Increasing Nutrient Solution pH Alleviated Aluminum-Induced Inhibition of Growth and Impairment of Photosynthetic Electron Transport Chain in Citrus sinensis Seedlings

Abstract

Although the physiological and molecular responses of Citrus to Al-toxicity or low pH have been examined in some details, little information is available on Citrus responses to pH and aluminum (Al) interactions. Citrus sinensis seedlings were irrigated for 18 weeks with nutrient solution at a concentration of 0 or 1 mM AlCl₃•6H₂O and a pH of 2.5, 3.0, 3.5, or 4.0. Thereafter, biomass, root, stem, and leaf concentrations of Al and nutrients, leaf gas exchange, chlorophyll a fluorescence (OJIP) transients, and related parameters were investigated to understand the physiological mechanisms underlying the elevated pH-induced alleviation of Citrus toxicity. Increasing the nutrient solution pH from 2.5 to 4.0 alleviated the Al-toxic effects on biomass, photosynthesis, OJIP transients and related parameters, and element concentrations, uptake, and distributions. In addition, low pH effects on the above physiological parameters were intensified by Al-toxicity. Evidently, a synergism existed between low pH and Al-toxicity. Increasing pH decreased Al uptake per root dry weight and its concentration in roots, stems, and leaves and increased nitrogen, phosphorus, calcium, magnesium, sulfur, and boron uptake per plant and their concentrations in roots, stems, and leaves. This might be responsible for the elevated pH-induced alleviation of growth inhibition and the impairment of the whole photosynthetic electron transport chain, thus preventing the decrease of CO₂ assimilation.

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1. Introduction

Aluminum (Al) exists mostly as silicate or oxide precipitates that are biologically inactive in neutral or moderately acidic soils. However, Al solubility increases greatly in acidic soils (pH < 5), resulting in the release of phytotoxic Al³⁺ from clay minerals into soil solution [1]. Micromolar concentration of Al³⁺ can cause a rapid inhibition of root elongation and subsequently impair the uptake of water and nutrients, leading to poor growth and yield loss of crops [2]. Therefore, Al-toxicity in acid soils has been regarded as a major factor limiting crop productivity worldwide, since ~ 30% of free ice land is acidic [3]. Furthermore, soil acidity is becoming an increasingly serious problem due to the improper farming practices and environmental deterioration [4]. In recent decades, many researchers have investigated Al-toxic effects on plant growth [5, 6], uptake of nutrients [6, 7], leaf CO₂ assimilation [8–11], and photosynthetic electron transport [12, 13].

The toxicity of Al to plants depends not only on their Al-tolerance, but also on the soil properties, primarily pH [14]. Hence, it is necessary to investigate the combination effects of Al and pH on plants to better understand the adaption of plants to acidic soils with high active Al. However, such data are very rare, because Al-toxicity and low pH are almost examined separately in different experiments. To our best knowledge, most of studies regarding pH effects on plant Al-toxicity have focused on the pH-induced alterations in toxic Al species and activities in solution, root growth, and root tissue (cell sap) Al concentration, and the results are not consistent [1, 14, 15]. Because Al-toxicity occurs mainly on acidic soils, it is generally believed that the lower the pH in the culture medium, the greater the toxicity of Al to plants. Degenhardt et al. [16] reported that an Al-induced increase in rhizosphere pH was responsible for the Al-resistance in the Arabidopsis alr-104 mutant and that increasing the solution pH from 4.4 to 4.5 improved root growth rate of both alr-104 mutant and wild type under Al-toxicity. Wang et al. [17] observed that the Al-tolerant wheat cultivar had higher capacity to keep higher rhizosphere pH relative to the Al-sensitive and that increasing the solution pH from 4.5 to 5.0 enhanced the Al-resistance of wheat. Using Eucalyptus trees, Yang et al. [18] found that raising the nutrient solution pH from 3.0 to 4.0 increased net photosynthesis and transpiration under Al-toxicity. However, Kinraide [19] found that the activities of Al³⁺ in soil solutions peaked at ~ pH 4.1 in the range of pH 3.5 to 5.5, implying that Al-toxicity in soils with pH 4.1 might be more severe than that in more acidic or alkaline soils. Al level in rice root cell sap increased as solution pH increased from 4.0 to 6.0 [20].

Citrus often display poor growth and a shortened lifetime in low pH soils with high active Al [21]. In China, Citrus are cultivated mainly on acidic soils. Moreover, soil acidity is becoming an increasing urgent problem in some Citrus orchards [22]. Although the physiological and molecular responses of Citrus to Al-toxicity or low pH have been investigated in some details [9, 23–25], very scarcity is known on Citrus responses to pH and Al interactions. Preliminary study showed that increasing nutrient solution pH prevented Al-toxic effects on Citrus growth and photosynthesis. Here, we used C. sinensis seedlings as materials and investigated the effects of pH and Al interactions on biomass; Al and nutrient elements in roots, stems, and leaves; gas exchange, chlorophyll (Chl) a fluorescence (OJIP) transients and related parameters in leaves. Our objective was to determine the physiological mechanisms underlying the increased pH-induced alleviation of Citrus Al-toxicity.

2. Materials and Methods

2.1. Culture and Treatments of Seedlings

Seedling culture and treatments were conducted according to Guo et al. [26] with some modifications. Five-week-old “Xuegan” (C. sinensis) seedlings were transplanted to 6L pots (two per pot) containing sand thoroughly washed with tap water, and then planted in a greenhouse under a natural photoperiod at Fujian Agriculture and Forestry University, Fuzhou with an annual average relative humidity, temperature, and sunlight of ~ 76%, 20°C and 1600 h, respectively. Six weeks after transplanting, each pot was replenished every day with freshly prepared nutrient solution [i.e., macronutrients (in mM): Ca(NO₃)₂,1; KNO₃, 1; MgSO₄, 0.5; KH₂PO₄, 0.1; and micronutrients (in μM): Fe-EDTA, 20; ZnSO₄, 2; MnCl₂, 2; CuSO₄, 0.5; and (NH₄)₆Mo₇O₂₄, 0.065] at a concentration of 0 or 1 mM AlCl₃•6H₂O and a pH of 2.5, 3.0, 3.5 or 4.0 for 18 weeks until a portion of nutrient solution began to leak out from a hole at the bottom of the pot (~ 500 mL). The pH of the solution was adjusted by NaOH or HCl before irrigation. At the end of the experiment, fully expanded (~ 7-week-old) leaves were used for the assays of all physiological parameters.

2.2. Measurements of Root, Stem, and Leaf Dry Weights

At the end of the experiment, ten seedlings per treatment from different pots were harvested and then divided into roots, stems, and leaves. Their dry weight (DW) was weighted after being dried to a constant weight at 70°C.

2.3. Measurements of Leaf OJIP Transients and Calculation of Related Parameters

Leaf OJIP transients were measured in 3 h dark-adapted seedlings at room temperature using a Handy Plant Efficiency Analyzer (Handy PEA, Hansatech Instruments Limited, Norfolk, UK). All fluorescence parameters were calculated according to Jiang et al. [27] and Banks [28].

2.4. Leaf Gas Exchange

Leaf gas exchange was measured with a CIRAS-2 portable photosynthesis system (PP systems, Herts, UK) at a leaf temperature of ~ 26°C, a controlled light intensity of ~ 1000 μmol m⁻² s⁻¹ and a controlled CO₂ concentration of ~ 380 μmol mol⁻¹ between 9 and 12 a.m. on a sunny day. The flow rate through the 2.5 cm² leaf chamber was kept at 200 μmol s⁻¹. Water use efficiency (WUE) was calculated as CO₂ assimilation/transpiration rate.

2.5. Assays of Elements, Calculation of Element Uptake, and Distributions in Roots, Stems, and Leaves

The small first- and second-order fibrous roots (< 2.0 mm diameter), the middle sections of stems, and the recent fully expanded mature (~ 7-week-old) leaves were used for the measurements of elements [26]. Al was assayed with a NexION 300X Inductively Coupled Plasma Mass Spectrometer (ICP-MS, PerkinElmer, CT, USA). Phosphorus (P) was measured colorimetrically as blue molybdate-phosphate complexes. Potassium (K) was assayed with a FP640 Flame Photometry (Shanghai Precision Scientific Instrument Co., Ltd., Shanghai, China). Calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) were determined with a PinAAcle 900F Atomic Absorption Spectrometer (Perkinelmer Singapore Pte Ltd., Singapore). Sulfur (S) was determined by the simple turbidimetric method. Nitrogen (N) was assayed by the Kjeldahl method with a Kjeltec 8200 Auto Distillation (FOSS Analytical AB, Höganäs, Sweden). Boron (B) was determined by the curcumin method [22, 24].

Element uptake and distributions were calculated as described previously [26].

2.6. Data Analysis

There were 12 pots (24 seedlings) per treatment in a completely randomized design. Results represented the mean ± SE of 4-12 replicates (one plant from different pot per treatment) except for the mean OJIP transients and the different expressions derived from the mean transients. Significant differences among the eight treatments were analysed by four (pH values) × two (Al levels) ANOVA and followed by the least significant difference (LSD) at P< 0.05 level.

Principal component analysis (PCA) was performed using a SPSS® statistical software (version 17.0, IBM, NY, USA) [26].

3. Results

3.1. Seedling Growth

Without Al-toxicity, only pH 2.5 decreased stem, leaf, shoot, and whole plant DW and increased root DW/shoot DW ratio relative to control (pH 4.0); with Al-toxicity, all the six parameters kept stable when pH decreased from 4.0 to 3.5, then root, stem, leaf, shoot, and whole plant DW decreased and root DW/shoot DW ratio increased with further decreasing pH. Al decreased or did not alter stem, leaf, shoot, and whole plant DW, but it increased or did not affect root DW and root DW/shoot DW ratio. The interactive effects of pH and Al were significant for root DW, whole plant DW, and root DW/shoot DW ratio. Many rotten and died fibrous roots were observed in the pH 2.5 + 1 mM Al-treated seedlings (Figure 1 and Figure S1).

[figure omitted; refer to PDF]

3.2. Leaf Gas Exchange

Without Al-toxicity, leaf stomatal conductance (${\mathrm{g}}_{\mathrm{s}}$) and transpiration rate (CO₂ assimilation) did not change as pH decreased from 4.0 to 3.0 (3.5) and then decreased with further decreasing pH, but intercellular CO₂ concentration (${\mathrm{C}}_{\mathrm{i}}$), the ratio of intercellular to ambient CO₂ concentration (${\mathrm{C}}_{\mathrm{i}}$/${\mathrm{C}}_{\mathrm{a}}$), and WUE did not alter in response to pH. With Al-toxicity, leaf CO₂ assimilation, ${\mathrm{g}}_{\mathrm{s}}$, transpiration rate, and WUE kept unchanged as pH decreased from 4.0 to 3.5 and then decreased with further decreasing pH, but both ${\mathrm{C}}_{\mathrm{i}}$ and ${\mathrm{C}}_{\mathrm{i}}$/${\mathrm{C}}_{\mathrm{a}}$ ratio were the highest at pH 2.5. Interactions between pH and Al on leaf gas exchange were significant only for CO₂ assimilation (Figure 2).

[figure omitted; refer to PDF]

Regression analysis showed that CO₂ assimilation increased with increasing ${\mathrm{g}}_{\mathrm{s}}$, but it increased with decreasing ${\mathrm{C}}_{\mathrm{i}}$ and ${\mathrm{C}}_{\mathrm{i}}$/${\mathrm{C}}_{\mathrm{a}}$ ratio in leaves (Figure S2).

3.3. Leaf OJIP Transients and Related Parameters

OJIP transients from the 0 mM Al-treated leaves displayed little alterations in response to pH except for a slight increase in O-step at pH 2.5 compared with OJIP transients from the pH 4.0 + 0 mM Al-treated leaves. Al increased the heterogeneity of samples, especially at pH 2.5. OJIP transients from the 1 mM Al-treated leaves displayed an increased O-step at pH 2.5-3.0 and a suppressed P-step at pH 2.5 compared with OJIP transients from the pH 4.0 + 1 mM Al-treated leaves (Figure S3). OJIP transients from the low pH and/or Al-treated leaves had positive ΔL-, ΔK-, ΔJ-, and ΔI-bands around 150 μs, 300 μs, 2 ms, and 30 ms compared with OJIP transients from the pH 4.0 + 0 mM Al-treated leaves, respectively. The positive ΔL-, ΔK-, ΔJ-, and ΔI-bands were the most pronounced from the leaves treated by pH 2.5 + 1 mM Al (Figure 3).

[figure omitted; refer to PDF]

Without Al-toxicity, minimum fluorescence (${\mathrm{F}}_{\mathrm{o}}$), maximum fluorescence (${\mathrm{F}}_{\mathrm{m}}$), maximum variable fluorescence (${\mathrm{F}}_{\mathrm{v}}$), maximum primary yield of photochemistry of photosystem II (PSII; ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{o}}$), fraction of oxygen evolving complex (OEC) relative to control (${\mathrm{F}}_{\mathrm{O}\mathrm{E}\mathrm{C}}$), maximum amplitude of IP phase, maximum PSII efficiency of dark-adapted leaves (${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{m}}$), quantum yield for energy dissipation (${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/ABS), quantum yield for electron transport (${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/ABS), quantum yield for the reduction of end acceptors of PSI per photon absorbed (${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/ABS), efficiency with which a trapped exciton can move an electron into the electron transport chain from ${{\mathrm{Q}}_{\mathrm{A}}}^{-}$ to the PSI end electron acceptors (${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$), absorption flux per reaction centre (RC, ABS/RC), dissipated energy flux per RC (${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/RC), electron transport flux per RC (${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/RC), reduction of end acceptors at PSI electron acceptor side (${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/RC), dissipated energy flux per cross section (CS, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$), reduction of end acceptors at PSI electron acceptor side (${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$), and total performance index (${\mathrm{P}\mathrm{I}}_{\mathrm{a}\mathrm{b}\mathrm{s},\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}$) displayed little change in response to pH except for increased ${\mathrm{F}}_{\mathrm{o}}$ and ${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$/RC and decreased ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{F}}_{\mathrm{O}\mathrm{E}\mathrm{C}}$, maximum amplitude of IP phase, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/ABS, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/ABS, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/RC, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/RC, and ${\mathrm{P}\mathrm{I}}_{\mathrm{a}\mathrm{b}\mathrm{s},\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}$ at pH 2.5. However, approximated initial slope (in ms⁻¹) of the fluorescence transient V = f(t) (${\mathrm{M}}_{\mathrm{o}}$) and trapped energy flux per RC (${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$/RC) increased, and probability that a trapped exciton moves an electron into the electron transport chain beyond ${{\mathrm{Q}}_{\mathrm{A}}}^{-}$ (${\psi }_{\mathrm{E}\mathrm{o}}$ or ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$) decreased with decreasing pH (Figure 4).

[figure omitted; refer to PDF]

With Al-toxicity, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/ABS, ABS/RC, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/RC, and ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$ (${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{M}}_{\mathrm{o}}$, and ${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$/RC) did not change as pH decreased from 4.0 to 3.0 (3.5) and then increased with further decreasing pH. By contrast, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{F}}_{\mathrm{O}\mathrm{E}\mathrm{C}}$, maximum amplitude of IP phase, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/ABS, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/ABS, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/RC, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$, and ${\mathrm{P}\mathrm{I}}_{\mathrm{a}\mathrm{b}\mathrm{s},\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}$ (${\mathrm{F}}_{\mathrm{m}}$, ${\mathrm{F}}_{\mathrm{v}}$, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{m}}$ and ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/RC) did not change as pH decreased from 4.0 to 3.5 (3.0) and then decreased with further decreasing pH (Figure 4).

Al-toxicity increased or did not affect ${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{M}}_{\mathrm{o}}$, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/ABS, ABS/RC, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/RC, ${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$/RC, and ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$ but decreased or did not alter the other fluorescence parameters. Interactions between pH and Al on Chl a fluorescence parameters were significant only for ${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{F}}_{\mathrm{m}}$, ${\mathrm{F}}_{\mathrm{v}}$, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{m}}$, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/ABS, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/ABS, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/ABS, ABS/RC, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/RC, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/RC, and ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$ (Figure 4).

Leaf CO₂ assimilation increased with increasing ${\mathrm{F}}_{\mathrm{m}}$, ${\mathrm{F}}_{\mathrm{v}}$, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{F}}_{\mathrm{O}\mathrm{E}\mathrm{C}}$, maximum amplitude of IP phase, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{m}}$, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/ABS, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/ABS, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/RC, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/RC, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$, or ${\mathrm{P}\mathrm{I}}_{\mathrm{a}\mathrm{b}\mathrm{s},\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}$, respectively, but it decreased with increasing ${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{M}}_{\mathrm{o}}$, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/ABS, ABS/RC, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/RC, ${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$/RC, or ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$, respectively (Figure S4).

3.4. Concentrations, Uptake, and Distributions of Elements

Without Al-toxicity, the levels of Al, S, and Mn in leaves, stems, and roots, Cu in leaves and roots, Zn in stems and leaves, and Fe in roots increased or did not change with decreasing pH, but the levels of P, N, Ca, K, Mg, and B in leaves, stems, and roots, Fe in leaves and stems, Cu in stems, and Zn in leaves decreased or did not alter with decreasing pH (Figures 5-6).

[figure omitted; refer to PDF] [figure omitted; refer to PDF]

With Al-toxicity, the levels of Al and Mn in leaves, stems, and roots, and Cu in leaves increased or did not change with decreasing pH. The levels of N, P, K, Ca, Mg, B, and Fe in leaves, stems, and roots, Cu in stems and roots, Zn in leaves and stems, and S in stems decreased or did not change with decreasing pH. The levels of S in leaves and Zn in roots (S in roots) increased as pH decreased from 4.0 to 3.5 (3.0) and then decreased with further decreasing pH (Figures 5-6).

Al-toxicity increased or did not alter the levels of Al and B in leaves, stems, and roots, Zn in leaves and stems, Mn in leaves, and S in roots at each given pH with the exceptions that the levels of Zn in leaves and S in roots were decreased by Al at pH 2.5, but decreased or did not alter the levels of N, P, K, Ca, Mg, Cu, and Fe in leaves, stems, and roots, S and Mn in stems, and Zn in roots with the exception that root Fe level was increased by Al at pH 4.0. The levels of S in leaves and Mn in roots were decreased by Al at pH 2.5-3.0, but increased or unaffected by Al at pH 3.5-4.0. Interactions between pH and Al on element levels were significant only for N, S, Mn, and Zn levels in leaves, stems, and roots, K level in leaves and roots, Fe level in stems and roots, P and Ca levels in leaves, B level in stems, and Mg, and Cu levels in roots (Figures 5-6).

Without Al-toxicity, the uptake of Al, S, Cu, Fe, Mn, and Zn per plant (root DW) increased or did not alter with decreasing pH with the exceptions that the uptake of Mn and Zn per plant was higher at pH 3.0 than that at pH 2.5, but the uptake of the other elements per plant (root DW) decreased or kept unchanged with decreasing pH. With Al-toxicity, the uptake of Al and Mn per plant (root DW) increased or did not alter with decreasing pH with the exception that the uptake of Mn per plant was slightly higher at pH 3.5 than that at pH 2.5, but the uptake of the other elements per plant (root DW) decreased or kept unchanged with decreasing pH with the exception that the uptake of S per plant (root DW) was higher at pH 3.5 than that at pH 4.0. Al-toxicity increased the uptake of Al per plant (root DW) and increased or did not alter the uptake of B per plant (root DW) with the exception that the uptake of B per plant was decreased by Al at pH 2.5, but it decreased the uptake of N, P, K, Ca, and Mg per plant (root DW) and decreased or did not change the uptake of S, Cu, Fe, and Zn per root DW. The uptake of S, Cu, Fe, Mn, and Zn per plant and of Mn per root DW was decreased by Al at pH 2.5-3.0, but increased or unaltered by Al at pH 3.5-4.0. Interactions between pH and Al on the uptake of elements were significant for the uptake of all elements except for the uptake of Al, N, and K per root DW (Figure 7).

[figure omitted; refer to PDF]

Whole plant (root) DW increased with increasing uptake of N, P, K, Ca, Mg, S, B, or Fe (S, B, or Fe) per plant (Figure S5).

Leaf CO₂ assimilation decreased with increasing leaf Al or Mn concentration, but it increased with increasing leaf N, K, Ca, Mg, Fe, or Zn concentrations. Leaf CO₂ assimilation increased with increasing uptake of N, P, K, Ca, Mg, S, B, or Fe per plant (Figure S6).

Leaf Al increased with increasing ${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{M}}_{\mathrm{o}}$, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/ABS, ABS/RC, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/RC, ${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$/RC, or ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$, respectively, but it decreased with increasing ${\mathrm{F}}_{\mathrm{m}}$, ${\mathrm{F}}_{\mathrm{v}}$, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{F}}_{\mathrm{O}\mathrm{E}\mathrm{C}}$, maximum amplitude of IP phase, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{m}}$, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/ABS, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/ABS, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/RC, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/RC, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$, or ${\mathrm{P}\mathrm{I}}_{\mathrm{a}\mathrm{b}\mathrm{s},\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}$, respectively (Figure S7).

Generally speaking, all element distributions in leaves and stems (roots) decreased (increased) or did not alter with decreasing pH with or without Al-toxicity. Al decreased (increased) or did not affect Al, N, P, K, S, B, Cu, Mn, and Zn distributions in leaves and stems (roots) with the exceptions that P distribution in stems and Cu distribution in leaves were increased by Al at pH 3.5. Al decreased Ca and Mg distributions in leaves and increased or did alter their distributions in stems and roots. At pH 2.5, Al increased (decreased) Fe distribution in leaves and stems (roots), but at pH 3.0-4.0, it decreased (increased) or did not alter Fe distribution in leaves and stems (roots). Interactions between pH and Al on element distributions were significant for all element distributions in leaves, stems, and roots except for the distributions of Ca, Mg, and B in leaves and Al, N, K, and Zn in stems (Figures 8-9).

[figure omitted; refer to PDF] [figure omitted; refer to PDF]

3.5. PCA Loading Plots

PCA was carried out to examine the physiological patterns of C. sinensis seedlings in response to pH with or without Al-toxicity (Figure 10 and Tables S1-S2). The first two components contributed to 60.6% and 68.3% of the total variation in the 0 and 1 mM Al-treated C. sinensis seedlings, respectively. These parameters were more highly clustered in the 1 mM Al-treated seedlings than those in the 0 mM Al-treated ones. For the 0 mM Al-treated seedlings, PC1 was heavily loaded on N uptake per root DW (0.985), N uptake per plant (0.982), B uptake per root DW (0.980), B uptake per plant (0.977), Mg uptake per root DW (0.975), leaf N concentration (0.974), Ca uptake per root DW (0.972), Ca uptake per plant (0.963), K uptake per root DW (0.959), and P uptake per root DW (0.952) (Table S1). For the 1 mM Al-treated seedlings, PC1 was the mostly affected by the alterations of stem N concentration (0.982), N uptake per root DW (0.981), Mg uptake per root DW (0.981), B uptake per root DW (0.980), Mg uptake per plant (0.978), B uptake per plant (0.977), N uptake per plant (0.977), Ca uptake per plant (0.967), Ca uptake per root DW (0.965), and leaf Mn concentration (-0.962) (Table S2).

[figures omitted; refer to PDF]

Also, we determined the physiological patterns of C. sinensis seedlings in response to Al at pH 2.5, 3.0, 3.5, or 4.0 (Figure 11 and Tables S3–S6). The contribution of PC1 and PC2 to the total variation displayed little change as pH decreased from pH 4.0 to 3.5 and then increased with further decreasing pH. For the pH 4.0-treated seedlings, Ca uptake per root DW(0.981), Ca uptake per plant (0.979), K uptake per root DW (0.974), Al uptake per plant (-0.974), stem Mn concentration (0.973), Mg uptake per root DW (0.972), N uptake per root DW (0.970), Mg distribution in roots (-0.970), P uptake per root DW (0.965), and Al uptake per root DW (-0.958) contributed largely to PC1 (Table S3). For the pH 3.5-treated seedlings, stem Mn concentration (0.985), N uptake per root DW (0.983), Mn distribution in stems (0.982), Mg distribution in leaves (0.982), Mn distribution in roots (-0.979), P uptake per root DW (0.977), leaf Mg concentration (0.974), root P concentration (0.970), Ca uptake per root DW (0.970), and Mn uptake per plant (-0.969) were the main contributors to PC1 (Table S4). For the pH 3.0-treated seedlings, PC1 was mostly influenced by the alterations of Mg uptake per root DW (0.995), Mg uptake per plant (0.994), P uptake per root DW (0.982), leaf Mg concentration (0.982), P uptake per plant (0.982), N uptake per plant (0.980), N uptake per root DW(0.979), leaf P concentration (0.979), leaf Ca concentration (0.978), and S uptake per plant (0.978) (Table S5). For the pH 2.5-treated seedlings, PC1 was largely accounted for by the modifications of S uptake per plant (0.991), N uptake per plant (0.991), S distribution in roots (-0.991), S uptake per root DW (0.990), N uptake per root DW(0.990), S distribution in leaves (0.987), Mg uptake per plant(0.985), leaf S concentration (0.984), Mg uptake per root DW(0.982), and K uptake per plant (0.980) (Table S6).

[figures omitted; refer to PDF]

4. Discussion

4.1. Interactive Effects of Al and Low pH on C. sinensis Seedlings Showed Synergism

The Al-induced alterations of most physiological parameters and OJIP transients became more pronounced with decreasing pH. Many parameters were altered by Al-toxicity only at pH 2.5-3.0, but unaffected at pH 3.5-4.0 (Figures 1–9 and S1 and S3). The exception was that the Al-induced increase in leaf and stem level of B was greater at pH 3.5-4.0 than that at pH 2.5-3.0 or similar between the two (Figure 6). These findings indicated that the Al-induced alterations of these physiological parameters were intensified by low pH. Obviously, increasing the nutrient solution pH from 2.5 to 4.0 alleviated Al-toxicity of C. sinensis seedlings. This agrees with the results obtained on Arabidopsis [16], wheat [17], and Eucalyptus [18]. We observed that both the level of Al in roots, stems, and leaves and Al uptake per plant (root DW) increased with decreasing pH with or without Al-toxicity with the exception that Al uptake per plant was basically unchanged in response to pH in 1 mM Al-treated seedlings (Figures 5 and 7). The increased pH-induced decreases in the level of Al in roots, stems, and leaves and Al uptake per root DW might be responsible for the elevated pH-induced alleviation of C. sinensis Al-toxicity. Also, we observed that the low pH-induced alterations of most physiological parameters were greater in the 1 mM Al-treated seedlings than those in the 0 mM Al-treated ones (Figures 1–9 and S1 and S3), demonstrating that the low pH-induced alterations of physiological parameters were enhanced by Al-toxicity. To conclude, there was a synergism between low pH and Al.

4.2. Al-Toxicity Increased Root Al Accumulation, Especially at Low pH

Plant Al-tolerance is associated not only with less uptake of Al by roots, but also with relatively less transport of Al from roots to shoots [29]. Previous studies indicated that the supply of S, B, and P decreased Al level in stems and leaves and increased or did not affect root Al level, thus alleviating Citrus Al-toxicity [6, 7, 23]. Thus, the Al-induced increase in root Al accumulation and decrease in leaf and stem Al accumulation (Figure 8) might be an adaptive strategy of C. sinensis to Al-toxicity. However, the increase in Al-tolerance due to the increased pH could not be explained in this way, because Al distribution in roots of the Al-treated seedlings was higher at pH 2.5-3.0 than that at pH 3.5-4.0. We found that increasing the nutrient solution pH from 2.5 to 4.0 decreased the level of Al in roots, stems, and leaves and the uptake of Al per root DW (Figures 5 and 7), which might play a key role in the increased pH-induced alleviation of Citrus Al-toxicity.

4.3. Increased Uptake and Levels of Nutrients Might Play a Role in the Elevated pH-Induced Alleviation of Al-Toxicity

Micromolar concentration of Al³⁺ can lead to a rapid inhibition of root growth and subsequently interfering with the uptake of nutrients [2, 20]. Al decreased the uptake of N, P, K, Ca, Mg, and Cu per plant at each given pH, especially at low pH with the exception that the uptake of Cu per plant was not altered by Al at pH 4.0. The uptake of S, B, Mn, and Zn per plant was increased and decreased by Al at pH 3.5-4 and pH 2.5-3, respectively (Figure 7). Regression analysis indicated that both whole plant DW and leaf CO₂ assimilation decreased with decreasing uptake of N, P, K, Ca, Mg, S, B, or Fe per plant (Figures S5-S6). Generally viewed, Al decreased the levels of N, P, K, Ca, Mg, and S in roots, stems, and leaves, especially at pH 2.5-3.0 with the exceptions of a few, but increased the level of B in leaves, stems, and roots at each given pH. Also, B level in roots, stems, and leaves increased with increasing pH with or without Al-toxicity (Figures 5-6). Regression analysis showed that leaf CO₂ assimilation decreased with decreasing leaf concentration of N, K, Ca, Mg, Fe, or Zn (Figure S6). The supply of Ca, Mg, S, B, and P can ameliorate Al-toxicity of plants [6, 7, 23, 30–35]. PCA showed that N, P, K, Ca, Mg, S, and B uptake per plant and/or per root DW was the main contributors to PC1 in the 0 mM Al-, 1 mM Al-, pH 4.0-, pH 3.5-, pH 3,0- and/or pH 2.5-treated seedlings (Tables S1-S6), demonstrating that the uptake of these elements might play a role in Citrus Al-toxicity (tolerance) and/or low pH-toxicity (tolerance). Thus, both the increased uptake per plant and/or root, stem, and leaf levels of N, P, Ca, K, Mg, S, and B might be involved in the increased pH-induced alleviation of Citrus Al-toxicity.

4.4. Causes for the Elevated pH-Induced Alleviation of Photosynthetic Decline in the Al-Treated Leaves

Although ${\mathrm{g}}_{\mathrm{s}}$ in the Al-treated leaves increased with increasing pH (Figure 2(b)) and leaf CO₂ assimilation increased with increasing ${\mathrm{g}}_{\mathrm{s}}$ (Figure S2), the ameliorative action of the increased pH against the inhibitory effect of Al on photosynthesis (Figure 2(a)) could not explained by the increased ${\mathrm{g}}_{\mathrm{s}}$ alone, because low pH increased or did not affect both ${\mathrm{C}}_{\mathrm{i}}$ and ${\mathrm{C}}_{\mathrm{i}}$/${\mathrm{C}}_{\mathrm{a}}$ ratio (Figure 2) and leaf CO₂ assimilation decreased with increasing ${\mathrm{C}}_{\mathrm{i}}$ or ${\mathrm{C}}_{\mathrm{i}}$/${\mathrm{C}}_{\mathrm{a}}$ ratio (Figure S2).

Previous studies showed that the impaired whole photosynthetic electron transport chain from the donor side of PSII to the reduction of PSI end acceptors was the main cause contributing to the Al-induced inhibition of photosynthesis in Citrus leaves [27] and that the Al-induced impairment of the whole photosynthetic electron transport chain and the subsequent decline in leaf CO₂ assimilation could be alleviated by the supply of B, S, and P [6, 7, 23]. We observed that Al-toxicity lowered ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{m}}$ (a good indicator of photoinhibitory effects on PSII) and ${\psi }_{\mathrm{E}\mathrm{o}}$ (${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$), increased ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/RC, and altered greatly OJIP transients in low pH treated leaves (Figures 3-4), together demonstrating that photoinhibition occurred in these leaves [36, 37]. Increasing the nutrient solution pH from 2.5 to 4.0 prevented the Al-induced alterations of OJIP transients and all the 21 fluorescence parameters (Figures 3-4 and S3). Regression analysis showed that there was a positive relationships between leaf CO₂ assimilation and ${\mathrm{F}}_{\mathrm{m}}$, ${\mathrm{F}}_{\mathrm{v}}$, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{F}}_{\mathrm{O}\mathrm{E}\mathrm{C}}$, maximum amplitude of IP phase, ${\mathrm{F}}_{\mathrm{v}}$/${\mathrm{F}}_{\mathrm{m}}$, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/ABS, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/ABS, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$, ${\mathrm{E}\mathrm{T}}_{\mathrm{o}}$/RC, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/RC, ${\mathrm{R}\mathrm{E}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$, or ${\mathrm{P}\mathrm{I}}_{\mathrm{a}\mathrm{b}\mathrm{s},\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}$, but a negative relationship between leaf CO₂ assimilation and ${\mathrm{F}}_{\mathrm{o}}$, ${\mathrm{M}}_{\mathrm{o}}$, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/ABS, ABS/RC, ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/RC, ${\mathrm{T}\mathrm{R}}_{\mathrm{o}}$/RC, or ${\mathrm{D}\mathrm{I}}_{\mathrm{o}}$/${\mathrm{C}\mathrm{S}}_{\mathrm{o}}$ (Figure S4). These results suggested that the increased pH alleviated the Al-toxic impairment on the whole electron transport chain, thus preventing the Al-induced inhibition of photosynthesis.

Evidence shows that the deficiencies of mineral nutrients (N, P, K, Ca, and Mg) can impair the whole photosynthetic electron transport chain and cause a marked decline in leaf photosynthesis [38–43]. Here, the increased pH-induced alleviation of the Al-induced decreases in leaf levels of N, P, K, Ca, and Mg and their uptake per plant might be one of the causes for preventing the Al-induced decline in leaf CO₂ assimilation, as indicated by the positive correlations between leaf CO₂ assimilation and leaf level of N, K, Ca, or Mg and uptake per plant of N, P, K, Ca, or Mg (Figure S6). Previous study showed that Al-toxicity increased or did not affect B concentration in Citrus grandis roots, stem and leaves, but supply B alleviated the Al-induced impairment occurring in the whole photosynthetic electron transport chain and inhibition of photosynthesis [23]. Our results showed that B concentration in roots, stems, and leaves and its uptake increased with increasing pH (Figures 6-7) and that leaf CO₂ assimilation increased with increasing B uptake per plant and displayed an increased trend with increasing leaf B concentration (Figure S6). These results indicated that the increased pH-induced increase in B uptake per plant might contribute to the alleviation of photosynthesis inhibition in Al-treated leaves. The antagonistic action of the increased pH against the inhibitory effect of Al-toxicity on leaf CO₂ assimilation might also involve the increased pH-induced a decrease in leaf Al concentration (Figure 5(a)), as indicated by the negative and significant relationship between leaf CO₂ assimilation and Al concentration (Figure S6) and the negative or positive relationships between leaf CO₂ assimilation and Chl a fluorescence parameters (Figure S4). Based on these results, we concluded that increasing the solution pH from 2.5 to 4.0 mitigated the Al-induced impairment occurring on the whole photosynthetic electron transport chain, thus preventing the Al-induced decline in CO₂ assimilation via decreasing the level of Al and increasing the uptake per plant of elements (N, P, K, Ca, Mg, and B) and their levels in leaves.

5. Conclusions

Our data clearly demonstrated that a synergism existed between low pH and Al and that increasing the nutrient solution pH from 2.5 to 4.0 alleviated the Al-toxicity of C. sinensis seedlings. Increasing pH decreased Al uptake per root DW and its level in roots, stems, and leaves and increased N, P, K, Ca, Mg, S, and B uptake per plant and their levels in roots, stems, and leaves. This might account for the increased pH-induced alleviation of Citrus Al-toxicity.

Disclosure

The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

References

[1] T. B. Kinraide, "Identity of the rhizotoxic aluminium species," Plant and Soil, vol. 134 no. 1, pp. 167-178, DOI: 10.1007/BF00010729, 1991.

[2] L. V. Kochian, "Cellular mechanisms of aluminum toxicity and resistance in plants," Annual Review of Plant Physiology and Plant Molecular Biology, vol. 46, pp. 237-260, DOI: 10.1146/annurev.pp.46.060195.001321, 1995.

[3] H. R. von Uexküll, E. Mutert, "Global extent, development and economic impact of acid soils," Plant and Soil, vol. 171 no. 1,DOI: 10.1007/BF00009558, 1995.

[4] J. H. Guo, X. J. Liu, Y. Zhang, J. L. Shen, W. X. Han, W. F. Zhang, P. Christie, K. W. Goulding, P. M. Vitousek, F. S. Zhang, "Significant acidification in major chinese croplands," Science, vol. 327 no. 5968, pp. 1008-1010, DOI: 10.1126/science.1182570, 2010.

[5] C. J. Graham, "The influence of nitrogen source and aluminum on growth and elemental composition of Nemaguard peach seedlings," Journal of Plant Nutrition, vol. 24 no. 3, pp. 423-439, DOI: 10.1081/PLN-100104970, 2001.

[6] H.-X. Jiang, N. Tang, J.-G. Zheng, Y. Li, L.-S. Chen, "Phosphorus alleviates aluminum-induced inhibition of growth and photosynthesis in Citrus grandis seedlings," Physiologia Plantarum, vol. 137 no. 3, pp. 298-311, DOI: 10.1111/j.1399-3054.2009.01288.x, 2009.

[7] P. Guo, Q. Li, Y.-P. Qi, L. Yang, X. Ye, H. Chen, L. Chen, "Sulfur-mediated-alleviation of aluminum-toxicity in Citrus grandis seedlings," International Journal of Molecular Sciences, vol. 18,DOI: 10.3390/ijms18122570, 2017.

[8] M. Moustakas, G. Ouzounidou, "Aluminum effects on photosynthesis and elemental uptake in an aluminum-tolerant and non-tolerant wheat cultivar," Journal of Plant Nutrition, vol. 18 no. 4, pp. 669-683, DOI: 10.1080/01904169509364930, 1995.

[9] W. E. Pereira, D. L. de Siqueira, C. A. Martínez, M. Puiatti, "Gas exchange and chlorophyll fluorescence in four Citrus rootstocks under aluminium stress," Journal of Plant Physiology, vol. 157 no. 5, pp. 513-520, DOI: 10.1016/S0176-1617(00)80106-6, 2000.

[10] H. P. Peixoto, F. M. Da Matta, J. C. Da Matta, "Responses of the photosynthetic apparatus to aluminum stress in two sorghum cultivars," Journal of Plant Nutrition, vol. 25 no. 4, pp. 821-832, DOI: 10.1081/PLN-120002962, 2002.

[11] X. Zhao, Q. Chen, Y. Wang, Z. Shen, W. Shen, X. Xu, "Hydrogen-rich water induces aluminum tolerance in maize seedlings by enhancing antioxidant capacities and nutrient homeostasis," Ecotoxicology and Environmental Safety, vol. 144, pp. 369-379, DOI: 10.1016/j.ecoenv.2017.06.045, 2017.

[12] Z. Li, F. Xing, D. Xing, "Characterization of target site of aluminum phytotoxicity in photosynthetic electron transport by fluorescence techniques in tobacco leaves," Plant & Cell Physiology, vol. 53 no. 7, pp. 1295-1309, DOI: 10.1093/pcp/pcs076, 2012.

[13] J. Moustaka, G. Ouzounidou, I. Sperdouli, M. Moustakas, "Photosystem II is more sensitive than photosystem I to Al 3+ induced phytotoxicity," Materials, vol. 11, 2018.

[14] T. B. Kinraide, P. R. Ryan, L. V. Kochian, "Interactive effects of Al 3+ , H + , and other cations on root elongation considered in terms of cell-surface electrical potential," Plant Physiology, vol. 99 no. 4, pp. 1461-1468, DOI: 10.1104/pp.99.4.1461, 1992.

[15] Y. Yang, Z. H. Guo, Z. J. Ye, "Changes of rhizosphere pH and the relationship with Al-resistant of different Al-tolerant wheat ( Triticum aestivum L.) under Al stress," Journal of Agro-Environment Science, vol. 29, pp. 636-641, 2010.

[16] J. Degenhardt, P. B. Larsen, S. H. Howell, L. V. Kochian, "Aluminum resistance in the arabidopsis mutant alr -104 is caused by an aluminum-induced increase in rhizosphere pH," Plant Physiology, vol. 117 no. 1, pp. 19-27, DOI: 10.1104/pp.117.1.19, 1998.

[17] P. Wang, S. Bi, L. Ma, W. Han, "Aluminum tolerance of two wheat cultivars (brevor and atlas66) in relation to their rhizosphere pH and organic acids exuded from roots," Journal of Agricultural and Food Chemistry, vol. 54 no. 26, pp. 10033-10039, DOI: 10.1021/jf0611769, 2006.

[18] M. Yang, L. Tan, Y. Xu, Y. Zhao, F. Cheng, S. Ye, W. Jiang, W. L. Araujo, "Effect of low pH and aluminum toxicity on the photosynthetic characteristics of different fast-growing eucalyptus vegetatively propagated clones," PLoS ONE, vol. 10,DOI: 10.1371/journal.pone.0130963, 2015.

[19] T. B. Kinraide, "Toxicity factors in acidic forest soils: attempts to evaluate separately the toxic effects of excessive Al 3+ and H + and insufficient Ca 2+ and Mg 2+ upon root elongation," European Journal of Soil Science, vol. 54 no. 2, pp. 323-333, DOI: 10.1046/j.1365-2389.2003.00538.x, 2003.

[20] J. Xia, N. Yamaji, T. Kasai, J. F. Ma, "Plasma membrane-localized transporter for aluminum in rice," Proceedings of the National Acadamy of Sciences of the United States of America, vol. 107 no. 43, pp. 18381-18385, DOI: 10.1073/pnas.1004949107, 2010.

[21] Z. Lin, D. L. Myhre, "Citrus root growth as affected by soil aluminum level under field conditions," Soil Science Society of America Journal, vol. 54 no. 5, pp. 1340-1344, DOI: 10.2136/sssaj1990.03615995005400050023x, 1990.

[22] Y. Li, M.-Q. Han, F. Lin, "Soil chemical properties, Guanximiyou pummelo leaf mineral nutrient status and fruit quality in the southern region of Fujian province, China," Journal of Soil Science and Plant Nutrition, vol. 15, pp. 615-628, 2015.

[23] H.-X. Jiang, N. Tang, J.-G. Zheng, L.-S. Chen, "Antagonistic actions of boron against inhibitory effects of aluminum toxicity on growth, CO 2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, and photosynthetic electron transport probed by the JIP-test, of Citrus grandis seedlings," BMC Plant Biology, vol. 9, article 102,DOI: 10.1186/1471-2229-9-102, 2009.

[24] A. Long, J. Zhang, L.-T. Yang, "Effects of low pH on photosynthesis, related physiological parameters and nutrient profile of Citrus," Frontiers in Plant Science, vol. 8, article 185, 2017.

[25] J. Zhang, Q. Li, Y.-P. Qi, W. Huang, L. Yang, N. Lai, X. Ye, L. Chen, "Low pH-responsive proteins revealed by a 2-DE based MS approach and related physiological responses in Citrus leaves," BMC Plant Biology, vol. 18, article 188,DOI: 10.1186/s12870-018-1413-3, 2018.

[26] P. Guo, Y.-P. Qi, Y.-T. Cai, T. Yang, L. Yang, Z. Huang, L. Chen, H. Rennenberg, "Aluminum effects on photosynthesis, reactive oxygen species and methylglyoxal detoxification in two Citrus species differing in aluminum tolerance," Tree Physiology, vol. 38 no. 10, pp. 1548-1565, DOI: 10.1093/treephys/tpy035, 2018.

[27] H.-X. Jiang, L.-S. Chen, J.-G. Zheng, S. Han, N. Tang, B. R. Smith, "Aluminum-induced effects on photosystem II photochemistry in Citrus leaves assessed by the chlorophyll a fluorescence transient," Tree Physiology, vol. 28 no. 12, pp. 1863-1871, DOI: 10.1093/treephys/28.12.1863, 2008.

[28] J. M. Banks, "Continuous excitation chlorophyll fluorescence parameters: a review for practitioners," Tree Physiology, vol. 37 no. 8, pp. 1128-1136, DOI: 10.1093/treephys/tpx059, 2017.

[29] P. B. Vose, P. J. Randall, "Resistance to aluminium and manganese toxicities in plants related to variety and cation-exchange capacity," Nature, vol. 196, pp. 85-86, DOI: 10.1038/196085a0, 1962.

[30] I. R. Silva, T. J. Smyth, D. W. Israel, C. D. Raper, T. W. Rufty, "Magnesium is more efficient than calcium in alleviating aluminum rhizotoxicity in soybean and its ameliorative effect is not explained by the Gouy-Chapman-Stern model," Plant & Cell Physiology, vol. 42 no. 5, pp. 538-545, DOI: 10.1093/pcp/pce066, 2001.

[31] I. R. Silva, T. J. Smyth, D. W. Israel, C. D. Raper, T. W. Rufty, "Magnesium ameliorates aluminum rhizotoxicity in soybean by increasing citric acid production and exudation by roots," Plant & Cell Physiology, vol. 42 no. 5, pp. 546-554, DOI: 10.1093/pcp/pce067, 2001.

[32] M. Reyes-Díaz, C. Meriño-Gergichevich, E. Alarcón, M. Alberdi, W. J. Horst, "Calcium sulfate ameliorates the effect of aluminum toxicity differentially in genotypes of highbush blueberry ( Vaccinium corymbosum L.)," Soil Science & Plant Nutrition, vol. 11 no. 4, pp. 59-78, DOI: 10.4067/S0718-95162011000400005, 2011.

[33] M. Riaz, L. Yan, X. Wu, S. Hussain, O. Aziz, Y. Wang, M. Imran, C. Jiang, "Boron alleviates the aluminum toxicity in trifoliate orange by regulating antioxidant defense system and reducing root cell injury," Journal of Environmental Management, vol. 208, pp. 149-158, DOI: 10.1016/j.jenvman.2017.12.008, 2018.

[34] L. Yan, M. Riaz, X. Wu, C. Du, Y. Liu, C. Jiang, "Ameliorative effects of boron on aluminum induced variations of cell wall cellulose and pectin components in trifoliate orange ( Poncirus trifoliate (L.) Raf.) rootstock," Environmental pollution, vol. 240, pp. 764-774, DOI: 10.1016/j.envpol.2018.05.022, 2018.

[35] C. Meriño-Gergichevich, G. Ondrasek, M. Zovko, D. Šamec, M. Alberdi, M. Reyes-Díaz, "Comparative study of methodologies to determine the antioxidant capacity of Al-toxified blueberry amended with calcium sulfate," Soil Science & Plant Nutrition, vol. 15 no. 4, pp. 965-978, 2015.

[36] L. Force, C. Critchley, J. J. van Rensen, "New fluorescence parameters for monitoring photosynthesis in plants. 1. The effect of illumination on the fluorescence parameters of the JIP-test," Photosynthesis Research, vol. 78 no. 1, pp. 17-33, DOI: 10.1023/A:1026012116709, 2003.

[37] K. Maxwell, G. N. Johnson, "Chlorophyll fluorescence—a practical guide," Journal of Experimental Botany, vol. 51 no. 345, pp. 659-668, DOI: 10.1093/jexbot/51.345.659, 2000.

[38] C. Bednarz, D. Oosterhuis, R. Evans, "Leaf photosynthesis and carbon isotope discrimination of cotton in response to potassium deficiency," Environmental and Experimental Botany, vol. 39 no. 2, pp. 131-139, DOI: 10.1016/S0098-8472(97)00039-7, 1998.

[39] L. Cheng, L. H. Fuchigami, P. J. Breen, "The relationship between photosystem II efficiency and quantum yield for CO 2 assimilation is not affected by nitrogen content in apple leaves," Journal of Experimental Botany, vol. 52 no. 362, pp. 1865-1872, DOI: 10.1093/jexbot/52.362.1865, 2001.

[40] N. Tang, Y. Li, L.-S. Chen, "Magnesium deficiency-induced impairment of photosynthesis in leaves of fruiting Citrus reticulata trees accompanied by up-regulation of antioxidant metabolism to avoid photo-oxidative damage," Journal of Plant Nutrition and Soil Science, vol. 175 no. 5, pp. 784-793, DOI: 10.1002/jpln.201100329, 2012.

[41] L. Cheng, L. H. Fuchigami, "Rubisco activation state decreases with increasing nitrogen content in apple leaves," Journal of Experimental Botany, vol. 51 no. 351, pp. 1687-1694, DOI: 10.1093/jexbot/51.351.1687, 2000.

[42] Z.-H. Lin, L.-S. Chen, R.-B. Chen, F.-Z. Zhang, H.-X. Jiang, N. Tang, "CO 2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, carbohydrates and photosynthetic electron transport probed by the JIP-test, of tea leaves in response to phosphorus supply," BMC Plant Biology, vol. 9, article 4,DOI: 10.1186/1471-2229-9-43, 2009.

[43] P. V. Rangnekar, "Effect of calcium deficiency on the carbon metabolism in photosynthesis and respiration of tomato leaf," Plant and Soil, vol. 42 no. 3, pp. 565-583, DOI: 10.1007/BF00009943, 1975.