PLANT GROWTH, DEVELOPMENT AND CHANGE IN GSH LEVEL IN SAFFLOWER (CARTHAMUS TINCTORIUS L.) EXPOSED TO COPPER AND LEAD

The effects of exposure to heavy metals, copper (Cu) and lead (Pb) in the soil, separately and in combination, were examined in Safflower (Carthamus tinctorius L.). Plant growth and development, GSH level and GSH2 expression at seedling, branching, and flowering stages were studied. Cu at lower concentrations had a stimulating effect on seedling height and root length. A significant positive correlation was observed between heavy metal concentrations and inhibition of plant growth. Plant height, root length and lateral root numbers decreased progressively with increasing concentrations of Cu and Pb. Except at the seedling stage, the metal mixture elicited a synergistic effect on safflower growth and development. The GSH content was significantly reduced in both safflower roots and leaves at increased concentrations of heavy metals, with the exception of the treatment with a low concentration of Cu that resulted in a slightl increase in GSH content at the seedling and branching stages. RT-PCR analysis revealed a negative correlation between GSH2 expression levels and metal concentration. Short exposure to low concentrations of Cu induce an increase in GSH synthesis to preserve normal plant growth, whereas prolonged exposure and large Cu and Pb concentrations affect the GSH metabolic chain, and are severely toxicity. The findings obtained in this study enhance our understanding of the role of the GSH pool in the response of plants to heavy metal-induced stress, and serve as a basis for improved cultivation of safflower.


INTRODUCTION
Agricultural soils in many parts of the world are contaminated by heavy metals in varying degrees, and heavy metal pollution of agricultural soils is one of the most serious ecological problems worldwide (Yadav 2010;Singh et al., 2007).Studying the effects and mechanisms of heavy metal stress is beneficial for agricultural production.Copper (Cu) is a heavy metal and an essential micronutrient that is critical in maintaining normal metabolism in higher plants.Although it is involved in a wide range of biochemical and physiological processes, an accumulation of excess Cu causes injury on the cellular level by forming reactive oxygen species (ROS) in plant cells (Upadhyay andPanda, 2009, Nzengue et al., 2011).Lead (Pb) is one of the most toxic heavy metals and is a human carcinogen.It has adverse effects on plant morphology, growth and photosynthetic processes.High levels of Pb can also inhibit enzymatic activities, alter membrane permeability, disrupt mineral nutrition (Sharma and Dubey 2005) and induce oxidative stress by increasing ROS production in plant cells (Reddy et al., 2005).To combat the oxidative damage induced by heavy metal stress, plants have an antioxidant defense system that may involve GSH (glutathione) (Speisky et al., 2009;Estrella-Gómez et al., 2012).
GSH is a tripeptide formed by a peptide bond condensation of L-glutamic acid, L-cysteine and glycine (Wachter et al., 2005).It is synthesized by two sequential reactions catalyzed by γ-glutamylcysteine synthetase (GSH1) and GSH synthetase (GSH2) (Okuma et al., 2011;Estrella-Gómez et al., 2012).GSH is a major intracellular thiol compound (Iwasaki et al., 2009) that plays important roles in many biological processes, such as intracellular reduction-oxidation meta-bolic cycles, transport, protein synthesis (Ensafi et al., 2013) and elimination of ROS (Foyer and Noctor 2005;Mullineaux and Rausch 2005;Morris et al., 2013) in plants.The glutathione molecule contributes to the regulation of the intracellular redox environment and the detoxification of xenobiotics caused by an excess of metals, including Cu and Pb, and it also functions as a crucial intracellular antioxidant (Roušar et al., 2012;Menon and Board, 2013).GSH1 and GSH2 are important enzymes for GSH synthesis and accumulation.GSH1 and GSH2 mRNAs are increased by conditions that require enhanced GSH abundance for metabolic functions, such as heavy metal sequestration (Maughan and Foyer, 2006;Xiang and Oliver, 1998).Recently, Blazhenko et al. (2014) reported that GSH2 transcription increased in the yeast Hansenula polymorpha under cadmium (Cd) treatment.However, unlike the extensive study of GSH1 (Rausch et al., 2007), GSH2 was less studied in plants, especially in safflower.
Safflower (Carthamus tinctorius L.) is a traditional medicinal plant and represents a new type of oilseed crop and industrial resource.To date, most studies have focused on the genetic resource (Patel et al., 1989;Yang et al., 2007), cultivation techniques (Kizel, 2002), chemical constituents (Takahashi et al., 1984;Zhang et al., 1997) and pharmacology (Chu et al., 2006;Jun et al., 2011) of safflower.Studies on heavy metal toxicity in safflower are limited (Nosrati et al., 2013).The levels of Cu and Pb that induce toxicity or growth alterations are not known.Moreover, antioxidants, such as GSH, and related gene expression responses to heavy metal stress are also not known.In this study, a greenhouse pot experiment was conducted to evaluate the effect of Cu and Pb on safflower development, GSH level and GSH2 gene expression.Safflowers were grown in soil with Cu, Pb, or both.We focused on the morphological, biochemical and molecular properties, such as plant height, root length, lateral root number, GSH content and GSH2 expression, to evaluate the effects of Cu and Pb on plant growth and development of safflower.
Safflower seeds (Variety 'Wei honghua', Xinxiang Forint Health Food Co., Ltd.) were surfacesterilized with 0.1% (w/v) HgCl 2 for 5 min and then washed thoroughly with sterile water.Sterilized seeds were germinated in Petri plates on sterile wet paper towels at 25°C in the dark for 3 days.Twenty-three-day-old germinated seeds were transferred to each of the pots containing soil treated with Cu, Pb or Cu-Pb.The experiment was conducted at a standard long day light (16 h) at 25°C-35°C during the day, and at 10°C-18°C during the night (8 h), at 65±5% relative humidity.The plants were cultivated for 240 days from 15 October 2011 to 15 June 2012.During cultivation, controlled irrigation using water was scheduled according to tensiometer measurements to maintain soil moisture content within field capacity.In the potted processing experiments, the growth of safflower was divided into seedling (approximately 60 days after transplantation), rosette (approximately 100 days after transplantation), elongation (approximately 140 day after transplantation), branching (approximately 170 days after transplantation), flowering (approximately 210 days after transplantation) and mature (about 240 days after transplantation) stages.We selected three stages, namely, the seedling, branching and flowering stages, for the measurement of the parameters of plant growth and GSH content.

Determination of plant height, root length and lateral root number
Plants were harvested at the end of each selected stage, and the height, root length and lateral root number were determined.Fifteen plants were evaluated for each treatment.

Assays of glutathione content
The GSH content was determined using the method of Ellman (1959) with modifications.In brief, 0.2 g of safflower samples (roots or leaves) were rinsed in cold water (0°C) and the excess water was removed using a filter paper.Each sample was grounded in 3 mL of 5% trichloroacetic acid solution by centrifugation for 20 min at 4°C; 0.1 mL of the resulting supernatant was mixed with 4.4 mL of PBS (0.1 mol/L) and 0.5 mL of 5.5-dithiobis (2-nitro-benzoic acid; 0.04% w/v).The samples were incubated at 30°C for 5 min in the dark.The absorbance was read at 412 nm using a Unico spectrophotometer (UV-4802).The concentration of GSH was from the calibration curve obtained with commercial standards (Sigma, Taufkirchen, Germany).

Statistical analysis
Data were subjected to one-way ANOVA using SPSS version 18.0 (PASW Statistics for Windows, 2009, Chicago: SPSS Inc.).Separate analyses were performed for each sampling, and significant differences (p <0.05 or p <0.01) were determined between the metal treatments and the control.

Safflower total RNA isolation and full-length GSH2 gene cloning
Total RNA was isolated from the control and heavy-metal-treated safflower seedlings using the Super Purified Total RNA Rapid Extraction Kit (Aidlab Biotech, Beijing, China).RNA samples were treated with RNase-free DNase I (Takara, Dalian, China) at 37°C for 30 min.The first-strand cDNA was synthesized from 2 μg of RNA template with TUREscript 1st Strand cDNA Synthesis Kit (Aidlab) according to the manufacturer's protocols with oligo (dT) 18 as a primer.The cDNA samples were used as templates in the subsequent degenerate PCR and semi-quantitative RT-PCR.
Degenerate primers were designed from the conserved amino acid regions of the GSH2 protein (Table 1).PCR was performed by denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and elongation at 72°C for 1 min with additional elongation at 72°C for 10 min after the last cycle.Amplified products were cloned into the vector pMD18-T (TaKaRa) and transformed into competent Escherichia coli DH5α.Positive clones were screened from Amp + /LB plates containing IPTG/X-Gal and then sent to SinoGeno-Max (Beijing, China) for DNA sequencing.
The full-length GSH2 cDNA was obtained by rapid amplification of cDNA ends (RACE) tech-nology.Based on the conserved regions of the cloned GSH2 gene, gene-specific primers (Table 1) were designed for 3´-RACE and 5´-RACE.Reverse transcription reactions were performed with 3 μg-5 μg of total RNA to produce 3´-and 5´-cDNA using the RACE Core Set (TakaRa) according to the manufacturer's instructions.The RACE products were cloned into the pMD18-T vector and sequenced (SinoGenoMax).

Semi-quantitative PCR analysis of GSH2 expression
Total RNA was extracted from the control and different concentrations of metal-treated safflower leaves in the seedling, branching and flowering stages.RNA samples were treated with RNasefree DNase I, and the first-strand cDNA was synthesized from 2 μg of RNA.primers used for detecting the safflower GSH2 gene.The housekeeping actin gene was used as the control in the RT-PCR reactions, and the primers are listed in Table 1 (Sugiyama et al., 2003).The reaction included denaturation at 95°C for 5 min, followed by 35 cycles (25 cycles for actin) of 30 s at 95°C, 30 s at 50°C, 1 min at 72°C and then 10 min at 72°C.The amplification products were separated by electrophoresis on 1.5% (w/v) agarose gel and visualized by a UV imaging system.

Effects of different metal treatments on safflower growth and development
To examine safflower growth and development response to Cu and Pb, we determined the plant height, root length and lateral root number.Comparisons between metal-treated and untreated plants at seedling, branching and flowering stages were carried out (Fig. 1).Results showed that plant height and root length increased after exposure to a low concentration of Cu (50 µg/g) at the seedling stage; however, the lateral root number of safflower plants showed no significant change compared with the control at the same stage.Safflower seedlings exposed to high Cu concentrations (≥400 µg/g) showed evident signs of growth inhibition.Plant height and lateral root number remarkably decreased when the concentration of Cu reached 100 µg/g, and root length significantly decreased when the concentration of Cu was more than 400 µg/g at the branching stage.Root length and lateral root number decreased after exposure to all of the examined concentrations of Cu (increasing "levels" of Cu stress) at the flowering stage.
Regarding the treatment with Pb, inhibition in plant growth was directly proportional to the Pb concentration in the soil.Root length and lateral root number significantly decreased at the seedling stage when the concentration was greater than 500 µg/g, and plant height significantly decreased when the concentration was 800 µg/g.Root length significantly decreased at the branching stage, even with the lowest Pb concentration (300 µg/g), and plant height and lateral root numbers decreased when the concentration of Pb ≥ 500 µg/g.Root length and lateral root number in the flowering stage were significantly inhibited, even with the lowest concentration of Pb.
As for the stress incurred by the combination of Cu and Pb, plant height, root length and lateral root number were all significantly inhibited in the branching and flowering stages.In addition, the degree of inhibition observed in the treatment with the Cu and Pb combination was larger than in the treatments with Cu or Pb alone.For example, plant height, root length and number of lateral roots decreased by 51.5%, 80.7% and 76.2%, respectively, under combined treatment of Cu (800 µg/g) and Pb (1000 µg/g), and 24.5%, 65.2% and 57.7% under the treatment with 800 µg/g Cu, and 31.8%,73.3% and 75.4% under the treatment with 1000 µg/g Pb.
Results also showed that the degree of inhibition of safflower growth was directly proportional to the length of exposure to Cu and Pb.For example, root length was reduced by 19.9%, 44.1% and 65.2% in the seedling, branching, and flowering stages, respectively.

Effect of different metal treatments on GSH content in safflower roots and leaves
The GSH contents in the roots and leaves were determined to evaluate the biochemical response of safflower to Cu and Pb.As shown in Fig. 2A, when the plants were exposed to 100 µg Cu/g and 300 µg Pb/g, the GSH content displayed a significantly (P <0.01) decreasing trend with the increase in metal concentration at all growth stages.Notably, the GSH content increased slightly under the lowest concentration of Cu (50 µg/g) at the seedling and branching stages (P <0.05), suggesting that short exposure to low concentrations of Cu could induce an increase in the GSH content in safflower roots.Furthermore, the effect of Cu-Pb stress on the GSH content of safflower roots was considerably greater than that of Cu or Pb alone.Under the treatment with the highest metal concentrations, at the flowering stage the GSH content of roots was significantly (P <0.01) reduced, by 55.27% (Cu 800 µg/g), 63.91% (Pb 1000 µg/g) and 72.62% (Cu 800 µg/g + Pb 1000 µg/g) as compared to the control.In general, the pattern of changes in the GSH content in safflower leaves at different concentrations of heavy metal was similar to that in safflower roots (Fig. 2B).The GSH content significantly (P <0.01) decreased in the leaves of safflower with increasing heavy metal concentration, except in the treatment with a low concentration of Cu (50 µg/g) at the seedling and branching stages.Interestingly, an increase in GSH content was also observed in safflower leaves under short exposure to low concentrations of Cu.The toxic effect of Cu-Pb on GSH content of safflower leaves was higher than that of Cu or Pb alone, suggesting a collaborative effect of Cu and Pb./g).1: CK; 2: Cu50; 3: Cu100; 4: Cu400; 5: Cu800; 6: CK; 7: Pb300; 8: Pb500; 9: Pb800; 10: Pb1000; 11: CK; 12: Cu50 + Pb300; 13: Cu100 + Pb500; 14: Cu400 + Pb800; 15: Cu800 + Pb1000.

Gene expression analysis of safflower GSH2 in response to different heavy metal treatments
The expression of GSH2 was analyzed to evaluate further the relationship between changes in GSH levels and gene expression.The 1263 bp transcript sequence of GSH2 was obtained by RACE-PCR.RT-PCR analysis showed a negative relationship between GSH2 expression levels and metal concentration.In the control and Cu-stressed leaves of safflower seedlings, GSH2 expression levels did not change, with the exception of leaves from plants exposed to 800 µg/g Cu.However, the expression levels of GSH2 in leaves decreased considerably at the branching and flowering stages (Fig. 3).Furthermore, the GSH2 gene of safflower leaves at the branching and flowering stages was barely expressed in plants that were exposed to the Cu-Pb combination.

DISCUSSION
Heavy metal contamination has adverse effects on plant growth and development, such as reduced root length, leaf area, plant height, dry weight, and a decline in photosynthesis (Vinit-Dunand et al., 2002;Oncel et al., 2000;Bradshaw and Chadwick, 1980).The present study revealed that short exposure to low concentrations of Cu and Pb had a minimal influence on safflower growth and development, whereas prlonged exposure and high concentrations exerted adverse effects.In the present study, both Cu and Pb were identified as effective elements for inhibiting safflower growth.A positive correlation was observed between the degree of toxicity and heavy metal concentration.Similar phytotoxic responses to Cu and Pb are observed in many plant species (Munzuroglu and Geckil, 2002;MacFarlane and Burchett, 2002;An et al., 2004;Shu et al., 2014).For example, Munzuroglu and Geckil (2002) studied the inhibitory effects of six metals, including Cu and Pb, on wheat and cucumber, and the results showed that both plants exhibit reduced seed germination rates, root, and hypocotyl or coleoptile length with increasing concentrations of heavy metals.The toxic symptomatology observed is speculated to be a consequence of tissue damage, alteration of membrane permeability, peroxidation of chloroplast membrane lipids, inhibition of photosynthetic electron transport and disruptions to carbohydrate metabolism and protein synthesis (Droppa and Horvath, 1990;Wozny and Krzeslowska, 1993;MacFarlane and Burchett, 2002).Notably, lower Cu concentrations stimulated plant height and root length in the seedling stage in this study.Shu et al. (2014) also reported that Pb has a stimulating effect on the seedling height and leaf area of Jatropha curcas L. at low concentrations.These findings suggest a positive effect of low concentrations of some heavy metals on plant growth.
Different compounds are often found as mixtures in the environment.Soil pollution by combinations of heavy metals has received considerable attention.The exposure of cells to a combination of metals may result in increased permeability of the plasma membrane accompanied by increased cellular uptake and toxicity (MacFarlane and Burchett, 2002).Several studies have been conducted to investigate the combined effects of heavy metals on some plant species.The combination of Pb and Zn was reported to increase toxicity to Avicennia marina (grey mangrove) (MacFarlane and Burchett, 2002).Another report showed that Cu, Cd and Pb have a combined effect on Cucumis sativus growth and inhibit or enhance the bioaccumulation of individual metals depending on metal combination (An et al., 2004).Interactions between Cu and Pb have been rarely reported.In this study, the combination of Cu and Pb exhibited higher toxicity to safflower growth and development compared to Cu or Pb alone, indicating an interaction between these two metals in transport or a reduction in the efficacy of exclusion from plantGSH is a prominent cellular an-tioxidant that plays a critical role in membrane protection against free radical damage in most eukaryotic organisms (Rennenberg and Brunold, 1994;Moran et al., 2000).Our results showed that short exposure and low concentrations of Cu could induce an increase in GSH content in safflower roots and leaves.However, when plants were exposed to 100 µg/g Cu and 300 µg/g Pb, the GSH content progressively decreased with increasing heavy metal concentrations.The results were apparently inconsistent with the majority of studies.Increased levels of GSH were observed with increasing Cu or Pb concentrations in plant species, such as Arabidopsis thaliana (Drążkiewicz et al., 2003), Sedum alfredii (Gupta et al., 2010) and Phanerochaete chrysosporium (Xu et al., 2014).However, a decline in GSH content in Brassica napus roots (Russo et al., 2008) and Scenedesmus bijugatus cells (Nagalakshmi and Prasad, 2001) has been reported under Cu stress.A Pb-induced decline in GSH was reported in Vicia faba, Phaseolus vulgaris (Piechalak et al., 2002) and Pisum sativum (Malecka et al., 2009).Furthermore, an unchanged GSH content was observed in the nodules of Glycine max under Cd stress (Balestrasse et al., 2001).These findings reveal that GSH levels in plants are metal-dependent (Israr et al., 2011), dose (Kalinowska and Pawlik-Skowronska, 2010), plant cultivars (Cruz de Carvalho et al., 2010) and even tissue type (Russo et al., 2008).García-Giménez et al. (2012) recently reported that GSH might be involved in epigenetic phenomena and the control of nuclear protein degradation by nuclear proteasome.These reports imply that a complex mechanism underlies the GSH response to heavy metal stress.Based on the consistency between GSH2 expression and changes in GSH content, our results indicated that GSH protects the normal growth of plant under short exposures to low concentrations of Cu, whereas an excess of Cu, Pb or Cu and Pb may alter the equilibrium between the synthesis and utilization of GSH, probably via the regulation of GSH2 gene expression.As previously discussed, the relationship between GSH and the stress response is complex, and further studies are necessary.

Fig. 2 .
Fig. 2. Effects of Cu, Pb and their combination on the GSH content in (A) roots and (B) leaves of safflower.The GSH content of roots and leaves was analyzed in control and heavy metal-treated safflower at the seedling, branching and flowering stages.The X axis and asterisk are as explained in Fig. 1.Values are expressed as mean ± SD based on three independent experiments and bars indicate SD.

Table 1 . Primers used in this study PCR Primer name Sequence (5′-3′)
Table 1 lists the PCR