Activation of phenylpropanoid pathway in legume plants exposed to heavy metals. Part I. Effects of cadmium and lead on phenylalanine ammonia-lyase gene expression, enzyme activity and lignin content.

Species-specific changes in expression of phenylalanine ammonia-lyase (PAL) and lignin content were detected in roots of soybean (Glycine max L.) and lupine (Lupinus luteus L.) seedlings treated with different concentrations of cadmium (Cd(2+), 0-25 mg/l) or lead (Pb(2+), 0-350 mg/l). The stimulatory effect of both metals was observed in mRNA coding for PAL in soybean. In the case of lupine, changes of PAL mRNA level were dependent on the metal used: Cd(2+) caused a decrease, whereas Pb(2+) an increase of PAL transcript level. The activity of PAL was enhanced in both plant species at higher metal concentrations (15-25 mg/l of Cd(2+) or 150-350 mg/l of Pb(2+)); however it was not directly correlated with PAL mRNA. This suggests a transcriptional and posttranscriptional control of PAL expression under heavy metals stress. In soybean, Cd(2+) or Pb(2+) treatment increased lignin content, while in lupine the effect was opposite. The decreased lignin accumulation in lupine roots in response to heavy metals, despite an increased PAL activity, suggests that the activated phenylpropanoid pathway was involved in the synthesis of secondary metabolites other than lignin.


INTRoduCTIoN
The contamination of soil with heavy metals, such as cadmium and lead, has become a serious environmental problem leading to toxic effects in plants and health hazard.Heavy metals trigger various stress reactions in plants, which depend on plant genotype, developmental stage as well as the concentration and the metal type.The plant responses may either lead to plant adaptation and resistance to the metal stress or result in severe plant damage and eventual death (Das et al., 1997;Sandalio et al., 2001;Seregin & Ivanov, 2001;Patra et al., 2004;Deckert, 2005;Sharma & Dubey, 2005).A coordinated network of molecular processes provides plant cells with multiple metal-detoxifying mechanisms and repair capabilities which allow plants to survive in metal-containing environments (Clemens, 2001;Cobbett & Goldsbrough 2002;Shingu et al., 2005).
Earlier studies performed in our laboratory showed that lead (Pb 2+ ) and cadmium (Cd 2+ ) caused a growth in-hibition of lupine and soybean seedlings which was correlated with accumulation of the metals within plant cells and various stress responses.They included activation of antioxidant system (Rucińska et al., 1999;Sobkowiak et al., 2004;Pawlak et al., 2009), perturbation of the cell cycle, DNA damage (Deckert & Gwóźdź, 1999;Sobkowiak & Deckert, 2003;2004;Rucińska et al., 2004) and induction of various proteins (Przymusiński & Gwóźdź, 1999;Sobkowiak & Deckert, 2006).The function of some of those proteins in the plant response to metals is still not known.One of the proteins induced by Cd 2+ in soybean was identified as chalcone synthase (CHS; EC 2.3.1.74)(Sobkowiak & Deckert, 2006).This result suggested an involvement of the phenylpropanoid pathway in the response of legume plants to metals.The gene coding for phenylalanine ammonia-lyase (PAL; EC 4.3.1.5),a key enzyme in the phenylpropanoid pathway, was also detected among Cd 2+ -induced genes in Brassica juncea (Fusco et al., 2005).On the other hand, a proteomic analysis of Arabidopsis thaliana exposed to Cd 2+ and a study on Pb 2+ -regulated genes in Sesbania drummondi failed to detect an induction of any proteins or genes corresponding to the phenylpropanoid pathway enzymes (Roth et al., 2006;Srivastava et al., 2007).Those results were unexpected, as activation of PAL and an increase in lignin content are considered as common plant responses to various stress factors, including heavy metals (Jbir et al., 2001;Dixon et al., 2002;Janas et al., 2002;Mandre, 2002;Winkel-Shirley, 2002;Jouili & Ferjani, 2003;Lin et al., 2005;Yang et al., 2007;Kovacik & Klejdus, 2008).The expression of genes coding for both enzymes, PAL and CHS, has been analyzed under various stress conditions, but the knowledge on their regulation by metals in agronomically important plant species is limited.
In this study the expression of the gene coding for PAL, the activity of the enzyme, and lignin content were assessed in root seedlings of two legume species: soybean (Glycine max) and lupine (Lupinus luteus) exposed to cadmium (Cd 2+ ) or lead (Pb 2+ ).Unexpectedly, we observed opposite reactions of the two plant species to heavy metals.This calls into question the common belief that PAL activation and increased lignin accumulation are universal plant responses to abiotic stress.

MATeRIALS ANd MeThodS
Plant material.Seeds of soybean (Glycine max L. cv.Nawiko) obtained from the Department of Genetics and Plant Breeding, Poznań University of Life Sciences, and yellow lupine (Lupinus luteus L. cv.Juno) were surface sterilized with 75 % ethanol for 5 min, followed by 1% sodium hypochloride for 10 min, washed in water and germinated for 48 h on water-moistened filter paper in Petri dishes.The seedlings were than transferred to dishes containing 5 ml of either distilled water (control), aqueous solution of CdCl 2 containing 5, 10, 15, 20 or 25 mg/l of Cd 2+ or aqueous solution of Pb(NO 3 ) 2 containing 50, 100, 150, 200 or 350 mg/l of Pb 2+ .The seedlings were incubated in the dark at 22 °C for 48 h.
Determination of transcript level of phenylalanine ammonia-lyase gene (PAL) by reverse transcription PCR (RT-PCR).Total RNA was extracted from 100 mg of frozen root tips (5-mm long) using the TRI ® Reagent (Sigma) according to the manufacturer′s instructions and then treated with deoxyribonuclease I, amplification grade (Sigma).Samples containing 1 µg of RNA were used for reverse transcription with the RevertAid TM First Strand cDNA Synthesis Kit (Fermentas) and oligo(dT) 18 primer according to the manufacturer′s procedure, in a total volume of 20 µl.
Extraction and assay of phenylalanine ammonialyase (PAL) activity.The PAL (EC 4.3.1.5)activity was measured with a modified method of Cahill and Mc-Comb (1992).Root tips were homogenized on ice in a mortar with 0.1 M Tris/HCl buffer at pH 8.9 containing 10 mM mercaptoethanol, in a ratio of 4 ml buffer for 300 mg of plant tissue.Extracts were centrifuged at 15 000 × g at 4 °C for 30 min.Supernatants were used for measurement.The incubation mixture contained 80 mM borate buffer (pH 8.9), 30 mM l-phenylalanine and 0.5 ml enzymatic extract in a volume of 1.5 ml.After one hour of incubation in a water bath at 30 °C the re-action was stopped by addition of 1.5 ml of 2 M HCl.The amount of trans-cinnamic acid formed was measured spectrophotometricaly at 290 nm using a UV-1202 Shimadzu spectrophotometer.The activity of the enzyme was expressed in µmole of cinnamic acid formation per 1 mg protein per hour.Protein content was assayed according to Bradford.
Determination of lignin content.Lignin content was measured according to a modified method of Doster and Bostock (1988).Root tips were extracted for 48 h with two one-milliliter portions of methanol per 1 g of tissue.Then the residue was dried in a desiccator and pulverized in a mortar.Twenty milligrams of dry tissue was mixed with 2 ml of 2 M HCl and 0.5 ml of thioglycolic acid.The samples were heated at 95 °C for 4 h and centrifuged at 3 000 × g for 20 minutes.Pellets were washed twice with deionized water, extracted with 2 ml of 0.5 M NaOH for 18 h at room temperature and separated by centrifugation at 15 000 × g.The NaOH extract was collected and the precipitate was washed with 2 ml of deionized water and centrifuged at 15 000 × g.The obtained supernatant was added to the NaOH extract.Then the extract was acidified with 1 ml of concentrated HCl, left at 5 °C overnight and centrifuged (15 000 × g).The pellet was dissolved in 10 ml of 0.5 M NaOH and centrifuged at 15 000 × g.The absorbance was measured at 280 nm in a spectrophotometer.Lignin content was expressed in OD 280 units obtained from 10 mg of dry matter.
Statistics.All results are presented as means ± S.D. (standard deviation) obtained from nine independent replicates derived from tree independent biological experiments.

Effects of cadmium and lead on steady-state level of PAL mRNAs and activity PAL enzyme in soybean and lupine
The relative transcript levels of PAL mRNA were established by semi-quantitative reverse transcription PCR (RT-PCR) of RNA isolated from roots incubated for 48 h in control conditions or in the presence of increasing concentrations of either Cd 2+ (Figs.1A and 2A) or Pb 2+ (Figs.1B and 2B).PAL mRNA amount was normalized to that of a housekeeping gene (ubiquitin -Ub) mRNA.
The PAL mRNA level was higher in soybean roots growing in the presence of Cd 2+ (Fig. 1A) or Pb 2+ (Fig. 1B) than in the control.The highest amount of mRNA coding for PAL was observed in the presence of 15 mg/l of Cd 2+ or 50 mg/l of Pb 2+ .At the higher metal concentrations (20-25 mg/l of Cd 2+ , 150-350 mg/l of Pb 2+ ) (Figs. 1A and 1B) the amount of PAL mRNA returned to the control value (for Cd 2+ ) or to a value by only 34% above the control one (for Pb 2+ ).
The activity of PAL was increased in soybean treated with Cd 2+ or Pb 2+ (Figs.1A and 1B).The activity was strongly stimulated in roots exposed to Cd 2+ and this effect was dose-dependent (Fig. 1A).At the highest concentration of Cd 2+ (25 mg/l) the activity was almost three times higher than in the control plants.The PAL activity was also increased in Pb 2+ -treated soybean roots.It reached the highest level, about 70 % higher than in control plants, in the presence of the highest concentration of Pb 2+ (350 mg/l) (Fig. 1B).
The amount of PAL mRNA was decreased in lupine seedlings treated with Cd 2+ as compared to the control.
The observed inhibitory effect was most pronounced at the highest metal concentration (25 mg/l of Cd 2+ ; Fig. 2A).In contrast Pb 2+ treatment led to the accumulation of PAL transcript.However, this effect was only detected at medium Pb 2+ concentrations (150-200 mg/l of Pb 2+ ).At the highest Pb 2+ concentration (350 mg/l of Pb 2+ ) the PAL mRNA level was below that of control (Fig. 2B).
The activity of PAL was stimulated in lupine roots treated with Cd 2+ or Pb 2+ (Figs.2A and 2B).The highest activity of the enzyme was observed in lupine roots exposed to 25 mg/l of Cd 2+ or 150 mg/l of Pb 2+ .At the highest concentration of Cd 2+ (25 mg/l) the PAL activity in lupine roots was increased by about 40%, whereas Pb 2+ (150 mg/l) caused the stimulation of the enzyme activity by about 50 % (Figs.2A and 2B).

effects of cadmium and lead on lignin content in soybean and lupine
Lignin content was analyzed in roots of soybean (Fig. 3A) and lupine (Fig. 3B) seedlings treated for 48 h with selected concentrations of Cd 2+ (10 or 25 mg/l) or Pb 2+ (150 or 350 mg/l).The effect of the heavy metals on lignin accumulation was opposite in the analyzed plant species.The treatment of soybean with Cd 2+ or Pb 2+ caused a significant, 2-fold, increase in lignin content, except for the lower concentration of Pb 2+ (150 mg/l) where it remained at the control level.In contrast, in Cd 2+ or Pb 2+ -treated lupine roots lignin level was decreased.This effect was more pronounced at the higher concentrations of the heavy metals (25 mg/l of Cd 2+ and 350 mg/l of Pb 2+ ; Figs. 3A and 3B).

dISCuSSIoN
In this study we analyzed cadmium-and lead-induced changes in the expression and activity of the key phenylpropanoid pathway enzyme (PAL) and lignin content in roots of soybean (Glycine max) and lupine (Lupinus luteus) seedlings.Both Cd 2+ and Pb 2+ affected PAL mRNA level in the analyzed plants (Fig. 1).The stimulatory effect of both metals was observed in case of mRNA coding for PAL in soybean.In the case of lupine, the changes of PAL mRNA level were dependent on the metal used: Cd 2+ caused a decrease, whereas Pb 2+ an increase of PAL transcript level.Transcription activation of PAL genes has been observed in various plant species affected by different environmental stress factors (Dixon et al., 2002;Winkel-Shirley, 2002;Taiz & Zeiger, 2006).In contrast, inhibition of PAL expression is a less known plant response to unfavorable conditions.So far the de-crease of PAL mRNA has been described in fresh-cut endive (Cichorium intybus) subjected to heat shock (Salman et al., 2008) and in tea plants (Camellia sinensis) under drought stress (Singh et al., 2009).
PAL activity was increased in both plant species treated with Cd 2+ or Pb 2+ .However, even when the level of PAL mRNA was increased as well, the two responses were not co-ordinated.The highest enzyme activity was observed in soybean and lupine treated with higher concentrations of the metals (25 mg/l of Cd 2+ or 150-350 mg/l of Pb 2+ ; Figs. 1A, 1B and 2A, 2B).The highest PAL transcript level in soybean was detected at medium concentrations of both metals (15 mg/l of Cd 2+ or 50-100 mg/l of Pb 2+ ) (Figs. 1A and 1B).These data indicate that lower concentrations of Cd 2+ or Pb 2+ are most effective in inducing PAL in soybean at the transcript level, whereas higher concentrations of the metals are needed to affect PAL expression post-transcriptionally, at the level of the enzyme activity.The post-transcriptional mode of PAL regulation may play a major role in the case of Cd 2+ -treated lupine roots, where actually a decrease in the transcript level was correlated with enhanced enzyme activity (Fig. 2A).The PAL activity is known to be affected by various stress factors (Dixon & Paiva, 1995;Booker & Miller, 1998;Sarma & Sharma, 1999).Stimulation of PAL activity has already been noted in plants exposed to copper and cadmium (Jouili & Ferjani, 2003;Kovacik & Klejdus, 2008).However, to our knowledge, Cu 2+ and Cd 2+ were the only metals whose effects on the phenylpropanoid pathway were analyzed so far.Our results indicate that in legume plants other heavy metals, such as Pb 2+ , also activate the expression of PAL at the level of transcription and posttranscriptionally.
It is generally accepted that the common plant responses to stress factors involve an enhancement of PAL activity and an increase of lignin content (Cahill & McComb, 1992;Jbir et al., 2001;Mandre, 2002;Jouili & Ferjani, 2003;Cabané et al., 2004;Lee et al., 2007).An increased lignin accumulation was reported in pepper and soybean treated with Cu 2+ or Cd 2+ (Diaz et al., 2001;Lin et al., 2005;Yang et al., 2007).However, our present data indicate that lignin synthesis is not a universal plant response to heavy metals.Our results confirmed that treatment of soybean seedlings with Cd 2+ or Pb 2+ caused an enhancement of lignin level.In contrast, lupine roots showed a decreased lignin content in response to either of the two heavy metals (Fig. 3B).Therefore, the increase of PAL expression in lupine exposed to heavy metals could lead to the synthesis of secondary metabolites which are not involved in lignin formation.It was shown that treatment of soybean with Hg 2+ is correlated with increased level of the glyceollins (Mithöfer et al., 2004), while in Cu 2+ -treated white lupine elevated levels of genistein and 2′-hydroxygenistein were observed (Gagnon & Ibrahim, 1997).Based on this information and data presented here, we can conclude that heavy metal stress imposed by Cd 2+ or Pb 2+ causes induction of the phenylpropanoid pathway in soybean and lupine.The heavy metal-induced stimulation of this pathway in soybean is correlated with increased lignin content.In contrast, in lupine seedlings under the same conditions a decrease in lignin accumulation is observed, which suggests that PAL activation is involved in the synthesis of secondary metabolites other than lignin.The nature and the function of the secondary metabolites induced in lupine in response to heavy metals need to be elucidated.
Our accompanying paper (Pawlak-Sprada et al., 2011) addresses this question.Taken together, our data suggest that species-specific metabolic pathways are activated in legume plants exposed to heavy metals stress.