Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

Elevated pH-mediated mitigation of aluminum-toxicity in sweet orange (Citrus sinensis) roots involved the regulation of energy-rich compounds and phytohormones.

For the first time, we used targeted metabolome to investigate the effects of pH-aluminum (Al) interactions on energy-rich compounds and their metabolites (ECMs) and phytohormones in sweet orange (Citrus sinensis) roots. The concentration of total ECMs (TECMs) was reduced by Al-toxicity in 4.0-treated roots, but unaffected significantly in pH 3.0-treated roots. However, the concentrations of most ECMs and TECMs were not lower in pH 4.0 + 1.0 mM Al-treated roots (P4AR) than in pH 3.0 + 1.0 mM Al-treated roots (P3AR). Increased pH improved the adaptability of ECMs to Al-toxicity in roots. For example, increased pH improved the utilization efficiency of ECMs and the conversion of organic phosphorus (P) from P-containing ECMs into available phosphate in Al-treated roots. We identified upregulated cytokinins (CKs), downregulated jasmonic acid (JA), methyl jasmonate (MEJA) and jasmonates (JAs), and unaltered indole-3-acetic acid (IAA) and salicylic acid (SA) in P3AR vs pH 3.0 + 0 mM Al-treated roots (P3R); upregulated JA, JAs and IAA, downregulated total CKs, and unaltered MEJA and SA in P4AR vs pH 4.0 + 0 mM Al-treated roots (P4R); and upregulated CKs, downregulated JA, MEJA, JAs and SA, and unaltered IAA in P3AR vs P4AR. Generally viewed, raised pH-mediated increments of JA, MEJA, total JAs, SA and IAA concentrations and reduction of CKs concentration in Al-treated roots might help to maintain nutrient homeostasis, increase Al-toxicity-induced exudation of organic acid anions and the compartmentation of Al in vacuole, and reduce oxidative stress and Al uptake, thereby conferring root Al-tolerance. In short, elevated pH-mediated mitigation of root Al-stress involved the regulation of ECMs and phytohormones.

Molecular and Physiological Responses of Citrus sinensis Leaves to Long-Term Low pH Revealed by RNA-Seq Integrated with Targeted Metabolomics.

Low pH-induced alterations in gene expression profiles and organic acids (OA) and free amino acid (FAA) abundances were investigated in sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] leaves. We identified 503 downregulated and 349 upregulated genes in low pH-treated leaves. Further analysis indicated that low pH impaired light reaction and carbon fixation in photosynthetic organisms, thereby lowering photosynthesis in leaves. Low pH reduced carbon and carbohydrate metabolisms, OA biosynthesis and ATP production in leaves. Low pH downregulated the biosynthesis of nitrogen compounds, proteins, and FAAs in leaves, which might be conducive to maintaining energy homeostasis during ATP deprivation. Low pH-treated leaves displayed some adaptive responses to phosphate starvation, including phosphate recycling, lipid remodeling, and phosphate transport, thus enhancing leaf acid-tolerance. Low pH upregulated the expression of some reactive oxygen species (ROS) and aldehyde detoxifying enzyme (peroxidase and superoxidase) genes and the concentrations of some antioxidants (L-tryptophan, L-proline, nicotinic acid, pantothenic acid, and pyroglutamic acid), but it impaired the pentose phosphate pathway and VE and secondary metabolite biosynthesis and downregulated the expression of some ROS and aldehyde detoxifying enzyme (ascorbate peroxidase, aldo-keto reductase, and 2-alkenal reductase) genes and the concentrations of some antioxidants (pyridoxine and γ-aminobutyric acid), thus disturbing the balance between production and detoxification of ROS and aldehydes and causing oxidative damage to leaves.

The aluminum distribution and translocation in two citrus species differing in aluminum tolerance.

BACKGROUND: Many citrus orchards of south China suffer from soil acidification, which induces aluminum (Al) toxicity. The Al-immobilization in vivo is crucial for Al detoxification. However, the distribution and translocation of excess Al in citrus species are not well understood. RESULTS: The seedlings of 'Xuegan' [Citrus sinensis (L.) Osbeck] and 'Shatianyou' [Citrus grandis (L.) Osbeck], that differ in Al tolerance, were hydroponically treated with a nutrient solution (Control) or supplemented by 1.0 mM Al3+ (Al toxicity) for 21 days after three months of pre-culture. The Al distribution at the tissue level of citrus species followed the order: lateral roots > primary roots > leaves > stems. The concentration of Al extracted from the cell wall (CW) of lateral roots was found to be about 8 to 10 times higher than in the lateral roots under Al toxicity, suggesting that the CW was the primary Al-binding site at the subcellular level. Furthermore, the Al distribution in CW components of the lateral roots showed that pectin had the highest affinity for binding Al. The relative expression level of genes directly relevant to Al transport indicated a dominant role of Cs6g03670.1 and Cg1g021320.1 in the Al distribution of two citrus species. Compared to C. grandis, C. sinensis had a significantly higher Al concentration on the CW of lateral roots, whereas remarkably lower Al levels in the leaves and stems. Furthermore, Al translocation revealed by the absorption kinetics of the CW demonstrated that C. sinensis had a higher Al retention and stronger Al affinity on the root CW than C. grandis. According to the FTIR (Fourier transform infrared spectroscopy) analysis, the Al distribution and translocation might be affected by a modification in the structure and components of the citrus lateral root CW. CONCLUSIONS: A higher Al-retention, mainly attributable to pectin of the root CW, and a lower Al translocation efficiency from roots to shoots contributed to a higher Al tolerance of C. sinensis than C. grandis. The aluminum distribution and translocation of two citrus species differing in aluminum tolerance were associated with the transcriptional regulation of genes related to Al transport and the structural modification of root CW.

Molecular mechanisms for pH-mediated amelioration of aluminum-toxicity revealed by conjoint analysis of transcriptome and metabolome in Citrus sinensis roots.

Little is known about the effects of pH-aluminum (Al) interactions on gene expression and/or metabolite profiles in plants. Eleven-week-old seedlings of Citrus sinensis were fertilized with nutrient solution at an Al level of 0 or 1 mM and a pH of 3.0 or 4.0 for 18 weeks. Increased pH mitigated Al-toxicity-induced accumulation of callose, an Al-sensitive marker. In this study, we identified more differentially expressed genes and differentially abundant metabolites in pH 4.0 + 1 mM Al-treated roots (P4AR) vs pH 4.0 + 0 mM Al-treated roots (P4R) than in pH 3.0 + 1 mM Al-treated roots (P3AR) vs pH 3.0 + 0 mM Al-treated roots (P3R), suggesting that increased pH enhanced root metabolic adaptations to Al-toxicity. Further analysis indicated that increased pH-mediated mitigation of root Al-toxicity might be related to several factors, including: enhanced capacity to maintain the homeostasis of phosphate and energy and the balance between generation and scavenging of reactive oxygen species and aldehydes; and elevated accumulation of secondary metabolites such as polyphenol, proanthocyanidins and phenolamides and adaptations of cell wall and plasma membrane to Al-toxicity.

CsiLAC4 modulates boron flow in Arabidopsis and Citrus via high-boron-dependent lignification of cell walls.

The mechanisms underlying plant tolerance to boron (B) excess are far from fully understood. Here we characterized the role of the miR397-CsiLAC4/CsiLAC17 (from Citrus sinensis) module in regulation of B flow. Live-cell imaging techniques were used in localization studies. A tobacco transient expression system tested modulations of CsiLAC4 and CsiLAC17 by miR397. Transgenic Arabidopsis were generated to analyze the biological functions of CsiLAC4 and CsiLAC17. CsiLAC4's role in xylem lignification was determined by mRNA hybridization and cytochemistry. In situ B distribution was analyzed by laser ablation inductively coupled plasma mass spectrometry. CsiLAC4 and CsiLAC17 are predominantly localized in the apoplast of tobacco epidermal cells. Overexpression of CsiLAC4 in Arabidopsis improves the plants' tolerance to boric acid excess by triggering high-B-dependent lignification of the vascular system's cell wall and reducing free B content in roots and shoots. In Citrus, CsiLAC4 is expressed explicitly in the xylem parenchyma and is modulated by B-responsive miR397. Upregulation of CsiLAC4 in Citrus results in lignification of the xylem cell walls, restricting B flow from xylem vessels to the phloem. CsiLAC4 contributes to plant tolerance to boric acid excess via high-B-dependent lignification of cell walls, which set up a 'physical barrier' preventing B flow.

Raised pH conferred the ability to maintain a balance between production and detoxification of reactive oxygen species and methylglyoxal in aluminum-toxic Citrus sinensis leaves and roots.

Little is known about interactive effects of pH-aluminum (Al) on reactive oxygen species (ROS) and methylglyoxal (MG) metabolisms in plants. Citrus sinensis seedlings were fertilized with nutrient solution at an Al concentration of 1 or 0 mM and a pH of 4.0, 3.5, 3.0 or 2.5 for 18 weeks. Thereafter, gas exchange and chlorophylls in leaves, H2O2 generation, electrolyte leakage, total soluble proteins, MG, malondialdehyde (MDA), antioxidants, sulfur-containing compounds, enzymes [viz., antioxidant enzymes, sulfur metabolism-related enzymes, ascorbate oxidase, phosphomannose isomerase, glyoxalase I and glyoxalase II] involved in ROS and MG detoxification in leaves and roots were measured. Effects of low pH and Al-toxicity on these parameters displayed obvious synergism. Without Al-toxicity, low pH increased H2O2 production, electrolyte leakage, MDA and MG concentrations by 45.7%-90.3% (52.4%-73.6%), 24.3%-74.5% (26.7%-86.2%), 18.6%-44.8% (35.6%-53.7%) and 16.3%-47.1% (13.8%-51.7%) in leaves (roots) relative to pH 4, respectively; low pH-induced upregulation of enzymes involved in ROS and MG detoxification and sulfur-containing compounds in leaves and/or roots could not protect them against oxidative damage. At pH 2.5-3.0, Al-toxicity increased H2O2 production, electrolyte leakage, MDA and MG concentrations by 34.2%-35.5% (23.9%-72.7%), 10.2%-29.5% (23.7%-56.8%), 15.6%-35.7% (27.5%-33.9%) and 21.5%-26.8% (21.0%-49.2%) in leaves (roots), respectively, and decreased total soluble protein concentration by 46.2%-47.4% (18.8%-20.8%) in leaves (roots); at pH 3.5-4.0, Al-toxicity did not affect significantly the five parameters in leaves and roots except for Al-induced increases in root MDA concentration at pH 3.5-4.0 and root electrolyte leakage at pH 3.5, and Al-induced decrease in root total soluble protein concentration at pH 4.0. Raised pH conferred the ability to maintain a balance between production and detoxification of ROS and MG in leaves and roots, thus protecting them against oxidative damage, and hence alleviating Al-induced increase in electrolyte leakage and decrease in total soluble protein level.

Illumina sequencing revealed roles of microRNAs in different aluminum tolerance of two citrus species.

Self-germinated seedlings of Citrus sinensis and C. grandis were supplied with nutrient solution with 0 mM AlCl3·6H2O (control, -Al) or 1 mM AlCl3·6H2O (+Al) for 18 weeks. The DW (Dry weights) of leaf, stem, shoot and the whole plant of C. grandis were decreased and the ratio of root DW to shoot DW in C. grandis were increased by Al, whereas these parameters of C. sinensis were not changed by Al. Al treatment dramatically decreased the sulfur (S) content in C. grandis roots and the phosphorus (P) content in both C. sinensis and C. grandis roots. More Al was transported to shoots and leaves in C. grandis than in C. sinensis under Al treatment. Al treatment has more adverse effects on C. grandis than on C. sinensis, as revealed by the higher production of superoxide anion (O2 ·-), H2O2 and thiobarbituric acid reactive substace (TBARS) content in C. grandis roots. Via the Illumina sequencing technique, we successfully identified and quantified 12 and 16 differentially expressed miRNAs responding to Al stress in C. sinensis and C. grandis roots, respectively. The possible mechanism underlying different Al tolerance of C. sinensis and C. grandis were summarized as having following aspects: (a) enhancement of adventitious and lateral root development (miR160); (b) up-regulation of stress and signaling transduction related genes, such as SGT1, PLC and AAO (miR477, miR397 and miR398); (c) enhancement of citrate secretion (miR3627); (d) more flexible control of alternative glycolysis pathway and TCA cycle (miR3627 and miR482); (e) up-regulation of S-metabolism (miR172); (f) more flexible control of miRNA metabolism. For the first time, we showed that root development (miR160) and cell wall components (cas-miR5139, csi-miR12105) may play crucial roles in Al tolerance in citrus plants. In conclusion, our study provided a comprehensive profile of differentially expressed miRNAs in response to Al stress between two citrus plants differing in Al tolerance which further enriched our understanding of the molecular mechanism underlying Al tolerance in plants.

Comparative transcriptome analysis reveals candidate genes related to cadmium accumulation and tolerance in two almond mushroom (Agaricus brasiliensis) strains with contrasting cadmium tolerance.

Cadmium (Cd) is a toxic metal occurring in the environment naturally. Almond mushroom (Agaricus brasiliensis) is a well-known cultivated edible and medicinal mushroom. In the past few decades, Cd accumulation in A.brasiliensis has received increasing attention. However, the molecular mechanisms of Cd-accumulation in A. brasiliensis are still unclear. In this paper, a comparative transcriptome of two A.brasiliensis strains with contrasting Cd accumulation and tolerance was performed to identify Cd-responsive genes possibly responsible for low Cd-accumulation and high Cd-tolerance. Using low Cd-accumulating and Cd-tolerant (J77) and high Cd-accumulating and Cd-sensitive (J1) A.brasiliensis strains, we investigated 0, 2 and 5 mg L-1 Cd-effects on mycelium growth, Cd-accumulation and transcriptome revealed by RNA-Seq. A total of 57,884 unigenes were obtained. Far less Cd-responsive genes were identified in J77 mycelia than those in J1 mycelia (e.g., ABC transporters, ZIP Zn transporter, Glutathione S-transferase and Cation efflux (CE) family). The higher Cd-accumulation in J1 mycelia might be due to Cd-induced upregulation of ZIP Zn transporter. Cd impaired cell wall, cell cycle, DNA replication and repair, thus decreasing J1 mycelium growth. Cd-stimulated production of sulfur-containing compounds, polysaccharides, organic acids, trehalose, ATP and NADPH, and sequestration of Cd might be adaptive responses of J1 mycelia to the increased Cd-accumulation. DNA replication and repair had better stability under 2 mg L-1 Cd, but greater positive modifications under 5 mg L-1 Cd. Better stability of DNA replication and repair, better cell wall and cell cycle stability might account for the higher Cd-tolerance of J77 mycelia. Our findings provide a comprehensive set of DEGs influenced by Cd stress; and shed light on molecular mechanism of A.brasiliensis Cd accumulation and Cd tolerance.

Interactive effects of pH and aluminum on the secretion of organic acid anions by roots and related metabolic factors in Citrus sinensis roots and leaves.

Low pH and aluminum (Al)-toxicity often coexist in acidic soils. Citrus sinensis seedlings were treated with nutrient solution at a pH of 2.5, 3.0, 3.5 or 4.0 and an Al concentration of 0 or 1 mM for 18 weeks. Thereafter, malate, citrate, isocitrate, acid-metabolizing enzymes, and nonstructural carbohydrates in roots and leaves, and release of malate and citrate from roots were measured. Al concentration in roots and leaves increased under Al-toxicity, but it declined with elevating nutrient solution pH. Al-toxicity increased the levels of glucose, fructose, sucrose and total soluble sugars in leaves and roots at each given pH except for a similar sucrose level at pH 2.5-3.0, but it reduced or did not alter the levels of starch and total nonstructural carbohydrates (TNC) in leaves and roots with the exception that Al improved TNC level in roots at pH 4.0. Levels of nonstructural carbohydrates in roots and leaves rose with reducing pH with a few exceptions with or without Al-toxicity. A potential model for the possible role of root organic acid (OA) metabolism (anions) in C. sinensis Al-tolerance was proposed. With Al-toxicity, the elevated pH upregulated the OA metabolism, and increased the flow of carbon to OA metabolism, and the accumulation of malate and citrate in roots and subsequent release of them, thus reducing root and leaf Al and hence eliminating Al-toxicity. Without Al-toxicity, low pH stimulated the exudation of malate and citrate, an adaptive response of Citrus to low pH. The interactive effects of pH and pH on OA metabolism were different between roots and leaves.

Phosphorus-mediated alleviation of aluminum toxicity revealed by the iTRAQ technique in Citrus grandis roots.

Citrus grandis seedlings were irrigated with nutrient solutions with four Al-P combinations [two Al levels (0 mM and 1.2 mM AlCl3·6H2O) × two P levels (0 µM and 200 µM KH2PO4)] for 18 weeks. Al dramatically inhibited the growth of C. grandis seedlings, as revealed by a decreased dry weight of roots and shoots. Elevating P level could ameliorate the Al-induced growth inhibition and organic acid (malate and citrate) secretion in C. grandis. Using a comparative proteomic approach revealed by the isobaric tags for relative and absolute quantification (iTRAQ) technique, 318 differentially abundant proteins (DAPs) were successfully identified and quantified in this study. The possible mechanisms underlying P-induced alleviation of Al toxicity in C. grandis were proposed. Furthermore, some DAPs, such as GLN phosphoribosyl pyrophosphate amidotransferase 2, ATP-dependent caseinolytic (Clp) protease/crotonase family protein, methionine-S-oxide reductase B2, ABC transporter I family member 17 and pyridoxal phosphate phosphatase, were reported for the first time to respond to Al stress in Citrus plants. Our study provides some proteomic details about the alleviative effects of P on Al toxicity in C. grandis, however, the exact function of the DAPs identified herein in response to Al tolerance in plants must be further investigated.

Analysis of Interacting Proteins of Aluminum Toxicity Response Factor ALS3 and CAD in Citrus.

Aluminum (Al) treatment significantly decreased the dry weight (DW) of stem, shoot and whole plant of both Citrus sinensis and C. grandis, but did not change that of root. Al significantly decreased leaf DW of C. grandis, increased the ratio of root to shoot and the lignin content in roots of both species. The higher content of Al in leaves and stems and lignin in roots of C. grandis than that of C. sinensis might be due to the over-expression of Al sensitive 3 (ALS3) and cinnamyl alcohol deaminase (CAD) in roots of C. grandis, respectively. By using yeast-two-hybridazation (Y2H) and bimolecular fluorescence complementation (BiFC) techniques, we obtained the results that glutathione S-transferase (GST), vacuolar-type proton ATPase (V-ATPase), aquaporin PIP2 (PIP2), ubiquitin carboxyl-terminal hydrolase 13 (UCT13), putative dicyanin blue copper protein (DCBC) and uncharacterized protein 2 (UP2) were interacted with ALS3 and GST, V-ATPase, Al sensitive 3 (ALS3), cytochrome P450 (CP450), PIP2, uncharacterized protein 1 (UP1) and UP2 were interacted with CAD. Annotation analysis revealed that these proteins were involved in detoxification, cellular transport, post-transcriptional modification and oxidation-reduction homeostasis or lignin biosynthesis in plants. Real-time quantitative PCR (RT-qPCR) analysis further revealed that the higher gene expression levels of most of these interacting proteins in C. grandis roots than that in C. sinensis ones were consistent with the higher contents of lignin in C. grandis roots and Al absorbed by C. grandis. In conclusion, our study identified some key interacting components of Al responsive proteins ALS3 and CAD, which could further help us to understand the molecular mechanism of Al tolerance in citrus plants and provide new information to the selection and breeding of tolerant cultivars, which are cultivated in acidic areas.

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

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.

Overexpression of the peanut CLAVATA1-like leucine-rich repeat receptor-like kinase AhRLK1 confers increased resistance to bacterial wilt in tobacco.

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease affecting hundreds of plant species, yet the host factors remain poorly characterized. The leucine-rich repeat receptor-like kinase gene AhRLK1, characterized as CLAVATA1, was found to be up-regulated in peanut upon inoculation with R. solanacearum. The AhRLK1 protein was localized in the plasma membrane and cell wall. qPCR results showed AhRLK1 was induced in a susceptible variety but little changed in a resistant cultivar after inoculated with R. solanacearum. Hormones such as salicylic acid, abscisic acid, methyl jasmonate, and ethephon induced AhRLK1 expression. In contrast, AhRLK1 expression was down-regulated under cold and drought treatments. Transient overexpression of AhRLK1 led to a hypersensitive response (HR) in Nicotiana benthamiana. Furthermore, AhRLK1 overexpression in tobacco significantly increased the resistance to R. solanacearum. Besides, the transcripts of most representative defense responsive genes in HR and hormone signal pathways were significantly increased in the transgenic lines. EDS1 and PAD4 in the R gene signaling pathway were also up-regulated, but NDR1 was down-regulated. Accordingly, AhRLK1 may increase the defense response to R. solanacearum via HR and hormone defense signaling, in particular through the EDS1 pathway of R gene signaling. These results provide a new understanding of the CLAVATA1 function and will contribute to genetic enhancement of peanut.

Proteome profile analysis of boron-induced alleviation of aluminum-toxicity in Citrus grandis roots.

Aluminum (Al)-toxicity and boron (B)-deficiency are two major factors limiting crop production in tropical and subtropical areas. Elevating B supply can alleviate the Al-induced inhibition of growth in Citrus grandis. Seedlings of C. grandis were irrigated for 18 weeks with nutrient solutions containing two B levels (2.5 and 20 µM H3BO3) and two Al levels (0 and 1.2 mM AlCl3·6H2O). By using 2-dimensional electrophoresis (2-DE) based MALDI-TOF/TOF-MS method, this study successfully identified and quantified sixty-one differentially abundant proteins in Citrus roots in response to B-Al interactions. The mechanisms underlying the B-induced alleviation of Al-toxicity unveiled by 2-DE technique could be summarized as follows: a) remodeling of cell wall by reducing the synthesis of lignin (sugar ATP Binding Cassette (ABC) transporter ATPase and cinnamyl alcohol dehydrogenase) and increasing the modification of cell wall (UDP-forming); b) enhancing the abundances of proteasomes and turnover of dysfunctional proteins (proteasome or protease); c) increasing the abundance of stress response proteins, such as alcohol dehydrogenase, S-adenosylmethionine synthetase (SAMS) and glycosyl hydrolase; d) reinforcing cellular biological regulation and signal transduction (calreticulin-1). For the first time, some proteins, such as cell division protein 48 (CDC48), calreticulin and phospholipase, which might be involved in the downstream signaling of Al in Citrus plants, were successfully identified.

Aluminum effects on photosynthesis, reactive oxygen species and methylglyoxal detoxification in two Citrus species differing in aluminum tolerance.

Citrus are mainly grown in low pH soils with high active aluminum (Al). 'Xuegan' (Citrus sinensis (L.) Osbeck) and 'Shatian pummelo' (Citrus grandis (L.) Osbeck) seedlings were fertilized for 18 weeks with nutrient solution containing either 0 mM (control) or 1 mM (Al toxicity) AlCl3·6H2O. Aluminum induced decreases of biomass, leaf photosynthesis, relative water content and total soluble protein levels, and increases of methylglyoxal levels only occurred in C. grandis roots and leaves. Besides, the Al-induced decreases of pigments and alterations of chlorophyll a fluorescence transients and fluorescence parameters were greater in C. grandis leaves than those in C. sinensis leaves. Aluminum-treated C. grandis had higher stem and leaf Al levels and similar root Al levels relative to Al-treated C. sinensis, but lower Al distribution in roots and Al uptake per plant. Aluminum toxicity decreased nitrogen, phosphorus, potassium, calcium, magnesium and sulfur uptake per plant in C. grandis and C. sinensis seedlings, with the exception of Al-treated C. sinensis seedlings exhibiting increased sulfur uptake per plant and unaltered magnesium uptake per plant. Under Al-stress, macroelement uptake per plant was higher in C. sinensis than that in C. grandis. Aluminum toxicity decreased the ratios of reduced glutathione/(reduced + oxidized glutathione) and of ascorbate/(ascorbate + dehydroascorbate) only in C. grandis roots and leaves. The activities of most antioxidant enzymes, sulfur metabolism-related enzymes and glyoxalases and the levels of S-containing compounds were higher in Al-treated C. sinensis roots and leaves than those in Al-treated C. grandis ones. Thus, C. sinensis displayed higher Al tolerance than C. grandis did. The higher Al tolerance of C. sinensis might involve: (i) more Al accumulation in roots and less transport of Al from roots to shoots; (ii) efficient maintenance of nutrient homeostasis; and (iii) efficient maintenance of redox homeostasis via detoxification systems of reactive oxygen species and methylglyoxal.

Aluminum-responsive genes revealed by RNA-Seq and related physiological responses in leaves of two Citrus species with contrasting aluminum-tolerance.

Little is known about the physiological and molecular responses of leaves to aluminum (Al)-toxicity. Seedlings of Al-intolerant Citrus grandis and Al-tolerant Citrus sinensis were supplied daily with nutrient solution containing 0 mM (control) and 1.0 mM (Al-toxicity) AlCl3·6H2O for 18 weeks. We found that Al-treatment only decreased CO2 assimilation in C. grandis leaves, and that the Al-induced alterations of gene expression profiles were less in C. sinensis leaves than those in C. grandis leaves, indicating that C. sinensis seedlings were more tolerant to Al-toxicity than C. grandis ones. Al concentration was similar between Al-treated C. sinensis and C. grandis roots, but it was higher in Al-treated C. grandis stems and leaves than that in Al-treated C. sinensis stems and leaves. Al-treated C. sinensis seedlings accumulated relatively more Al in roots and transported relatively little Al to shoots. This might be responsible for the higher Al-tolerance of C. sinensis. Further analysis showed that the following several aspects might account for the higher Al-tolerance of C. sinensis, including: (a) Al-treated C. sinensis leaves had higher capacity to maintain the homeostasis of energy and phosphate, the stability of lipid composition and the integrity of cell wall than did Al-treated C. grandis leaves; (b) Al-triggered production of reactive oxygen species (ROS) and the other cytotoxic compounds was less in Al-treated C. sinensis leaves than that in Al-treated C. grandis leaves, because Al-toxicity decreased CO2 assimilation only in C. grandis leaves; accordingly, more upregulated genes involved in the detoxifications of ROS, aldehydes and methylglyoxal were identified in Al-treated C. grandis leaves; in addition, flavonoid concentration was increased only in Al-treated C. grandis leaves; (c) Al-treated C. sinensis leaves could keep a better balance between protein phosphorylation and dephosphorylation than did Al-treated C. grandis leaves; and (d) both the equilibrium of hormones and hormone-mediated signal transduction were greatly disrupted in Al-treated C. grandis leaves, but less altered in Al-treated C. sinensis leaves. Finally, we discussed the differences in Al-responsive genes between Citrus roots and leaves.

Sulfur-Mediated-Alleviation of Aluminum-Toxicity in Citrus grandis Seedlings.

Limited data are available on the sulfur (S)-mediated-alleviation of aluminum (Al)-toxicity in higher plants. Citrus grandis seedlings were irrigated for 18 weeks with 0.5 mM MgSO4 or 0.5 mM MgSO4 + 0.5 mM Na2SO4, and 0 (-Al) or 1 mM AlCl3·6H2O (+Al, Al-toxicity). Under Al-toxicity, S decreased the level of Al in leaves; increased the relative water content (RWC) of roots and leaves, the contents of phosphorus (P), calcium (Ca) and magnesium (Mg) per plant, the dry weights (DW) of roots and shoots, the ratios of root DW/shoot DW, and the Al-induced secretion of citrate from root; and alleviated the Al-induced inhibition of photosynthesis via mitigating the Al-induced decrease of electron transport capacity resulting from the impaired photosynthetic electron transport chain. In addition to decreasing the Al-stimulated H2O2 production, the S-induced upregulation of both S metabolism-related enzymes and antioxidant enzymes also contributed to the S-mediated-alleviation of oxidative damage in Al-treated roots and leaves. Decreased transport of Al from roots to shoots and relatively little accumulation of Al in leaves, and increased leaf and root RWC and P, Ca, and Mg contents per plant might also play a role in the S-mediated-alleviation of Al-toxicity.

Magnesium-deficiency-induced alterations of gas exchange, major metabolites and key enzymes differ among roots, and lower and upper leaves of Citrus sinensis seedlings.

Magnesium (Mg)-deficiency is a widespread problem adversely affecting the quality and yield of crops, including citrus. 'Xuegan' [Citrus sinensis (L.) Osbeck] seedlings were irrigated every other day with nutrient solution at an Mg concentration of 0 mM (Mg-deficiency) or 1 mM (Mg-sufficiency) for 16 weeks. Thereafter, biomass, leaf mass per area, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), pigments in the upper and lower leaves, Mg, gas exchange, organic acids, nonstructural carbohydrates, total soluble proteins, amino acids, phenolics and anthocyanins, and key enzymes related to organic acid, amino acid and phenolic metabolisms in the roots, and upper and lower leaves were assayed in order to test the hypothesis that Mg-deficiency-induced alterations of gas exchange, major metabolites and key enzymes may differ among the roots, and upper and lower leaves. Magnesium-deficiency affected the most measured parameters more in the lower than in the upper leaves except for the nonstructural carbohydrates, but the variation trends were similar between the two. Despite increased accumulation of nonstructural carbohydrates, the lower CO2 assimilation in the Mg-deficient leaves was not caused by the feedback inhibition mechanism via sugar accumulation. Both dark respiration and organic acid metabolism were elevated in the Mg-deficient lower leaves to 'consume' the excess carbohydrates, and inhibited in the Mg-deficient roots with less accumulation of nonstructural carbohydrates to keep the balance of net carbon. More total phenolics and fewer anthocyanins were accumulated in the Mg-deficient lower leaves, whereas the accumulation of both total phenolics and anthocyanins was reduced in the Mg-deficient roots. Interestingly, amino acid biosynthesis was repressed in the Mg-deficient roots and lower leaves, thus lowering the level of total free amino acids in these roots and leaves. To conclude, great differences existed in the Mg-deficiency-induced alterations of gas exchange, major metabolites and key enzymes among the roots, and upper and lower leaves.

Effects of Low pH on Photosynthesis, Related Physiological Parameters, and Nutrient Profiles of Citrus.

Seedlings of "Xuegan" (Citrus sinensis) and "Sour pummelo" (Citrus grandis) were irrigated daily with a nutrient solution at a pH of 2.5, 3, 4, 5, or 6 for 9 months. Thereafter, the following responses were investigated: seedling growth; root, stem, and leaf concentrations of nutrient elements; leaf gas exchange, pigment concentration, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and chlorophyll a fluorescence; relative water content, total soluble protein level, H2O2 production and electrolyte leakage in roots and leaves. This was done (a) to determine how low pH affects photosynthesis, related physiological parameters, and mineral nutrient profiles; and (b) to understand the mechanisms by which low pH may cause a decrease in leaf CO2 assimilation. The pH 2.5 greatly inhibited seedling growth, and many physiological parameters were altered only at pH 2.5; pH 3 slightly inhibited seedling growth; pH 4 had almost no influence on seedling growth; and seedling growth and many physiological parameters reached their maximum at pH 5. No seedlings died at any given pH. These results demonstrate that citrus survival is insensitive to low pH. H+-toxicity may directly damage citrus roots, thus affecting the uptake of mineral nutrients and water. H+-toxicity and a decreased uptake of nutrients (i.e., nitrogen, phosphorus, potassium, calcium, and magnesium) and water were likely responsible for the low pH-induced inhibition of growth. Leaf CO2 assimilation was inhibited only at pH 2.5. The combinations of an impaired photosynthetic electron transport chain, increased production of reactive oxygen species, and decreased uptake of nutrients and water might account for the pH 2.5-induced decrease in CO2 assimilation. Mottled bleached leaves only occurred in the pH 2.5-treated C. grandis seedlings. Furthermore, the pH 2.5-induced alterations of leaf CO2 assimilation, water-use efficiency, chlorophylls, polyphasic chlorophyll a fluorescence (OJIP) transients and many fluorescence parameters, root and leaf total soluble proteins, H2O2 production, and electrolyte leakage were all slightly greater in C. grandis than in C. sinensis seedlings. Hence, C. sinensis was slightly more tolerant to low pH than C. grandis. In conclusion, our findings provide novel insight into the causes of low pH-induced inhibition of seedling growth and leaf CO2 assimilation.