The Alleviation of Nutrient Deficiency Symptoms in Changbai Larch (Larix olgensis) Seedlings by the Application of Exogenous Organic Acids

Exogenous organic acids are beneficial in protecting plants from the stress of heavy metal toxins (e.g., Pb) in soils. This work focuses on the potential role of organic acids in protecting Changbai larch (Larix olgensis) seedlings from the stress of growing in nutrient deficient soil. The seedlings were planted in a nutrient rich or deficient soil (A1 horizon of a Haplic Cambisol without organic acid as the nutrient rich control, or fully-mixed A1 + B horizons in a proportion of 1:2 as deficient) in pots in a greenhouse. In A1 + B horizons the seedlings were treated daily with concentrations of oxalic or citric acid (OA or CA) at a rate approximately equivalent to 0, 0.04, 0.2, 1.0, or 2.0 mmol·kg−1 of soil for 10, 20, and 30 days. Nutrient deficiency stressed the seedlings as indicated by lipid peroxidation and malondialdehyde (MDA) content in leaves significantly increasing, and superoxide dismutase (SOD) activities, proline, photosynthetic pigment contents, and chlorophyll fluorescence (Fv/Fm) decreasing. The stress increased in controls over the application periods. When nutrient deficient plants were exposed to an organic acid (especially 5.0 or 10.0 mmol·L−1 for 20 days), the stress as indicated by the physiological parameters was reversed, and survival rate of seedlings, and biomass of root, stem, and leaf significantly increased; CA was more effective than OA. The results demonstrate that exogenous organic acids alleviate nutrient deficiency-induced oxidative injuries and improve the tolerance of L. olgensis seedlings to nutrient deficiency.


Introduction
Soil nutrient availability and uptake play an important role in regulating plant growth, development, and biomass accumulation [1,2]. When plants suffer from nutrient deficiency, metabolic changes in the plants and a decrease in photosynthetic efficiency often can be found, and consequently reduce growth rate [3]. Understanding how plants adapt to the stress of nutrient deficiency is of interest. Some studies have demonstrated that organic acids play an important role in the mobilization, absorption, and transportation of nutrients in plants [4,5]. These acids are also involved in regulating physiological processes of plants, including decreasing lipid peroxidation, improving antioxidant enzyme activity, increasing contents of osmotic regulation substances, and consequently being beneficial to plant growth and biomass accumulation. As such, these acids increase the resistance of plants to a nutrient deficient environment [6,7]. One month after transplanting the seedlings, organic acid treatments were initiated. OA or CA at 0, 0.2, 1.0, 5.0, and 10.0 mmol·L −1 was produced from organic salt solutions. The pH of these solutions (~5. 16) was modified with NaOH solution to match the average pH of local soil. There were three sampling and analysis times (i.e., 10, 20, and 30 days) after organic acid treatments. For each sampling time, there were 10 treatments, 10 pots per treatment, and 30 seedlings per pot or 900 seedlings total in each treatment. The experimental layout is described in Table 2. The 10 treatments sampled included Ck, organic acid at 0 mmol·L −1 , OA at 0.2, 1.0, 5.0, and 10.0 mmol·L −1 , and CA at 0.2, 1.0, 5.0, and 10.0 mmol·L −1 . The Ck treatment was comprised of A 1 horizon only to serve as a high nutrient control (without organic acid) to compare to the effects of nutrient deficiency in the A 1 plus B horizon mixture. The A 1 and B horizons were mixed fully in a proportion of 1:2 and included a zero organic acid (0 mmol·L −1 ) treatment (T1). Thereafter, OA or CA was added in the concentrations shown above as the other eight treatments (T2-T9) to investigate the effects of different kinds and concentrations of organic acids on L. olgensis seedlings under nutrient deficiency. For T2 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. In addition, due to the incomplete root development of the seedlings foliar applications of organic acid solutions were done to enhance organic acid uptake, a common approach used with foliar fertilization [17]. Organic acids were applied to the upper and lower surfaces of L. olgensis leaves using a sprinkling can until the leaf surfaces were uniformly wet, and liquid drops fell from the leaf surfaces. Application to upper and lower surfaces further enhances uptake as solutions mainly enter into the mesophyll cells of plant leaves through the pores and many more pores usually lie in the lower surface of leaves [18]. During the root irrigation and foliar spraying, Control and T1 were both treated with the same amount of daily organic acid-free watering. Organic acid treatments were applied once daily at 8:00 AM, seven days a week. At 10, 20, and 30 days after organic acid treatments, sampling and analysis of seedlings were conducted by harvesting 10 pots per treatment.  Treatment  Ck  T1  T2  T3  T4  T5  T6  T7  T8

Measurements of Biomass
At 10, 20, and 30 days after organic acid treatments, seedling survival was measured in all pots. Thereafter, a total of 15 seedlings were carefully harvested with a shovel to avoid root damage from across the 10 pots within each treatment, washed thoroughly with running tap water, and divided into leaves, stems, and roots. The dry weights were measured after drying at 80 • C until a constant weight was reached.

Nutrient Element Analysis
Mature needles and fine roots (diameter less than 2 mm) were randomly collected from across the 10 pots within each treatment, washed with de-ionized water, dried, de-enzymed for 15 min at 105 • C, and dried to constant weight at 70 • C. The samples were then powdered and microwave digested prior to determination of Mg, K, and Fe concentrations by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS, SCIEX ELAN 6000, Perkin Elmer, Waltham, MA, USA). All measurements were performed in triplicate.

Physiological Parameter Measurements
Chlorophyll fluorescence was measured on intact needles placed in the dark for 20 min [19] using the LI-6400 XT Portable Photosynthesis System (LI-COR, Lincoln, OR, USA). From each treatment, 10 seedlings total from across the 10 pots were randomly selected and measurements were made on needles in the middle of the seedling crown. Based on F 0 (initial fluorescence) and F m (maximal fluorescence), F v (variable fluorescence, F v = F m − F 0 ) and F v /F m (maximal photochemical efficiency of photosystemII (PSII) in the dark) were calculated.
In addition, from the 10 pots within each treatment, mature middle needles of seedlings were randomly collected and combined (about 3.0 g totally). The samples were frozen and ground in liquid N 2 , and the following parameters were immediately determined. The level of lipid peroxidation was expressed as malondialdehyde (MDA, the final product of lipid peroxidation) content and was determined as 2-thiobarbituric acid (TBA) reactive metabolites, superoxide dismutase (SOD) activity was determined using a nitro blue tetrazolium (NBT) photochemical reduction, proline content was determined by the acidic ninhydrin reaction, and total chlorophyll and carotenoid contents were determined by 80% acetone extraction-spectrophotometry. All of these assays referred to the methods of Li et al. [20], and the details were the same as Song et al. [21]. The analyses were all replicated three times.

Data Analysis
Analysis of variance for a completely randomized design used the main effects of organic acid and concentration with a repeated measure with SPSS 18.0. Although there were ten pots per organic acid (acid) × concentration of organic acid (Conc) × length of application (or Day) not all measurements were made in all ten pots; sample sizes are listed explicitly in Table 3. Also, many leaf level metrics of stress were correlated, and although all attributes were analyzed correlations are noted. Table 3. The p values of the main effects of application period (Day), organic acid (Acid), concentration of organic acid (Conc), and their interactions on the survival rate (Survival), biomass of leaf, stem, and root (Bio leaf , Bio stem , and Bio root , respectively), contents of Mg, K, and Fe in roots and leaves (Mg root , K root , Fe root , Mg leaf , K leaf , and Fe leaf , respectively), malondialdehyde (MDA), superoxide dismutase (SOD), proline, carotenoid (Car), and total chlorophyll (Chl) contents and F v /F m in leaves.

Survival, Biomass, and Nutrient Element Contents of L. olgensis Seedlings
Organic acid (citric or oxalic) type, concentration, and application period (Day) all had an important influence on most of the parameters in this study, including survival rate, biomass of different parts of seedlings, and nutrient contents ( Table 3). The survival rate of L. olgensis decreased over the 30 days in all treatments. In comparing the nutrient sufficient control (i.e., A 1 soil without organic acid inputs) with the T1 treatment (A 1 :B soil without organic acid inputs) the survival rate decreased slightly and non-significantly (p > 0.05) by 0.76, 0.77, and 0.41% at 10, 20, and 30 days, respectively. Compared with the T1 treatment, the survival rate increased with organic acid application except in the 0.2 mmol·L −1 organic acid treatments at 10 and 20 days. The greatest increases in survival were at 5.0 mmol·L −1 organic acid ( Figure 1; Table S1).

Survival, Biomass, and Nutrient Element Contents of L. olgensis Seedlings
Organic acid (citric or oxalic) type, concentration, and application period (Day) all had an important influence on most of the parameters in this study, including survival rate, biomass of different parts of seedlings, and nutrient contents ( Table 3). The survival rate of L. olgensis decreased over the 30 days in all treatments. In comparing the nutrient sufficient control (i.e., A1 soil without organic acid inputs) with the T1 treatment (A1:B soil without organic acid inputs) the survival rate decreased slightly and non-significantly (p > 0.05) by 0.76, 0.77, and 0.41% at 10, 20, and 30 days, respectively. Compared with the T1 treatment, the survival rate increased with organic acid application except in the 0.2 mmol•L −1 organic acid treatments at 10 and 20 days. The greatest increases in survival were at 5.0 mmol•L −1 organic acid ( Figure 1; Table S1). Compared with the nutrient sufficient control, the root, leaf, and stem dry biomasses decreased in T1. At 10 or 20 days the decrease was slight and non-significantly (p > 0.05), except for stem biomass that had declined at 20 days, but all declines were significant at 30 days. Under OA and CA treatments, most biomass components increased, although there were exceptions (e.g., stem and root biomass of 0.2 mmol•L −1 OA at 20 days or stem biomasses of 0.2, 1.0, and 5.0 mmol•L −1 CA at 30 days). The increases were greatest at 10.0 mmol•L −1 organic acid ( Figure 2; Table S2). Compared with the T1 treatment, most of the organic acid treatments had significantly greater biomass and there was a significant acid × concentration interaction as the magnitude of differences varied with concentration. For example, the biomass increases in response to CA were generally greater than those of OA at most concentrations ( Figure 2; Table S2).
Nutrient concentration also varied with treatment. Compared with the nutrient sufficient control (Ck: A1 soil only) the A1:B without acid treatment (T1) often had lower K concentrations for roots and leaves, while Fe contents were greater. The Mg contents in roots and leaves were not significantly different, though the values were often lower. With acid additions in the A1:B soil mix, most root concentrations of Mg and K declined and Fe contents increased with effects of acid type and concentration being significant (except Fe and Mg concentrations of 0.2 mmol•L −1 CA at 20 days was not significant). In the case of leaves, the response in Mg concentrations was consistent with roots. For K and Fe concentrations, although patterns were similar, the effect of acid type on concentrations was not significant. Furthermore, although the effect of organic acid concentration on leaf K and Fe  Compared with the nutrient sufficient control, the root, leaf, and stem dry biomasses decreased in T1. At 10 or 20 days the decrease was slight and non-significantly (p > 0.05), except for stem biomass that had declined at 20 days, but all declines were significant at 30 days. Under OA and CA treatments, most biomass components increased, although there were exceptions (e.g., stem and root biomass of 0.2 mmol·L −1 OA at 20 days or stem biomasses of 0.2, 1.0, and 5.0 mmol·L −1 CA at 30 days). The increases were greatest at 10.0 mmol·L −1 organic acid ( Figure 2; Table S2). Compared with the T1 treatment, most of the organic acid treatments had significantly greater biomass and there was a significant acid × concentration interaction as the magnitude of differences varied with concentration. For example, the biomass increases in response to CA were generally greater than those of OA at most concentrations ( Figure 2; Table S2).
Nutrient concentration also varied with treatment. Compared with the nutrient sufficient control (Ck: A 1 soil only) the A 1 :B without acid treatment (T1) often had lower K concentrations for roots and leaves, while Fe contents were greater. The Mg contents in roots and leaves were not significantly different, though the values were often lower. With acid additions in the A 1 :B soil mix, most root concentrations of Mg and K declined and Fe contents increased with effects of acid type and concentration being significant (except Fe and Mg concentrations of 0.2 mmol·L −1 CA at 20 days was not significant). In the case of leaves, the response in Mg concentrations was consistent with roots. For K and Fe concentrations, although patterns were similar, the effect of acid type on concentrations was not significant. Furthermore, although the effect of organic acid concentration on leaf K and Fe was significant, the impact on leaf Mg was not a strongly decreasing trend as observed in roots ( Figure 3; Table S3).
Forests 2016, 7, 213 6 of 16 was significant, the impact on leaf Mg was not a strongly decreasing trend as observed in roots ( Figure 3; Table S3).

Lipid Peroxidation
The MDA contents in nutrient deficient L. olgensis leaves (T1) were higher than those of the control (Ck), and the difference increased with Day. With organic acid treatments, the MDA concentration decreased, and most of the concentrations were below Ck. The most prominent effect was observed at 10.0 mmol•L −1 organic acid, the effects of CA were stronger than that of OA, and the order was 20 > 10 > 30 days. After organic acid treatments most MDA contents at 30 days were higher than 20 days, and much higher than 10 days (Figure 4; Table S4).

Lipid Peroxidation
The MDA contents in nutrient deficient L. olgensis leaves (T1) were higher than those of the control (Ck), and the difference increased with Day. With organic acid treatments, the MDA concentration decreased, and most of the concentrations were below Ck. The most prominent effect was observed at 10.0 mmol·L −1 organic acid, the effects of CA were stronger than that of OA, and the order was 20 > 10 > 30 days. After organic acid treatments most MDA contents at 30 days were higher than 20 days, and much higher than 10 days (Figure 4; Table S4).

Activities of SOD
Compared with Ck, the SOD activities in T1 decreased slightly, and the decrease was enhanced with application period. OA and CA treatments at 10, 20, and 30 days generally increased the SOD activities, and the biggest increases were found at 0.  Table S4).

Proline Contents
Compared with the control (Ck), the T1 treatment decreased the proline contents by 3.85, 4.15, and 5.34% at 10, 20, and 30 days, respectively. Compared with T1, the proline contents were higher

Activities of SOD
Compared with Ck, the SOD activities in T1 decreased slightly, and the decrease was enhanced with application period. OA and CA treatments at 10, 20, and 30 days generally increased the SOD activities, and the biggest increases were found at 0.  Table S4).

Activities of SOD
Compared with Ck, the SOD activities in T1 decreased slightly, and the decrease was enhanced with application period. OA and CA treatments at 10, 20, and 30 days generally increased the SOD activities, and the biggest increases were found at 0.  Table S4).

Proline Contents
Compared with the control (Ck), the T1 treatment decreased the proline contents by 3.85, 4.15, and 5.34% at 10, 20, and 30 days, respectively. Compared with T1, the proline contents were higher

Photosynthetic Pigment Contents and Chlorophyll Fluorescence
In comparing Ck to the T1 treatment, the total chlorophyll and carotenoid contents in L. olgensis leaves were not significantly different, but their values all decreased across all days (Table 4). Within the nutrient deficient soil (A1:B) acid type did not significantly affect total chlorophyll or carotenoids. Acid concentration and day, however, were significant and the peak values were at 10.0, 5.0, and 10.0 mmol•L −1 organic acids at 10, 20, and 30 days, respectively. Nutrient deficiency also decreased Fv/Fm ratios in leaves, however, they increased significantly under organic acid treatments with effects of acid type and concentration being significant, and the peak values were generally found at 10.0 mmol•L −1 (Figure 7; Table S4).

Photosynthetic Pigment Contents and Chlorophyll Fluorescence
In comparing Ck to the T1 treatment, the total chlorophyll and carotenoid contents in L. olgensis leaves were not significantly different, but their values all decreased across all days (Table 4). Within the nutrient deficient soil (A 1 :B) acid type did not significantly affect total chlorophyll or carotenoids. Acid concentration and day, however, were significant and the peak values were at 10.0, 5.0, and 10.0 mmol·L −1 organic acids at 10, 20, and 30 days, respectively. Nutrient deficiency also decreased F v /F m ratios in leaves, however, they increased significantly under organic acid treatments with effects of acid type and concentration being significant, and the peak values were generally found at 10.0 mmol·L −1 (Figure 7; Table S4).

Discussion
Tree growth under natural conditions can be usually restricted by limited availability of nutrients in soils and it is well-recognized that applying fertilizers (e.g., N or P) to soils can promote growth [22]. It is less well-known that applications of organic acids can also significantly promote seed germination, aboveground and belowground biomass accumulation, and plant growth and development. The beneficial role of organic acids for plants under natural conditions or under cold stress, water stress, or salinity stress has been established in Picea abies, cucumber, durum wheat, and cabbage treated with salicylic acid (SA) and other organic acids [6,23,24]. Plant height, leaf length, leaf width, and leaf area all increased in these studies, and the effects vary with the kind and concentration of organic acids, plant species, and plant stress. The current study builds on these previous organic acid studies but applies organic acids to L. olgensis seedlings, a critical tree crop for reforesting nutrient deficient soils in northeast China. Overall, L. olgensis seedling responses were consistent with previous results suggesting that seedling stress declined in the presence of organic acids. In this study, CA was more effective than OA in reducing stress under nutrient deficiency and higher concentrations (i.e., 5 to 10 mmol•L −1 ) were most effective (Figures 1-7; Table 4).
MDA, SOD, and proline can be considered as major parameters of physiological plant stress, which is used to indicate the metabolic activities and resistance of plants to adverse environments. Lipid peroxidation (demonstrated by MDA content) is generally an indicator of oxidative injury in stressed plants [25,26]. MDA can crosslink with substances such as proteins, nucleic acids, or amino acids to form insoluble compounds (lipofuscin) that interfere in normal cell activities [27]. Various environmental stresses can induce MDA accumulation, and in severe cases this can lead to plant death. Previous research has shown increasing MDA concentrations in Lupinus albus with P deficiency [28]. This study, using low nutrient soil, found decreasing MDA concentrations of L. olgensis leaves (Figure 4). These lower MDA concentrations suggest exogenous organic acids are involved in protecting the cell membrane of L. olgensis seedlings against oxidative stress.
SOD is one of the most effective antioxidant enzymes scavenging oxygen radicals in plants [29] and can convert superoxide radicals into less toxic agents [30]. Furthermore, the concentration of SOD activity is directly related to the degree of cell damage and plant resistance [31]. SOD responses to soil stress have been observed. For example, SOD activities of Avicennia marina (a common mangrove species in South China) were decreased by Pb stress [32] and the activities of SOD decreased in Cd-

Discussion
Tree growth under natural conditions can be usually restricted by limited availability of nutrients in soils and it is well-recognized that applying fertilizers (e.g., N or P) to soils can promote growth [22]. It is less well-known that applications of organic acids can also significantly promote seed germination, aboveground and belowground biomass accumulation, and plant growth and development. The beneficial role of organic acids for plants under natural conditions or under cold stress, water stress, or salinity stress has been established in Picea abies, cucumber, durum wheat, and cabbage treated with salicylic acid (SA) and other organic acids [6,23,24]. Plant height, leaf length, leaf width, and leaf area all increased in these studies, and the effects vary with the kind and concentration of organic acids, plant species, and plant stress. The current study builds on these previous organic acid studies but applies organic acids to L. olgensis seedlings, a critical tree crop for reforesting nutrient deficient soils in northeast China. Overall, L. olgensis seedling responses were consistent with previous results suggesting that seedling stress declined in the presence of organic acids. In this study, CA was more effective than OA in reducing stress under nutrient deficiency and higher concentrations (i.e., 5 to 10 mmol·L −1 ) were most effective (Figures 1-7; Table 4).
MDA, SOD, and proline can be considered as major parameters of physiological plant stress, which is used to indicate the metabolic activities and resistance of plants to adverse environments. Lipid peroxidation (demonstrated by MDA content) is generally an indicator of oxidative injury in stressed plants [25,26]. MDA can crosslink with substances such as proteins, nucleic acids, or amino acids to form insoluble compounds (lipofuscin) that interfere in normal cell activities [27]. Various environmental stresses can induce MDA accumulation, and in severe cases this can lead to plant death. Previous research has shown increasing MDA concentrations in Lupinus albus with P deficiency [28]. This study, using low nutrient soil, found decreasing MDA concentrations of L. olgensis leaves (Figure 4). These lower MDA concentrations suggest exogenous organic acids are involved in protecting the cell membrane of L. olgensis seedlings against oxidative stress.
SOD is one of the most effective antioxidant enzymes scavenging oxygen radicals in plants [29] and can convert superoxide radicals into less toxic agents [30]. Furthermore, the concentration of SOD activity is directly related to the degree of cell damage and plant resistance [31]. SOD responses to soil stress have been observed. For example, SOD activities of Avicennia marina (a common mangrove species in South China) were decreased by Pb stress [32] and the activities of SOD decreased in Cd-treated L. olgensis leaves, possibly due to the binding of Cd to the thiol groups of the enzymes [21]. Previous studies have shown that spraying SA and other organic acids on tobacco [33], rice (Oryza sativa) [34], or perennial ryegrass [35] plants increases SOD activity. Similar responses in SOD activity were observed in maize leaves at six and then days after 1.0 mmol·L −1 SA treatment, where the copper couple superoxide dismutase (Sod4) transcription increased [36]. In this study the lower nutrient soil (A 1 :B) resulted in a decrease of SOD activities that increased with application period. OA and CA treatments increased SOD, especially CA ( Figure 5). This SOD increase likely contributed to the removal of reactive oxygen species such as MDA (Figure 4).
Proline is one of the most effective substances controlling osmotic adjustment [37] and also plays a protective role in protein stability. The use of proline as an osmolyte was first reported by Cavalieri and Huang [38] in Juncus roemerianus, and since has been shown to be a common compound accumulated by plants as a response to salt, water, or cold stress [39]. Under soil stress, the proline contents in plant tissues are greatly increased in order to improve plant resistance [29,40]. This study found that proline contents of L. olgensis leaves in the A 1 :B soil were lower than those of Ck, which suggested that nutrient stress inhibited proline synthesis and disturbed their metabolisms. In A 1 :B soil proline concentrations increased after most organic acid treatments ( Figure 6). This increase suggests that organic acids may have induced proline synthesis and accumulation, thereby enhancing the ability of L. olgensis seedlings in osmotic regulation.
In contrast to the increases in proline contents and SOD activities noted above, there were a few concentrations of organic acids that resulted in contents or activities below the levels of the T1 control ( Figures 5 and 6). These observations might suggest that low concentrations of organic acids can have beneficial effects while high concentrations may have some harmful effects [41]. A possible explanation is that the initial enhanced growth from high organic acid treatments leads to increased nutrient demand and stress, which eventually surpasses the benefits of the acid treatments in relieving stress. In other words, the beneficial effects of organic acids failed to compensate for the growth induced nutrient stress.
In addition to indicators of stress, photosynthetic pigments also can be viewed as an adaptive response to adverse environments. Chlorophyll is highly sensitive to stress and indicates the resistance of plants to stressed environments [41]. Environmental stress may harm the chloroplast structure of plants, inhibiting chlorophyll synthesis [35]. Carotenoid, a cell endogenous antioxidant, also plays an important role in scavenging reactive oxygen species and preventing membrane lipid peroxidation [40]. Studies have shown that organic acids can increase the pigment contents of plants growing in both normal and stressed environments. Pigment increases in normal environments have been reported for malic acid, CA, tartaric acid, lactic acid, and SA across many plant species, including cabbage, tobacco, and others [8]. Under adverse environments, one example of positive response is SA treatments for three and five days that increased total chlorophyll contents of maize seedlings under osmotic stress by 7.5 and 45.3%, respectively [42]. In the current study, results were similar with organic acid treatments increasing both pigments, although there was no difference between the acids (Table 4).
Chlorophyll fluorescence measures the absorption, transfer, consumption, and distribution of light energy by the optical system in the process of photosynthesis, and can be used to evaluate relative photosynthetic performance of plants [43]. F v /F m (described in the methods) is a sensitive metric of stress in plants and plant tolerance to environmental factors [44]. Decreases in F v /F m indicate photoinhibition or photooxidation, which can be caused by photodamage to PSII in response to environmental stresses [45]. Significant reductions in the F v /F m ratio have been found in Mn-treated 'Kothreiki' and 'FS-17' olive cultivars [46], and in Caulerpa lentillifera in response to high salinity [47]. In C. lentillifera, F v /F m was >0.80, indicating the plant was healthy (<0.80 indicated stress, and values <0.70 resulted in death) [47]. The reduction of foliar F v /F m also has been reported in strawberry [48], red spruce, and balsam fir [49], and Norway spruce [50] and radiata pine [45] in response to low nutrient supply or an imbalance in nutrients. In this study the F v /F m ratio declined in L. olgensis leaves in low nutrient soils but was increased with organic acid treatments (Figure 7). In this study, changes in F v /F m were caused by an increase of F 0 and at high acid concentrations some seedlings had ratios close to or higher than those of healthy leaves, indicating that the seedlings no longer suffered damages from nutrient deficiency.
There are several possible mechanisms for how organic acids in low nutrient soils may be reducing stress and promoting plant growth, although all cannot be definitively addressed in this study. (1) Organic acids could promote root growth or uptake activity, thus indirectly promoting aboveground plant growth [51]; (2) Organic acids could effectively enhance chlorophyll content and photosynthesis rates in plants, which would not only promote the accumulation of organic matter, but also facilitate the transportation of water and inorganic salts, and thus indirectly promote plant growth [52]; (3) Organic acid injection into soils could significantly improve the solubilization and availability of soil nutrients (e.g., P, Fe, K), and promote plant absorption and transportation of nutrients [53]. Root biomass of the L. olgensis seedlings did increase with acid treatment and was associated with greater aboveground growth. It is not clear, however, if root growth was the cause of aboveground growth or was itself a response to some other mechanism induced by the organic acids.
As to the mechanism of organic acids to increase chlorophyll contents, it is clear that the formation of chlorophyll is affected by soil nutrient status. Mg is an essential element composing chlorophyll while Fe and Zn may be catalysts to certain enzymes during chlorophyll formation. In this study, Mg concentrations in roots and leaves declined with acid concentration but Fe concentrations increased (Figure 3), as did chlorophyll contents (Table 4). This suggests that the changes in chlorophyll contents are more responsive to Fe concentration increases, which may reflect a greater percent increase (i.e., (sampling date − initial)/initial concentration) in Fe concentration (0.07%-1.52%, 0.01%-0.33%, and 0.11%-0.75% at 10, 20, and 30 days, respectively) being greater than the declining range of Mg concentrations (0.02%-0.21%, 0.00%-0.28%, and 0.01%-0.10% at 10, 20, and 30 days, respectively).
Finally, organic acids can significantly increase the availability of some mineral elements including Fe, Zn, or Mg in the soil by mechanisms of acidification, chelation, redox reactions, or inhibition of adsorption to minerals [54]. In the case of organic acids the release of nutrients from Haplic Cambisols is mainly by the mechanism of complexation. The complexation ability of CA is greater than that of OA [55], and the dissociation constant of CA (pKa 3.14) is also greater than that of OA (pKa 1.23). As such, CA should be able to release more nutrients from these soils [56] and, in fact, the response in stress indicators is generally greater to CA than OA.
In an earlier study, Song et al. [56] showed that OA and CA could mobilize P and Fe from Haplic Cambisols, especially the A 1 horizon used here, and promote their absorption and transportation by L. olgensis seedlings. P accumulation and uptake efficiency in L. olgensis fine roots and leaves were both increased [55]. In the current study, OA and CA increased Fe contents of L. olgensis roots and leaves ( Figure 3). So from the perspective of plant nutrient supply, exogenous OA and CA could contribute to P and Fe absorption and accumulation in seedlings, and indirectly increase plant biomass. For improving nutrient release from the study soils in northeast China and increasing the subsequent P accumulation and P uptake efficiency in L. olgensis seedlings, CA > OA [55], a result consistent with the increase in biomass accumulation observed in this study (Figure 2).
Although the exact mechanism of organic acid treatment response is difficult to infer, this study clearly demonstrates the beneficial value of organic acids for reducing plant stress. Furthermore, since L. olgensis seedlings are planted in local forest soils rather than nutrient solution or hydroponic conditions, this study is closer to the plant growth media in an operational condition, and thus has practical application value for future nursery or reforestation undertakings in the region.

Conclusions
Soil nutrient deficiency had harmful effects on measures of plant stress in L. olgensis leaves. Survival rate and root, leaf, and stem biomass accumulation were all inhibited, MDA contents increased, and SOD activities, proline, total chlorophyll and carotenoid contents, and F v /F m ratios all decreased. These changes generally increased with the length of the application period. Nutrient deficiency decreased K concentrations in roots and leaves, while Fe contents were greater. The Mg contents in roots and leaves were not significantly different, although the values were often lower.
Compared with the nutrient deficiency treatment T1 (i.e., A 1 :B soil with no organic acid inputs) the application of organic acids reversed the measures of plant stress in L. olgensis leaves. Organic acid (citric or oxalic) type, concentration, and application period all had an important influence on most of the parameters in this study. The most prominent effects were observed at 20 days under the 5.0 or 10.0 mmol·L −1 treatments, and CA was more effective than OA. Organic acids increase survival rate and biomass accumulation of root, leaf, and stem, and MDA, SOD, proline, chlorophyll, and carotenoid measures of plant condition all improve. Hence, exogenous organic acids alleviate the damage of nutrient deficiency to L. olgensis seedlings, and improve their resistance to nutrient deficiency.
Supplementary Materials: The following are available online at www.mdpi.com/1999-4907/7/10/213/s1, Table S1: Statistical difference results of survival rates of L. olgensis seedlings induced by root irrigation and foliar spraying with different concentrations of organic acids and different application periods; Table S2: Statistical difference results of dry weights of different parts of L. olgensis seedlings induced by root irrigation and foliar spraying with different concentrations of organic acids and different application periods; Table S3: Statistical difference results of Mg, K and Fe contents in roots and leaves of L. olgensis seedlings induced by root irrigation and foliar spraying with different concentrations of organic acids and different application periods. Table S4: Statistical difference results of MDA contents, SOD activities, proline contents and Fv/Fm ratios of L. olgensis leaves for seedlings induced by root irrigation and foliar spraying with different concentrations of organic acids and different application periods.