glutathione and phytochelatins (PCs). Rana et al. (2008) observed that the expression of cytosolic GS was induced by Cd and salt stress. Studies have found that beta-tubulin and alpha-1,4- glucan-protein synthase are involved in plant cell wall metabolism (SPOKEVICIUS et al., 2007; JIN et al., 2011). Lee et al. (2010) also found that expression of alpha-1,4-glucan-protein synthase (A6) was significantly elevated. Membrane-bound ATPases provide energy and an H+ gradient for the co-transport of copper, cobalt, lead, and Cd, with protons used for the detoxification of these metal ions in plants (CAO et al. 2014). CAO et al. (2014) found that transcripts of V-ATPase were significantly elevated in a Cd-tolerant barley genotype but were not affected in a Cd-sensitive genotype. Kieffer et al. (2008) found that pathogenesis-related proteins showed a dramatic increase in expression. In our study, these proteins were all significantly up-regulated under Cd stress. Plants have evolved sophisticated defense mechanisms as an adaption to Cd stress. The results of this study suggest that Cd may induce signal molecules (ET) that subsequently activate Cd- responsive proteins, elevate the expression of antioxidant-related proteins that could scavenge the Cd-induced accumulation of ROS, and transport Cd into vacuoles and sequestrate Cd in cell walls, which would contribute to a decrease in the available Cd2+ of the cytosol. The negative impact of Cd on photosynthesis is well documented (CLIJSTERS; VAN ASSCHE 1985). Rubisco catalyzes the first step in net photosynthetic CO2 assimilation and photorespiratory carbon oxidation (SPREITZER and SALVUCCI 2002). Ferredoxin NADP(H) oxidoreductases (FNRs) catalyze electron transfer between NADP(H) and ferredoxin (LINTALA et al., 2007). An absence of AtLFNR1 led to a decrease in green leaf size, lower chlorophyll content and fewer light-harvesting complex proteins, and a markedly lower PSI/PSII ratio in the mutant than the wild type (LINTALA et al., 2007). In the present study, expression of rubisco (B1, B2), ferredoxin- NADP reductase (B7) and ferritin-1 (B8) were all down-regulated after Cd treatment. These results indicate that Cd toxicity damaged the photosynthesis system in saffron. Razavizadeh et al. 718 Proteomic analysis analysys… RAO, J.; LV, W.;YANG, J. Biosci. J., Uberlândia, v. 33, n. 3, p. 713-720, May/June. 2017 (2009) found that the stromal 70 kDa heat shock protein was significantly down-regulated under salinity in tobacco leaves. The authors believed that this down-regulation was likely the cause of the failure to protect cells against salinity. Similar results were also found in this study. Heat shock protein and molecular chaperonin have been found to play vital roles in protein stabilization, folding, assembly and translocation under abiotic stress (KOMATSU; HOSSAIN, 2013). Stromal 70 kDa heat shock-related protein (B3) and chaperonin (B6) were significantly down-regulated (Table 1). The 30S ribosomal protein 2 was also down-regulated after Cd treatment. These results demonstrate that Cd stress affected the normal functioning of the chloroplast. Moreover, down-regulation of initiation factor 4A also affected the level of translation in saffron. In summary, Cd stress affected the normal functioning of the chloroplast, reducing photosynthesis and translation. ACKNOWLEDGEMENTS This work was supported by the Star Program of the State Ministry of Science & Technology (2015GA700053), the Zhejiang Natural Science Foundation (LY13B020001) and the Agricultural Project of Zhejiang Public Technology Research (2015C32104). RESUMO: O cádmio (Cd) é um poluente ambiental global altamente prejudicial. As plantas desenvolveram mecanismos de defesa complexos como uma adaptação contra a toxicidade por Cd. Neste estudo, realizou-se um experimento em vaso para avaliar o perfil proteico do açafrão em resposta ao estresse por Cd. Foi descoberto que quinze proteínas foram supra-reguladas (up-regulated) nas folhas de plantas sob estresse por Cd e foram principalmente relacionados ao metabolismo, transdução de sinal, estresse e resposta de defesa e energia. 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