Exogenous Oxalic Acid and Citric Acid Improve Lead ( Pb ) Tolerance of Larix olgensis A . Henry Seedlings

We investigated the beneficial role of different concentrations of exogenous oxalic acid (OA) or citric acid (CA) for improving Pb tolerance and mitigating Pb-induced physiological toxicity in Changbai larch (Larix olgensis A. Henry) seedlings in northeast China. The seedlings were exposed to 100 mg·kg−1 Pb in soil alone or in combination with OA or CA irrigation for 10, 20, or 30 days. Pb-induced damage in L. olgensis was evident from elevated lipid peroxidation that significantly inhibited plant growth. Malondialdehyde (MDA) contents also increased in the presence of elevated Pb; however, superoxide dismutase (SOD) and peroxidase (POD) activities, as well as proline and pigment contents, all decreased. The damage increased in controls over the application periods. Pb contents in fine roots and leaves generally decreased with low-concentration organic acids (<1.0 mmol·L−1), but often increased at 5.0 and 10.0 mmol·L−1. Alternatively, when Pb-stressed plants were exposed to an organic acid (especially 5.0 or 10.0 mmol·L−1 for 10 days), the damage, as indicated by the physiological parameters, was reversed, and plant growth was promoted; CA was more effective in inducing these changes than OA. Therefore, exogenous organic acids have the potential to alleviate Pb-induced oxidative injuries, and can improve the tolerance of L. olgensis seedlings to Pb stress. Under lower OA and CA concentrations, the detoxification mechanism appears to be an external resistance mechanism; however, under higher concentrations (5.0–10.0 mmol·L−1) internal resistance mechanisms appear dominant. It is also possible that the two mechanisms work in tandem.


Introduction
Heavy-metal pollution has become a growing environmental problem in many countries around the world due to anthropogenic activities such as mining and smelting, the excessive use of chemical fertilizers, long-term wastewater irrigation, and vehicle emissions [1].This pollution resulted in soil contamination that is a hazard to human health through soil or edible plants.Lead (Pb) is one of the most toxic heavy-metal contaminants that easily accumulates in the soil, bioaccumulates in biological tissues, and has irreversible health effects [2].Its high toxicity affects plant physiological processes and growth, both of which are related to negative effects on nutrient uptake, respiration, photosynthesis, and antioxidant enzymes [3].
At the cellular level, a high concentration of Pb was shown to enhance oxidative stress in various plant species via the generation of reactive oxygen species (ROS).ROS-induced lipid membrane peroxidation can disturb the redox equilibrium [4], interrupt normal metabolic activities, and lead to cellular destruction or cell death [5].In general, increasing Pb concentration and exposure time induces a gradual increase in stress level [6].Improving the understanding of Pb stress and identifying management options for enhancing Pb tolerance of plants growing in Pb-stressed soil can facilitate restoration and reforestation in these areas.
Studies indicate that various sources of organic acids can act as a kind of natural chelating agent in soils, significantly affecting the migration and transformation behaviors of heavy metals [7].These organic acids also control plant availability and the biological toxicity of heavy metals.Relative to Pb, exogenous organic acids were demonstrated to play an important role in alleviating Pb-induced oxidative stress in various plant species and ameliorating detrimental effects of Pb on plants [8].There are two heavy-metal detoxification mechanisms for plants: external resistance and internal resistance.Organic acids can play a vital role in both mechanisms [9].
In external resistance, organic acids prevent metal ions from entering into the plant or reduce the metal availability in sensitive root zones by forming stable complexes with free metal ions.In internal resistance, organic acids chelate with metal ions already in plants, and change metal ions into non-toxic or less toxic forms [10].Plants generally adopt a variety of mechanisms to detoxify heavy metals, and the detoxification mechanisms vary with plant species [9,11].
The responses of plants in a Pb-stressed environment include physiological changes, which can inhibit toxicity and facilitate the recovery of impaired systems [2].Organic acids may augment these physiological responses, as well as the growth or biomass distribution patterns of plants in a Pb-stressed environment [8].This beneficial role of organic acids was established in species such as Boehmeria nivea (L.) Gaud [12], Brassica napus [11], Iris lactea var.chinensis [2,13], and Sesbania drummondii [14].
Beneficial organic acids studied in these species include oxalic acid (OA), citric acid (CA), ethylene diamine tetraacetic acid (EDTA), hydroxyethyl EDTA (HEDTA), diethylene triamine pentaacetic acid (DTPA), and nitrilotriacetic acid (NTA) [11,13,14].The protective roles of organic acids are affected by acid concentration and type, by metal (i.e., Pb) exposure concentration and time, as well as by plant species and plant-specific attributes of Pb absorption and accumulation [15].The focus of the above studies investigating organic acids, metal contamination, and plant species was on agronomic crops, while research about plantation grown forestry tree species remains little investigated.
Changbai larch (Larix olgensis A. Henry, belonging to Pinaceae) is one of the most extensively planted and productive commercial forestry species [16], and a useful species for the afforestation of Pb-polluted soils in northeast China, where areas of Pb-polluted soils need to be reclaimed.However, under severe Pb stress, larch survival and growth are restricted.The role of organic acids in L. olgensis Pb tolerance is unknown, although previous studies found that OA and CA are generally present in forest ecosystems of northeast China, especially in forest stands of L. olgensis.In the presence of L. olgensis, OA and CA could be leached from forest litters continuously at considerable concentrations [17].We, thus, hypothesized that exogenous OA and CA can play a role in improving the Pb tolerance of L. olgensis seedlings by mitigating Pb-induced physiological toxicity.If true, this study might help guide the future use of organic acids in restoration, reforestation, or afforestation efforts with L. olgensis seedlings in Pb-stressed soil areas of northeast China.Our study may also apply to other metal-or Pb-contaminated soil areas.Here, we applied OA and CA in concentrations bracketing those measured in L. olgensis litter leachates of northeast China [17] to L. olgensis seedlings growing in Pb-contaminated soil.We then measured seedling physiological responses to test our hypothesis.
The specific objectives of this study were to (1) investigate the physiological effects of Pb on L. olgensis seedlings during different Pb exposure times, (2) compare the effects of different concentrations and treatment times of exogenous OA or CA on seedling growth, Pb accumulation, and physiological characteristics of Pb-stressed seedlings; and (3) evaluate the interrelationships between organic acids and the Pb tolerance of L. olgensis seedlings.

Seed Harvest and Seedling Culture
L. olgensis seeds used in the study were harvested from L. olgensis plantations (stand age: 35 a) in the Maoershan Forest Research Station of the Northeast Forestry University, Harbin, Heilongjiang province, China, in September 2015.The L. olgensis plantations were growing in Typic Bori-Udic Cambosols corresponding to Haplic Cambisols (Greyic, Dystric) [18], which are zonal forest soils with high organic matter contents and well-developed horizons.The place of seed harvest is located at 127 • 30 -127 • 34 E and 45 • 21 -45 • 25 N with a continental temperate monsoon climate and an annual average temperature of 2.4 • C (range: −40 to 34 • C).Mean annual precipitation is 700 mm and the frost-free period is about 125 days with a growing season ranging between 120 and 140 days.When the seeds were harvested, the local daily average highest temperature was 21 • C and the lowest was 9 • C, and total amount of precipitation was 62 mm.Ripe cones were randomly picked from the trees of L. olgensis, and full seeds were selected, completely dried, put into clean and dry sands, and stored in a cold, dry, well-ventilated room.The sands had to be flipped every day to ensure the seeds were well ventilated until seedling culture.
The research was conducted under natural light in the greenhouse of this station.The daytime temperature in the greenhouse was 21-35 • C and the nighttime temperature was 8-18 • C; the daytime humidity was 50%-65% and the nighttime humidity was above 85%.Full and uniform L. olgensis seeds were collected, disinfected, stratified, and sown in late April 2016 in pots with a volume of 10,237.5 cm 3 .The pots were filled with the loam A 1 horizon of the Haplic Cambisols free from impurities (5.0 kg per pot, Table S1).A total of 60 seeds were planted in each pot, and 300 pots were planted in total.In late May, some seedlings were removed and only 30 were left per pot.The seedlings were acclimatized to ambient light and daily watering for one month.

Pb Treatment and Organic Acid Addition
After pre-culturing L. olgensis for one month, the soils in pots were treated with Pb(NO 3 ) 2 solution to achieve a soil Pb 2+ content of 100 mg•kg −1 .This concentration simulates soil Pb concentrations measured in the lead/zinc mining area of Yichun, Heilongjiang province, China, and the 100 mg•kg −1 was at the very high end.After Pb exposure for 10 days, which has previously been shown to be sufficient time for Pb uptake [2,19], organic acid treatments were initiated.OA or CA at 0, 0.2, 1.0, 5.0, and 10.0 mmol•L −1 concentrations was produced from organic salt solutions (pH 5.16) [17].Simulating the average pH of local soil was designed to prevent soil acidification caused by organic acid application, especially at high concentrations.Ten treatments were included (Ck: 0 mmol•L −1 acid without Pb addition; T1: 0 mmol•L −1 acid + Pb; T2: 0.2 mmol•L −1 OA + Pb; T3: 1.0 mmol•L −1 OA + Pb; T4: 5.0 mmol•L −1 OA + Pb; T5: 10.0 mmol•L −1 OA + Pb; T6: 0.2 mmol•L −1 CA + Pb; T7: 1.0 mmol•L −1 CA + Pb; T8: 5.0 mmol•L −1 CA + Pb; T9: 10.0 mmol•L −1 CA + Pb), and 30 pots were employed per treatment for 300 pots in total.For T2 to T5 and T6 to T9, root irrigation was performed by saturating soils with the different concentrations of OA or CA solutions, and applying solution at a rate approximately equivalent to 0.04, 0.2, 1.0, and 2.0 mmol•kg −1 of soil for each acid concentration, respectively.The organic acid addition to soil was estimated based on the concentration of organic acids in solution, the amount of solution, and the soil weight in each pot.OA or CA solutions were also applied to the upper and lower surfaces of L. olgensis leaves using a sprinkling can until the leaf surfaces were uniformly wet.During root irrigation and foliar spraying, control (Ck) and T1 were treated with the same amount of deionized water without organic acid.
OA and CA root irrigation and foliar spraying treatments were done once daily at 8:00 a.m. for a week.A constant application time was used to avoid changes in uptake due to plant physiological activity with time of day.The seven-day application was based on our previous research [20] that found that when organic acids such as OA and CA were added to the soils of this site sorption via anion exchange and biodegradation by microorganisms were evident.To ensure that we exceeded this sorption and biodegradation capacity we did some preliminary tests on L. olgensis seedlings and found that when organic acids were added consecutively for seven days the optimal effects of organic acids on L. olgensis seedlings were obtained (data not shown).At 10, 20, and 30 days after organic acid treatments started, the sampling and analysis of seedlings were conducted (Supplementary 1).Previous preliminary studies indicated that the 10-day sampling interval was appropriate for the analytical capacity of the laboratory and provided sufficient time for plant response.Harvest included 10 pots for each treatment at each time.The Ck treatment served as a no-Pb contamination control to compare to the effects of Pb stress (T1), and T2 to T9 were used to investigate the effects of different kinds and concentrations of organic acids on L. olgensis seedlings under Pb stress.Control and T1-T9 were sampled by complete harvest after 10, 20, and 30 days.

Plant Assays
At 10, 20, and 30 days after organic acid applications, seedling survival, and aboveground and belowground biomass were measured in all pots.Both before and at 10, 20, and 30 days after organic acid treatments, seedling height (with a meter rule, accuracy: 1.0 mm) and ground line diameter (GLD, with a vernier caliper, accuracy: 0.02 mm) were measured.A total of 30 random seedlings were measured for each treatment at each time across 10 pots, and the absolute growth rates were calculated with the formula below: Absolute growth = Value after treatment − Value before treatment At 10, 20, and 30 days after organic acid treatments, 10 seedlings per treatment from 10 pots were harvested, washed thoroughly with running tap water, and divided into leaf, stem, and root.Dry weights of all parts were determined after drying to a constant weight at 80 • C. Aboveground (stem + leaf) and belowground (root) biomass were calculated by treatment.
Fresh leaf samples were collected from each treatment across the 10 pots (about 3.0 g), frozen, and ground immediately in liquid nitrogen to determine physiological assays including lipid peroxidation (expressed as malondialdehyde (MDA) content), superoxide dismutase (SOD) and peroxidase (POD) activities, proline, carotenoid, and total chlorophyll contents.All of these were assayed according to the methods of Li et al. [21], and the details and methods were the same as Song et al. [22] (Supplementary 2).Each assay was replicated three times.
Finally, leaf and fine root (diameter ≤ 2 mm) samples were randomly collected from 10 pots within each treatment, washed with de-ionzed water, dried, de-enzymed for 15 min at 105 • C, and dried to a constant weight at 70 • C.Then, the samples were powdered, sifted through 2 mm nylon screens, mixed, microwave digested, and analyzed for Pb content on an inductively coupled plasma mass spectrometer (ICP-MS; SCIEX ELAN 6000, Perkin Elmer, Waltham, MA, USA).All samples were digested and measured in triplicate.

Data Analysis
Analysis of variance for a completely randomized design used the main effects of organic acid type and concentration with a repeated measure using the SPSS 18.0 software (IBM Corporation, Armonk, NY, USA).Although there were 10 pots per type of organic acid (type) × concentration of organic acid (conc) × treatment time (day), not all attributes were analyzed across all 10 pots; as such, sample sizes are listed explicitly in Table S2.Pearson correlations of all the parameters also were analyzed using the SPSS 18.0 software.Although all physiological assays were analyzed independently, correlations are noted, as many leaf-level metrics of stress were correlated.

Plant Growth Attributes
Type and concentration of organic acid, and treatment time (day) all affected the parameters measured in this study (Tables S2 and S3).Pb stress significantly decreased the survival rate at all treatment times.Compared with the T1 treatment, organic acid application increased the survival rate with the exception of the 0.2 and 1.0 mmol•L −1 OA and 0.2 and 1.0 mmol•L −1 CA treatments at 10 days, and 1.0 mmol•L −1 OA at 20 days.The greatest increases in survival were at 10.0 mmol•L −1 , but the effect of treatment time was not significant, and there were not significant day × type, type × conc, and day × conc interactions (Figure 1).days, and 1.0 mmol•L −1 OA at 20 days.The greatest increases in survival were at 10.0 mmol•L −1 , but the effect of treatment time was not significant, and there were not significant day × type, type × conc, and day × conc interactions (Figure 1).
Treatment time (days) Compared with Ck, the absolute growth of seedling height and GLD, and aboveground and belowground biomass all significantly decreased in Pb-treated soils (T1) (except for belowground biomass at 20 days), and the inhibition increased with increasing Pb exposure time (10, 20, and 30 days since initiation of organic acid treatments).In the presence of organic acid application, absolute seedling height growth (except for 0.2 and 1.0 mmol•L −1 OA at 10 days, and 1.0 and 5.0 mmol•L −1 OA at 20 days) and belowground biomass (except for 0.2 and 1.0 mmol•L −1 OA at 10 and 20 days, and 0.2, 1.0, and 10.0 mmol•L −1 CA at 20 days) both increased with effects of acid type, concentration, and analysis day all being significant.The most positive effects were generally found at 5.0 or 10.0 mmol•L −1 and the effects of CA were greater than those of OA.For belowground biomass, there were no significant day × type and type × conc interactions; however, for absolute seedling height growth, there were significant day × type, type × conc, and day × conc interactions.Organic acid applications increased absolute GLD growth (except for 0.2 mmol•L −1 OA at 10 days) and aboveground biomass (except for 1.0 mmol•L −1 OA and 0.2 mmol•L −1 CA at 10 days, 0.2 and 1.0 mmol•L −1 OA and CA at 20 days, and 0.2, 1.0, and 5.0 mmol•L −1 OA and 0.2 mmol•L −1 CA at 30 days) with effects of acid concentration and analysis day being significant.For absolute GLD growth, there was a significant day × conc interaction (Figures 2 and 3; Table S2).Compared with Ck, the absolute growth of seedling height and GLD, and aboveground and belowground biomass all significantly decreased in Pb-treated soils (T1) (except for belowground biomass at 20 days), and the inhibition increased with increasing Pb exposure time (10, 20, and 30 days since initiation of organic acid treatments).In the presence of organic acid application, absolute seedling height growth (except for 0.2 and 1.0 mmol•L −1 OA at 10 days, and 1.0 and 5.0 mmol•L −1 OA at 20 days) and belowground biomass (except for 0.2 and 1.0 mmol•L −1 OA at 10 and 20 days, and 0.2, 1.0, and 10.0 mmol•L −1 CA at 20 days) both increased with effects of acid type, concentration, and analysis day all being significant.The most positive effects were generally found at 5.0 or 10.0 mmol•L −1 and the effects of CA were greater than those of OA.For belowground biomass, there were no significant day × type and type × conc interactions; however, for absolute seedling height growth, there were significant day × type, type × conc, and day × conc interactions.Organic acid applications increased absolute GLD growth (except for 0.2 mmol•L −1 OA at 10 days) and aboveground biomass (except for 1.0 mmol•L −1 OA and 0.2 mmol•L −1 CA at 10 days, 0.2 and 1.0 mmol•L −1 OA and CA at 20 days, and 0.2, 1.0, and 5.0 mmol•L −1 OA and 0.2 mmol•L −1 CA at 30 days) with effects of acid concentration and analysis day being significant.For absolute GLD growth, there was a significant day × conc interaction (Figures 2 and 3; Table S2).

Physiological Responses
MDA contents of leaves in Pb-treated soils (T1) were higher than those of Ck (except for 10 days); however, Pb treatment (T1) decreased SOD and POD activities, as well as proline and total chlorophyll contents, relative to Ck, and the increments or declines grew with increasing Pb exposure time (Figures 4-7; Table S4).

Physiological Responses
MDA contents of leaves in Pb-treated soils (T1) were higher than those of Ck (except for 10 days); however, Pb treatment (T1) decreased SOD and POD activities, as well as proline and total chlorophyll contents, relative to Ck, and the increments or declines grew with increasing Pb exposure time (Figures 4-7; Table S4).MDA contents decreased under organic-acid treatments with the effects of some treatments being not significant, including 0.2 and 1.0 mmol•L −1 OA at 10 and 30 days, 0.2 mmol•L −1 OA at 20 days, 0.2 and 1.0 mmol•L −1 CA at 10 and 30 days, and 5.0 mmol•L −1 CA at 30 days, and often had concentrations below Ck.However, organic acid significantly increased SOD and POD activities, as well as proline and pigment contents, though the effects of some treatments were not significant, such as 0.2 mmol•L −1 OA at 10 days for SOD activities (Figures 4-7; Table S4).Days since treatment initiation and organic acid concentration both significantly affected physiological parameters measured.For MDA contents, at 10, 20, and 30 days, the most effective concentrations were 5.0, 5.0, and 5.0 mmol•L −1 for OA and 5.0, 10.0, and 10.0 mmol•L −1 for CA, respectively (Figure 4; Table S4).SOD and POD activities were usually most effective at 10.0 mmol•L −1 ; for proline, most of the treatments reached a maximum at 1.0 mmol•L −1 .For carotenoid contents, at 10, 20, and 30 days, the most effective concentrations were 10.0, 1.0, and 10.0 mmol•L −1 for OA and 1.0, 1.0, and 10.0 mmol•L −1 for CA, respectively; for total chlorophyll contents, at 10, 20, and 30 days, the most effective concentrations were 10.0, 5.0, and 5.0 mmol•L −1 for OA and 5.0, 1.0, and 10.0 mmol•L −1 for CA, respectively (Figures 5-7; Table S4).MDA contents decreased under organic-acid treatments with the effects of some treatments being not significant, including 0.2 and 1.0 mmol•L −1 OA at 10 and 30 days, 0.2 mmol•L −1 OA at 20 days, 0.2 and 1.0 mmol•L −1 CA at 10 and 30 days, and 5.0 mmol•L −1 CA at 30 days, and often had concentrations below Ck.However, organic acid significantly increased SOD and POD activities, as well as proline and pigment contents, though the effects of some treatments were not significant, such as 0.2 mmol•L −1 OA at 10 days for SOD activities (Figures 4-7; Table S4).Days since treatment initiation and organic acid concentration both significantly affected physiological parameters measured.For MDA contents, at 10, 20, and 30 days, the most effective concentrations were 5.0, 5.0, and 5.0 mmol•L −1 for OA and 5.0, 10.0, and 10.0 mmol•L −1 for CA, respectively (Figure 4; Table S4).SOD and POD activities were usually most effective at 10.0 mmol•L −1 ; for proline, most of the treatments reached a maximum at 1.0 mmol•L −1 .For carotenoid contents, at 10, 20, and 30 days, the most effective concentrations were 10.0, 1.0, and 10.0 mmol•L −1 for OA and 1.0, 1.0, and 10.0 mmol•L −1 for CA, respectively; for total chlorophyll contents, at 10, 20, and 30 days, the most effective concentrations were 10.0, 5.0, and 5.0 mmol•L −1 for OA and 5.0, 1.0, and 10.0 mmol•L −1 for CA, respectively (Figures 5-7; Table S4).MDA contents decreased under organic-acid treatments with the effects of some treatments being not significant, including 0.2 and 1.0 mmol•L −1 OA at 10 and 30 days, 0.2 mmol•L −1 OA at 20 days, 0.2 and 1.0 mmol•L −1 CA at 10 and 30 days, and 5.0 mmol•L −1 CA at 30 days, and often had concentrations below Ck.However, organic acid significantly increased SOD and POD activities, as well as proline and pigment contents, though the effects of some treatments were not significant, such as 0.2 mmol•L −1 OA at 10 days for SOD activities (Figures 4-7; Table S4).Days since treatment initiation and organic acid concentration both significantly affected physiological parameters measured.For MDA contents, at 10, 20, and 30 days, the most effective concentrations were 5.0, 5.0, and 5.0 mmol•L −1 for OA and 5.0, 10.0, and 10.0 mmol•L −1 for CA, respectively (Figure 4; Table S4).SOD and POD activities were usually most effective at 10.0 mmol•L −1 ; for proline, most of the treatments reached a maximum at 1.0 mmol•L −1 .For carotenoid contents, at 10, 20, and 30 days, the most effective concentrations were 10.0, 1.0, and 10.0 mmol•L −1 for OA and 1.0, 1.0, and 10.0 mmol•L −1 for CA, respectively; for total chlorophyll contents, at 10, 20, and 30 days, the most effective concentrations were 10.0, 5.0, and 5.0 mmol•L −1 for OA and 5.0, 1.0, and 10.0 mmol•L −1 for CA, respectively (Figures 5-7; Table S4).

Concentrations of organic acids (mmol
Decreasing MDA contents for both acids followed the order of 10 > 30 > 20 days.SOD activity for OA and CA increased for 30 > 20 > 10 days, while for POD activity with OA was 30 > 10 > 20 days and activity with CA was 10 > 20 > 30 days.The order of maximum proline contents for OA was 20 > 10 > 30 days, while for CA it was 10 > 20 > 30 days (Figures 4-7; Table S4).CA-mediated increases in POD activities, as well as proline and carotenoid contents, were more prominent than those of OA.For MDA contents, SOD and POD activities, and proline, carotenoid, and total chlorophyll contents, there were significant day × type, type × conc, and day × conc interactions, except for the day × type interaction for MDA contents (Figures 5-7; Table S4).

Pb Accumulation and Distribution
Pb contents in L. olgensis fine roots and leaves of Ck were low.In Pb-treated soil (T1), however, Pb concentrations in fine roots and leaves were significantly elevated, and Pb concentrations in roots were higher than those in leaves.Under organic acid treatments, Pb contents in fine roots and leaves generally decreased with low concentration organic acids (<1.0 mmol•L −1 ) but then often increased at 5.0 and 10.0 mmol•L −1 .Effects of acid type and concentration, as well as day since treatment initiation, were all significant (except for Pb contents in leaves under 0.2 mmol•L −1 CA at 10 days).CA exhibited a more prominent mitigating effect than OA (Figure 8; Table S4).Decreasing MDA contents for both acids followed the order of 10 > 30 > 20 days.SOD activity for OA and CA increased for 30 > 20 > 10 days, while for POD activity with OA was 30 > 10 > 20 days and activity with CA was 10 > 20 > 30 days.The order of maximum proline contents for OA was 20 > 10 > 30 days, while for CA it was 10 > 20 > 30 days (Figures 4-7; Table S4).CA-mediated increases in POD activities, as well as proline and carotenoid contents, were more prominent than those of OA.For MDA contents, SOD and POD activities, and proline, carotenoid, and total chlorophyll contents, there were significant day × type, type × conc, and day × conc interactions, except for the day × type interaction for MDA contents (Figures 5-7; Table S4).

Pb Accumulation and Distribution
Pb contents in L. olgensis fine roots and leaves of Ck were low.In Pb-treated soil (T1), however, Pb concentrations in fine roots and leaves were significantly elevated, and Pb concentrations in roots were higher than those in leaves.Under organic acid treatments, Pb contents in fine roots and leaves generally decreased with low concentration organic acids (<1.0 mmol•L −1 ) but then often increased at 5.0 and 10.0 mmol•L −1 .Effects of acid type and concentration, as well as day since treatment initiation, were all significant (except for Pb contents in leaves under 0.2 mmol•L −1 CA at 10 days).CA exhibited a more prominent mitigating effect than OA (Figure 8; Table S4).

Discussion
We found support for our hypothesis that exogenous organic acids may protect L. olgensis seedlings against Pb toxicity, as evidenced by the reversal in physiological characteristics indicating stress.In this study, though the effects of some treatments were not significant, organic acid treatments increased survival rate, absolute growth of seedling height and GLD, and belowground and aboveground biomass of seedlings in Pb-treated soil (Figures 1-3).For survival rate, the absolute growth of seedling height, belowground biomass, POD activities, proline and carotenoid contents in leaves, and Pb contents in fine roots and leaves, the effects of CA were all greater than OA (Figures 1, 2a, 3b, 5b, 6, 7a, and 8).
The protective role of organic acids, as demonstrated, for example, by the decrease in MDA contents, may contribute to this increased growth (Figures 1-4).It is also possible that organic acid irrigation of soil may have facilitated L. olgensis growth by exchanging nutrients (such as P and Fe) from soils and promoting nutrient absorption.Nutrient mobilization by CA is generally more effective than that of OA [23], a result consistent with greater plant-growth increases observed presently (Figures 1, 2a, and 3b).In the seedling stage of L. olgensis plants, because of the incomplete root growth and plant development, leaf surface fertilization is usually used rather than root fertilization [24].Here, foliar applications of OA or CA solutions were also used to enhance organic

Discussion
We found support for our hypothesis that exogenous organic acids may protect L. olgensis seedlings against Pb toxicity, as evidenced by the reversal in physiological characteristics indicating stress.In this study, though the effects of some treatments were not significant, organic acid treatments increased survival rate, absolute growth of seedling height and GLD, and belowground and aboveground biomass of seedlings in Pb-treated soil (Figures 1-3).For survival rate, the absolute growth of seedling height, belowground biomass, POD activities, proline and carotenoid contents in leaves, and Pb contents in fine roots and leaves, the effects of CA were all greater than OA (Figure 1, Figure 2a, Figure 3b, Figure 5b, Figure 6, Figure 7a, and Figure 8).
The protective role of organic acids, as demonstrated, for example, by the decrease in MDA contents, may contribute to this increased growth (Figures 1-4).It is also possible that organic acid irrigation of soil may have facilitated L. olgensis growth by exchanging nutrients (such as P and Fe) from soils and promoting nutrient absorption.Nutrient mobilization by CA is generally more effective than that of OA [23], a result consistent with greater plant-growth increases observed presently (Figure 1, Figure 2a, and Figure 3b).In the seedling stage of L. olgensis plants, because of the incomplete root growth and plant development, leaf surface fertilization is usually used rather than root fertilization [24].Here, foliar applications of OA or CA solutions were also used to enhance organic acid uptake.Solutions, such as nutrient liquids, mainly enter into the plant mesophyll cells through stomata, and there are usually more stomata on the under surface of leaves.As such, solutes pass through the lower surface more easily than the upper surface, and the absorption efficiency of the lower surface to solutions is higher.Spraying foliar fertilizers on the upper and lower surfaces of leaves is best to enhance the uptake of nutrients [25].Here, spraying OA and CA solutions were also carried out to enhance the uptake of organic acids.
One interesting observation in many attributes (e.g., SOD, proline, or Pb concentrations) is the lack of a monotonic increase or decrease in response to acid concentration.For example, Pb concentrations in roots initially declined; however, with increasing acid treatment concentration they increased at the highest concentrations (Figure 8).A previous study on the soils of this region [26] demonstrated that OA and CA both stimulated Pb release from the A 1 horizon.Although the exact concentrations of Pb-organic acid complexes in L. olgensis were not measured, this study may demonstrate that at low concentrations of organic acids (<1.0 mmol•L −1 ) reduced Pb contents in fine roots and leaves may result from an external resistance mechanism (i.e., complexes of OA or CA and Pb in soils inhibited Pb adsorption into plants or soluble Pb may be fixed on soil solid particles).However, at high acid concentrations (5.0-10.0mmol•L −1 ), Pb concentrations in fine roots and leaves often increased, but growth was high and many stress metrics were low (Figures 1-3 and 8).This response suggests an internal resistance mechanism that enhances Pb tolerance in the presence of elevated internal Pb.It is, of course, possible that the two kinds of mechanisms work together such that organic acid chelation in soils diminishes Pb absorption and accumulation in plants, while simultaneously, the toxicity of Pb that enters into plants is reduced by forming Pb-anion complexes.
Despite plant mechanisms to avoid or detoxify heavy metals, many metals can still have substantial negative effects on seed germination and plant growth.Pb, especially in high concentrations, leads to slow plant growth, root and leaf structural damage, and possibly death [2].Harmful effects were found on germination rate, seedling height, leaf number, shoot and root biomass, root and shoot length, and lateral root number [27].These effects vary with plant species.For example, Salsola passerina exhibited a higher Pb tolerance than Chenopodium album, which was partly due to Pb precipitation in S. passerina within the cell wall of the apoplast [19].The possible reasons for Pb-induced inhibition of plant growth include the use of more energy to deal with the metal stress than to produce biomass, or that Pb stress prevents plant water absorption [19].Pb stress was also demonstrated to decrease root activity, root respiration, root nutrient absorption, ATP production, and POD activity [12].This study was consistent with the above observations in finding decreased growth and an increase in lipid peroxidation with Pb stress (Figures 1-4).
Pb stress also induces an increase in the number of free radicals, which is associated with an increase in MDA content and membrane permeability, as observed previously in B. napus L. [11], sunflower [6], and maize [28].Generally, this damage increases with increasing Pb exposure time and Pb concentration [29].A protective response from CA application was observed in Juncus effuses L. [30] and B. napus L. [11], while both OA and CA benefits were demonstrated in I. lactea var.chinensis [13].A significant increase in MDA content with Pb stress was also found in this study with L. olgensis.Irrigation with organic acids decreased the MDA level of the seedlings (Figure 4) demonstrating their protective role against Pb toxicity.
SOD and POD are two important antioxidant enzymes and can effectively remove excessive free radicals and peroxide in Pb-stressed plants [11].There is considerable debate about the effects of Pb on SOD and POD activities since in some species (S. passerina Bunge and C. album L. [19], B. napus L. [11], tall fescue (Festuca arundinacea) [31] and I. lactea var.chinensis [13]) increases have been observed in leaves or roots, while in other species (Avicennia marina (a common mangrove species in South China)) [6] a decrease was observed.In this study, 100 mg kg −1 Pb decreased SOD and POD activities of L. olgensis leaves (Figure 5).
An increase in SOD and POD activities of Pb-stressed L. olgensis leaves was reversed under organic acid treatments suggesting that CA and OA both might improve antioxidant defense mechanisms in this species (Figure 5).Similar positive responses to organic acids under Pb stress were reported in B. nivea (L.) Gaud treated with CA and OA [12], and Juncus effusus L. [30] and B. napus L. [11] treated with CA.Positive responses to organic acids under Cd stress were also observed with salicylic acid-treated ryegrass (Lolium perenne L.) [32] and sulfosalicylic acid-treated rice seedlings (Oryza sativa) [33].Not all responses to organic acids are positive, however, with 5.0 mmol•L −1 CA and 0.5 and 5.0 mmol•L −1 OA treatment to different levels of Pb-stress significantly decreasing SOD activity of I. lactea var.chinensis leaves [13].In cases of positive response, the effects of CA in increasing POD activities were generally stronger than those of OA (Figure 5b), as CA has a stronger complexing capacity with metals and a larger dissociation constant than OA [23].Li et al. [12] also found a similar influence of CA over OA under Cd-stress.
Proline is a dominant precursor in amino acid synthesis often used to measure the tolerance of plants to adversity including metal stress.For example, in the presence of Pb proline contents of maize increased in the elongation stage, although this increase declined later in flowering and dough stages [28].Proline increases in response to CA have also been observed in B. napus L. [11] and B. nivea (L.) Gaud leaves and roots [12].We detected proline decline in Pb-stressed L. olgensis leaves and a distinct increase under OA and CA treatments (Figure 6).This increase suggested that with organic acid treatments L. olgensis may modify metabolism to mitigate harmful effects induced by Pb stress.
Chlorophyll is a key indicator of plant photosynthetic capacity, and its content is often used to evaluate tolerance of plants to metal stress [34].Heavy-metal stress, such as Pb or Cd, was typically demonstrated to decrease chlorophyll synthesis by inhibiting photosynthetic electron transport and protochlorophyllide reductase [12,32].Decreases were observed in B. napus L. [11], oilseed rape (B.napus) [35], S. passerina Bunge, and C. album L. [19].However, Shu et al. [27] found a stimulating effect in Jatropha curcas L. In the current study, chlorophyll contents decreased with increasing Pb exposure time (Figure 7).
OA and CA treatments beneficially affected pigment synthesis in Pb-stressed L. olgensis leaves (Figure 7), a response observed previously in B. napus L. treated with CA [11].The beneficial role of organic acids on pigment synthesis could be explained by the prevention of free metal ion transmission in the cytosol [12].Cd-stress, for example, destroyed plant cells through cytoplasmic shrinkage and metal deposition, while CA treatment restored the structure and shape of cells, and eliminated plasmolysis [12,30].Organic acids also contribute to nutrient mobilization (including Mg, Fe, and Zn) involved in chlorophyll formation via mechanisms such as complexation, oxidation-reduction reaction and acidification [36].Previous research on Cd-stressed L. olgensis seedlings in the same soil as the current work demonstrated that CA and OA both mobilized nutrients and were associated with increasing chlorophyll levels in leaves [22].Here, the reason for OA and CA increasing chlorophyll content under Pb stress might be similar to that observed under Cd stress [22].It should be emphasized that the research of Song et al. [22] is from the same investigator and had a similar aim as the current study (i.e., test the effects of exogenous organic acids on ecological resistance of L. olgensis seedlings under a stressed environment) as well as a similar methodological approach.In both studies, the kinds and concentrations of organic acid utilized were the same and the measured responses shared many similarities.The main difference between the studies is that the former soil was treated with Cd to induce stress while the current soil was treated with Pb.
Metal uptake and accumulation in roots, stems, and leaves of plants usually increases in a dose-dependent manner [32].Such accumulations were found in B. napus L. [11] and J. curcas L. [27].In J. curcas, cuttings accumulated much higher Pb in roots than aboveground seedling components [27].Pb uptake can be affected by the cultivation method as Pb accumulation of I. lactea var.chinensis planted in Pb tailings was much lower than that in an artificial culture experiment [2].In the current study, we planted L. olgensis seedlings in local forestry soils treated with Pb solution rather than using a hydroponics culture; as such, this study is closer to the plant growth media in real world conditions.Under these conditions, Pb increased in L. olgensis roots and leaves, with greater Pb accumulation in roots than leaves (Figure 8).Increases in Pb concentrations were associated with significant decreases in antioxidant enzyme activities, as well as proline and pigment contents (Figures 5-7), although accumulation in roots is a potential mechanism for plants to minimize Pb-induced damages.
Heavy metals in different parts of plants can have different organic forms.For example, S. passerina Bunge transformed free Pb into cell walls of the apoplast where it precipitated into an anchorage state [19].Enhanced Pb translocation from root to shoot was observed in B. napus L. treated with CA [11] and in I. lactea var.chinensis treated with CA and EDTA [2].Cd uptake was similarly promoted by CA and OA into aerial and belowground parts of Indian mustard and B. nivea (L.) Gaud, and Cd transport from roots to shoots was enhanced in B. nivea (L.) Gaud [12].In this study, under OA and CA treatments, more Pb was accumulated in roots than leaves (Figure 8), indicating that organic acids could protect L. olgensis from leaf-level oxidative stress.This result also found that the role of CA in enhancing the tolerance of L. olgensis seedlings was more prominent than OA (Figure 1, Figure 2b, Figure 3b, Figure 5b, Figure 6, and Figure 7a), maybe due to its stronger ligand affinity for chelating with Pb [23].

Conclusions
Exogenous OA and CA demonstrated a clear benefit in improving L. olgensis growth under Pb stress, and alleviating signs of physiological toxicity.In this study, increased soil Pb levels reduced plant growth and survival rate, inhibited antioxidant enzyme activities (SOD and POD), proline synthesis, and pigment contents, and enhanced lipid peroxidation.However, compared with the Pb-only treatment, organic acid applications reversed the physiological characteristics of Pb-stressed L. olgensis leaves, and the Pb-induced inhibition of survival rate, seedling height, GLD, aboveground and belowground biomass; all attributes were significantly mitigated.The most prominent effects were observed at 10 days and 5.0 or 10.0 mmol•L −1 , and CA was more effective than OA at the same concentration.Pb contents in fine roots and leaves generally decreased with low-concentration organic acid treatments (<1.0 mmol•L −1 ) but often increased at 5.0 and 10.0 mmol•L −1 ; thus, at low concentrations of organic acids, the detoxification mechanism of the seedlings in response to Pb stress may be mainly an external resistance mechanism, while, at high concentrations, an internal resistance mechanism may be used.Likely, both mechanisms work in tandem.L. olgensis accumulated more Pb in roots than in leaves, also possibly demonstrating a mechanism for tolerance in order to help it survive Pb toxicity.This work is among the first experimental evidence that reveals the interrelationships between exogenous OA and CA, and the tolerance of L. olgensis seedlings to Pb toxicity.As a native pioneer plant species, L. olgensis in combination with organic acids, especially CA, has promise to be applied in the afforestation of abandoned Pb-contaminated mining areas of northeast China.

Supplementary Materials:
The following are available online at http://www.mdpi.com/1999-4907/9/9/510/s1. Table S1: Selected properties of A1 horizon soil used in the experiment from a Haplic Cambisol collected in the Maoershan Forest Research Station of Northeast Forestry University, Heilongjiang province, China in 2015, Table S2: The p-values of the main effects of treatment time (Day), type of organic acid (Type), and concentration of organic acid (Conc), and their interactions on survival rate (survival), the absolute growth of seedling height (Height) and ground line diameter (GLD), belowground (Biobelow) and aboveground (Bioabove) biomass, MDA contents, SOD and POD activities, proline, carotenoid (Car), and total chlorophyll (Chl) contents in leaves, and Pb contents in fine roots and leaves (Pbroot and Pbleaf), Table S3: Pearson correlations of survival rate (survival), the absolute growth of seedling height (Seeding height growth), ground line diameter (GLD growth), belowground (Biobelow) and aboveground (Bioabove) biomass, MDA contents, SOD and POD activities, proline, carotenoid (Car), and total chlorophyll (Chl) contents in leaves, and Pb contents in fine roots and leaves (Pbroot and Pbleaf), Table S4: Post hoc tests of statistical difference by treatments.Treatments included root irrigation and foliar spraying with different concentrations of oxalic acid (OA) and citric acid (CA) over different application periods on Larix olgensis growing in Pb-contaminated soils.Response variables are MDA contents, SOD and POD activities, proline, carotenoid (Car), and total chlorophyll (Chl) contents, and Pb accumulation in fine roots and leaves (Pbroot and Pbleaf).Ck is a check treatment of seedlings grown in soil without organic acid treatment and no Pb addition.T1 to T9 are treatments of seedlings grown in Pb-stressed soils with addition of 0 acid, 0.2 mmol•L −1 OA, 1.0 mmol•L −1 OA, 5.0 mmol•L −1 OA, 10.0 mmol•L −1 OA, 0.2 mmol•L −1 CA, 1.0 mmol•L −1 CA, 5.0 mmol•L −1 CA, and 10.0 mmol•L −1 CA, respectively.Values followed by the same letter for the same

Figure 1 .
Figure 1.Survival rates of L. olgensis seedlings grown in Pb-stressed soils treated with different concentrations of organic acids over three treatment times (mean ± 1 S.D., n = 10).Values followed by the same letter are not statistically different at p < 0.05 by ANOVA (Tukey).

Figure 1 .
Figure1.Survival rates of L. olgensis seedlings grown in Pb-stressed soils treated with different concentrations of organic acids over three treatment times (mean ± 1 S.D., n = 10).Values followed by the same letter are not statistically different at p < 0.05 by ANOVA (Tukey).

Forests 2018, 9 ,Figure 2 .
Figure 2. Absolute growth of seedling height (a) and ground line diameter (b) of L. olgensis seedlings treated with different concentrations of organic acids over three treatment times (mean ± 1 S.D., n = 10).Values followed by the same letter are not statistically different at p < 0.05 determined from ANOVA (Tukey).

Figure 2 .Figure 3 .
Figure 2. Absolute growth of seedling height (a) and ground line diameter (b) of L. olgensis seedlings treated with different concentrations of organic acids over three treatment times (mean ± 1 S.D., n = 10).Values followed by the same letter are not statistically different at p < 0.05 determined from ANOVA (Tukey).

Figure 3 .
Figure 3. Aboveground (a) and belowground (b) biomass of L. olgensis seedlings treated with different concentrations of organic acids over three treatment times (mean ± 1 S.D., n = 10).Values followed by the same letter are not statistically different at p < 0.05 determined from ANOVA (Tukey).

Figure 7 .
Figure 7. Carotenoid (a) and total chlorophyll (b) contents of L. olgensis leaves treated with different concentrations of organic acids over three treatment times (mean ± 1 S.D., n = 3).FW = fresh weight.

Figure 8 .
Figure 8. Pb accumulation in L. olgensis fine roots (a) and leaves (b) treated with different concentrations of organic acids and different treatment times (mean ± 1 S.D., n = 3).DW = dry weight.

Figure 8 .
Figure 8. Pb accumulation in L. olgensis fine roots (a) and leaves (b) treated with different concentrations of organic acids and different treatment times (mean ± 1 S.D., n = 3).DW = dry weight.