Boron Reduced Copper Excess-Induced Oxidative Damage in Citrus sinensis by Modulating Reactive Oxygen Species and Methylglyoxal Formation and Their Detoxification Systems.

Citrus is mainly cultivated in acid soil with low boron (B) and high copper (Cu). In this study, Citrus sinensis seedlings were submitted to 0.5 (control) or 350 µM Cu (Cu excess or Cu exposure) and 2.5, 10, or 25 µM B for 24 weeks. Thereafter, H2O2 production rate (HPR), superoxide production rate (SAPR), malondialdehyde, methylglyoxal, and reactive oxygen species (ROS) and methylglyoxal detoxification systems were measured in leaves and roots in order to test the hypothesis that B addition mitigated Cu excess-induced oxidative damage in leaves and roots by reducing the Cu excess-induced formation and accumulation of ROS and MG and by counteracting the impairments of Cu excess on ROS and methylglyoxal detoxification systems. Cu and B treatments displayed an interactive influence on ROS and methylglyoxal formation and their detoxification systems. Cu excess increased the HPR, SAPR, methylglyoxal level, and malondialdehyde level by 10.9% (54.3%), 38.9% (31.4%), 50.3% (24.9%), and 312.4% (585.4%), respectively, in leaves (roots) of 2.5 µM B-treated seedlings, while it only increased the malondialdehyde level by 48.5% (97.8%) in leaves (roots) of 25 µM B-treated seedlings. Additionally, B addition counteracted the impairments of Cu excess on antioxidant enzymes, ascorbate-glutathione cycle, sulfur metabolism-related enzymes, sulfur-containing compounds, and methylglyoxal detoxification system, thereby protecting the leaves and roots of Cu-exposed seedlings against oxidative damage via the coordinated actions of ROS and methylglyoxal removal systems. Our findings corroborated the hypothesis that B addition alleviated Cu excess-induced oxidative damage in leaves and roots by decreasing the Cu excess-induced formation and accumulation of ROS and MG and by lessening the impairments of Cu excess on their detoxification systems. Further analysis indicated that the pathways involved in the B-induced amelioration of oxidative stress caused by Cu excess differed between leaves and roots.

Boron-mediated amelioration of copper toxicity in Citrus sinensis seedlings involved reduced concentrations of copper in leaves and roots and their cell walls rather than increased copper fractions in their cell walls.

Little information is available on how boron (B) supplementation affects plant cell wall (CW) remodeling under copper (Cu) excess. 'Xuegan' (Citrus sinensis) seedlings were submitted to 0.5 or 350 µM Cu × 2.5 or 25 µM B for 24 weeks. Thereafter, we determined the concentrations of CW materials (CWMs) and CW components (CWCs), the degree of pectin methylation (DPM), and the pectin methylesterase (PME) activities and PME gene expression levels in leaves and roots, as well as the Cu concentrations in leaves and roots and their CWMs (CWCs). Additionally, we analyzed the Fourier transform infrared (FTIR) and X-ray diffraction (XRD) spectra of leaf and root CWMs. Our findings suggested that adding B reduced the impairment of Cu excess to CWs by reducing the Cu concentrations in leaves and roots and their CWMs and maintaining the stability of CWs, thereby improving leaf and root growth. Cu excess increased the Cu fractions in leaf and root pectin by decreasing DPM due to increased PME activities, thereby contributing to citrus Cu tolerance. FTIR and XRD indicated that the functional groups of the CW pectin, hemicellulose, cellulose, and lignin could bind and immobilize Cu, thereby reducing Cu cytotoxicity in leaves and roots.

Physiological and Ultrastructural Responses to Excessive-Copper-Induced Toxicity in Two Differentially Copper Tolerant Citrus Species.

Over-applied copper (Cu)-based agrochemicals are toxic to citrus trees. However, less information is available discussing the ultrastructural alterations in Cu-stressed citrus species. In the present study, seedlings of Citrus sinensis and Citrus grandis that differed in Cu-tolerance were sandy-cultured with nutrient solution containing 0.5 µM Cu (as control) or 300 µM Cu (as Cu toxicity) for 18 weeks. At the end of the treatments, the physiological parameters and ultrastructural features of the citrus leaves and roots were analyzed. The results indicate that Cu toxicity significantly decreased the ratio of shoot biomass to dry weight, the Cu translocation factor and the total chlorophyll of two citrus species. The anatomical and ultrastructural alterations verified that excessive Cu resulted in starch granules accumulated in the leaves and roots of the two citrus species. Under Cu toxicity, increased root flocculent precipitate and thickened root cell wall might reduce the Cu translocation from citrus roots to the shoots. Compared with C. sinensis, C. grandis maintained a relatively integral root cellular structure under Cu toxicity, which provided a structural basis for a higher Cu tolerance than C. sinensis. The present results increase our understanding of the physiological and ultrastructural responses to Cu toxicity in citrus species.

High pH Alleviated Sweet Orange (Citrus sinensis) Copper Toxicity by Enhancing the Capacity to Maintain a Balance between Formation and Removal of Reactive Oxygen Species and Methylglyoxal in Leaves and Roots.

The contribution of reactive oxygen species (ROS) and methylglyoxal (MG) formation and removal in high-pH-mediated alleviation of plant copper (Cu)-toxicity remains to be elucidated. Seedlings of sweet orange (Citrus sinensis) were treated with 0.5 (non-Cu-toxicity) or 300 (Cu-toxicity) µM CuCl2 × pH 4.8, 4.0, or 3.0 for 17 weeks. Thereafter, superoxide anion production rate; H2O2 production rate; the concentrations of MG, malondialdehyde (MDA), and antioxidant metabolites (reduced glutathione, ascorbate, phytochelatins, metallothioneins, total non-protein thiols); and the activities of enzymes (antioxidant enzymes, glyoxalases, and sulfur metabolism-related enzymes) in leaves and roots were determined. High pH mitigated oxidative damage in Cu-toxic leaves and roots, thereby conferring sweet orange Cu tolerance. The alleviation of oxidative damage involved enhanced ability to maintain the balance between ROS and MG formation and removal through the downregulation of ROS and MG formation and the coordinated actions of ROS and MG detoxification systems. Low pH (pH 3.0) impaired the balance between ROS and MG formation and removal, thereby causing oxidative damage in Cu-toxic leaves and roots but not in non-Cu-toxic ones. Cu toxicity and low pH had obvious synergistic impacts on ROS and MG generation and removal in leaves and roots. Additionally, 21 (4) parameters in leaves were positively (negatively) related to the corresponding root parameters, implying that there were some similarities and differences in the responses of ROS and MG metabolisms to Cu-pH interactions between leaves and roots.

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.

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.

Boron-mediated amelioration of copper-toxicity in sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] seedlings involved reduced damage to roots and improved nutrition and water status.

'Xuegan' (Citrus sinensis) seedlings were fertilized 6 times weekly for 24 weeks with 0.5 or 350 µM CuCl2 and 2.5, 10 or 25 µM H3BO3. Cu-toxicity increased Cu uptake per plant (UPP) and Cu concentrations in leaves, stems and roots, decreased water uptake and phosphorus, nitrogen, calcium, magnesium, potassium, sulfur, boron and iron UPP, and increased the ratios of magnesium, potassium, calcium and sulfur UPP to phosphorus UPP and the ratios of leaf magnesium, potassium and calcium concentrations to leaf phosphorus concentration. Many decaying and dead fibrous roots occurred in Cu-toxic seedlings. Cu-toxicity-induced alterations of these parameters and root damage decreased with the increase of boron supply. These results demonstrated that B supplementation lowered Cu uptake and its concentrations in leaves, stems and roots and subsequently alleviated Cu-toxicity-induced damage to root growth and function, thus improving plant nutrient (decreased Cu uptake and efficient maintenance of the other nutrient homeostasis and balance) and water status. Further analysis indicated that the improved nutrition and water status contributed to the boron-mediated amelioration of Cu-toxicity-induced inhibition of seedlings, decline of leaf pigments, large reduction of leaf CO2 assimilation and impairment of leaf photosynthetic electron transport chain revealed by greatly altered chlorophyll a fluorescence (OJIP) transients, reduced maximum quantum yield of primary photochemistry (Fv/Fm), quantum yield for electron transport (ETo/ABS) and total performance index (PIabs,total), and elevated dissipated energy per reaction center (DIo/RC). To conclude, our findings corroborate the hypothesis that B-mediated amelioration of Cu-toxicity involved reduced damage to roots and improved nutrient and water status. Principal component analysis showed that Cu-toxicity-induced changes of above physiological parameters generally decreased with the increase of B supply and that B supply-induced alterations of above physiological parameters was greater in 350 µM Cu-treated than in 0.5 µM Cu-treated seedlings. B and Cu had a significant interactive influence on C. sinensis seedlings.

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.

Metabolomics combined with physiology and transcriptomics reveals how Citrus grandis leaves cope with copper-toxicity.

Limited data are available on metabolic responses of plants to copper (Cu)-toxicity. Firstly, we investigated Cu-toxic effects on metabolomics, the levels of free amino acids, NH4+-N, NO3--N, total nitrogen, total soluble proteins, total phenolics, lignin, reduced glutathione (GSH) and malondialdehyde, and the activities of nitrogen-assimilatory enzymes in 'Shatian' pummelo (Citrus grandis) leaves. Then, a conjoint analysis of metabolomics, physiology and transcriptomics was performed. Herein, 59 upregulated [30 primary metabolites (PMs) and 29 secondary metabolites (SMs)] and 52 downregulated (31 PMs and 21 SMs) metabolites were identified in Cu-toxic leaves. The toxicity of Cu to leaves was related to the Cu-induced accumulation of NH4+ and decrease of nitrogen assimilation. Metabolomics combined with physiology and transcriptomics revealed some adaptive responses of C. grandis leaves to Cu-toxicity, including (a) enhancing tryptophan metabolism and the levels of some amino acids and derivatives (tryptophan, phenylalanine, 5-hydroxy-l-tryptophan, 5-oxoproline and GSH); (b) increasing the accumulation of carbohydrates and alcohols and upregulating tricarboxylic acid cycle and the levels of some organic acids and derivatives (chlorogenic acid, quinic acid, d-tartaric acid and gallic acid o-hexoside); (c) reducing phospholipid (lysophosphatidylcholine and lysophosphatidylethanolamine) levels, increasing non-phosphate containing lipid [monoacylglycerol ester (acyl 18:2) isomer 1] levels, and inducing low-phosphate-responsive gene expression; and (d) triggering the biosynthesis of some chelators (total phenolics, lignin, l-trytamine, indole, eriodictyol C-hexoside, quercetin 5-O-malonylhexosyl-hexoside, N-caffeoyl agmatine, N'-p-coumaroyl agmatine, hydroxy-methoxycinnamate and protocatechuic acid o-glucoside) and vitamins and derivatives (nicotinic acid-hexoside, B1 and methyl nicotinate). Cu-induced upregulation of many antioxidants could not protect Cu-toxic leaves from oxidative damage. To conclude, our findings corroborated the hypothesis that extensive reprogramming of metabolites was carried out in Cu-toxic C. grandis leaves in order to cope with Cu-toxicity.

Effects of phosphorus deficiency on the absorption of mineral nutrients, photosynthetic system performance and antioxidant metabolism in Citrus grandis.

Phosphorus (P) is an essential macronutrient for plant growth, development and production. However, little is known about the effects of P deficiency on nutrient absorption, photosynthetic apparatus performance and antioxidant metabolism in citrus. Seedlings of 'sour pummelo' (Citrus grandis) were irrigated with a nutrient solution containing 0.2 mM (Control) or 0 mM (P deficiency) KH2PO4 until saturated every other day for 16 weeks. P deficiency significantly decreased the dry weight (DW) of leaves and stems, and increased the root/shoot ratio in C. grandis but did not affect the DW of roots. The decreased DW of leaves and stems might be induced by the decreased chlorophyll (Chl) contents and CO2 assimilation in P deficient seedlings. P deficiency heterogeneously affected the nutrient contents of leaves, stems and roots. The analysis of Chl a fluorescence transients showed that P deficiency impaired electron transport from the donor side of photosystem II (PSII) to the end acceptor side of PSI, which showed a greater impact on the performance of the donor side of PSII than that of the acceptor side of PSII and photosystem I (PSI). P deficiency increased the contents of ascorbate (ASC), H2O2 and malondialdehyde (MDA) as well as the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in leaves. In contrast, P deficiency increased the ASC content, reduced the glutathione (GSH) content and the activities of SOD, CAT, APX and monodehydroascorbate reductase (MDHAR), but did not increase H2O2 production, anthocyanins and MDA content in roots. Taking these results together, we conclude that P deficiency affects nutrient absorption and lowers photosynthetic performance, leading to ROS production, which might be a crucial cause of the inhibited growth of C. grandis.

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.

Excess Copper-Induced Alterations of Protein Profiles and Related Physiological Parameters in Citrus Leaves.

This present study examined excess copper (Cu) effects on seedling growth, leaf Cu concentration, gas exchange, and protein profiles identified by a two-dimensional electrophoresis (2-DE) based mass spectrometry (MS) approach after Citrus sinensis and Citrus grandis seedlings were treated for six months with 0.5 (control), 200, 300, or 400 µM CuCl2. Forty-one and 37 differentially abundant protein (DAP) spots were identified in Cu-treated C. grandis and C. sinensis leaves, respectively, including some novel DAPs that were not reported in leaves and/or roots. Most of these DAPs were identified only in C. grandis or C. sinensis leaves. More DAPs increased in abundances than DAPs decreased in abundances were observed in Cu-treated C. grandis leaves, but the opposite was true in Cu-treated C. sinensis leaves. Over 50% of DAPs were associated with photosynthesis, carbohydrate, and energy metabolism. Cu-toxicity-induced reduction in leaf CO2 assimilation might be caused by decreased abundances of proteins related to photosynthetic electron transport chain (PETC) and CO2 assimilation. Cu-effects on PETC were more pronounced in C. sinensis leaves than in C. grandis leaves. DAPs related to antioxidation and detoxification, protein folding and assembly (viz., chaperones and folding catalysts), and signal transduction might be involved in Citrus Cu-toxicity and Cu-tolerance.

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.

Excess copper effects on growth, uptake of water and nutrients, carbohydrates, and PSII photochemistry revealed by OJIP transients in Citrus seedlings.

Seedlings of 'Shatian pummelo' (Citrus grandis) and 'Xuegan' (Citrus sinensis) were supplied daily with nutrient solution at a concentration of 0.5 (control), 100, 200, 300, 400, or 500 µM CuCl2 for 6 months. Thereafter, seedling growth; leaf, root, and stem levels of nutrients; leaf gas exchange; levels of pigments; chlorophyll a fluorescence (OJIP) transients and related parameters; leaf and root relative water content; levels of nonstructural carbohydrates; H2O2 production rate; and electrolyte leakage were comprehensively examined (a) to test the hypothesis that Cu directly damages root growth and function, thus impairing water and nutrient uptake and hence inhibiting shoot growth; (b) to establish whether the Cu-induced preferential accumulation of Cu in the roots is involved in Cu tolerance of Citrus; and (c) to elucidate the possible causes for the Cu-induced decrease in photosynthesis. Most of the growth and physiological parameters were greatly altered only at 300-500 µM (excess) Cu-treated seedlings. Cu supply increased the level of Cu in the roots, stems, and leaves, with a greater increase in the roots than that in the stems and leaves. Many of the fibrous roots became rotten and died under excess Cu. These findings support the hypothesis that Cu directly damages root growth and function, thus impairing water and nutrient uptake and hence inhibiting shoot growth, and the conclusion that the preferential accumulation of Cu in the roots under excess Cu is involved in the tolerance of Citrus to Cu toxicity. The lower CO2 assimilation in excess Cu-treated leaves was caused mainly by nonstomatal factors, including structural damage to thylakoids, feedback inhibition due to increased accumulation of nonstructural carbohydrates, decreased uptake of water and nutrients, increased production of reactive oxygen species, and impaired photosynthetic electron transport chain. Also, we discussed the possible causes for the excess Cu-induced decrease in leaf pigments and accumulation of nonstructural carbohydrates in the roots and leaves.

Responses of reactive oxygen species and methylglyoxal metabolisms to magnesium-deficiency differ greatly among the roots, upper and lower leaves of Citrus sinensis.

BACKGROUND: Magnesium (Mg)-deficiency is one of the most prevalent physiological disorders causing a reduction in Citrus yield and quality. 'Xuegan' (Citrus sinensis) seedlings were irrigated for 16 weeks with nutrient solution containing 2 mM (Mg-sufficiency) or 0 mM (Mg-deficiency) Mg(NO3)2. Thereafter, we investigated the Mg-deficient effects on gas exchange and chlorophyll a fluorescence in the upper and lower leaves, and Mg, reactive oxygen species (ROS) and methylglyoxal (MG) metabolisms in the roots, lower and upper leaves. The specific objectives were to corroborate the hypothesis that the responses of ROS and MG metabolisms to Mg-deficiency were greater in the lower leaves than those in the upper leaves, and different between the leaves and roots. RESULTS: Mg level was higher in the Mg-deficient upper leaves than that in the Mg-deficient lower leaves. This might be responsible for the Mg-deficiency-induced larger alterations of all the measured parameters in the lower leaves than those in the upper leaves, but they showed similar change patterns between the Mg-deficient lower and upper leaves. Accordingly, Mg-deficiency increased greatly their differences between the lower and upper leaves. Most of parameters involved in ROS and MG metabolisms had similar variation trends and degrees between the Mg-deficient lower leaves and roots, but several parameters (namely glutathione S-transferase, sulfite reductase, ascorbate and dehydroascorbate) displayed the opposite variation trends. Obviously, differences existed in the Mg-deficiency-induced alterations of ROS and MG metabolisms between the lower leaves and roots. Although the activities of most antioxidant and sulfur metabolism-related enzymes and glyoxalase I and the level of reduced glutathione in the Mg-deficient leaves and roots and the level of ascorbate in the leaves were kept in higher levels, the levels of malonaldehyde and MG and/or electrolyte leakage were increased in the Mg-deficient lower and upper leaves and roots, especially in the Mg-deficient lower leaves and roots. CONCLUSIONS: The ROS and MG detoxification systems as a whole did not provide sufficient detoxification capacity to prevent the Mg-deficiency-induced production and accumulation of ROS and MG, thus leading to lipid peroxidation and the loss of plasma membrane integrity, especially in the lower leaves and roots.

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.

Effects of High Toxic Boron Concentration on Protein Profiles in Roots of Two Citrus Species Differing in Boron-Tolerance Revealed by a 2-DE Based MS Approach.

Citrus are sensitive to boron (B)-toxicity. In China, B-toxicity occurs in some citrus orchards. So far, limited data are available on B-toxicity-responsive proteins in higher plants. Thirteen-week-old seedlings of "Sour pummelo" (Citrus grandis) and "Xuegan" (Citrus sinensis) was fertilized every other day until dripping with nutrient solution containing 10 µM (control) or 400 µM (B-toxicity) H3BO3 for 15 weeks. The typical B-toxic symptom only occurred in 400 µM B-treated C. grandis leaves, and that B-toxicity decreased root dry weight more in C. grandis seedlings than in C. sinensis ones, demonstrating that C. sinensis was more tolerant to B-toxicity than C. grandis. Using a 2-dimensional electrophoresis (2-DE) based MS approach, we identified 27 up- and four down-accumulated, and 28 up- and 13 down-accumulated proteins in B-toxic C. sinensis and C. grandis roots, respectively. Most of these proteins were isolated only from B-toxic C. sinensis or C. grandis roots, only nine B-toxicity-responsive proteins were shared by the two citrus species. Great differences existed in B-toxicity-induced alterations of protein profiles between C. sinensis and C. grandis roots. More proteins related to detoxification were up-accumulated in B-toxic C. grandis roots than in B-toxic C. sinensis roots to meet the increased requirement for the detoxification of the more reactive oxygen species and other toxic compounds such as aldehydes in the former. For the first time, we demonstrated that the active methyl cycle was induced and repressed in B-toxic C. sinensis and C. grandis roots, respectively, and that C. sinensis roots had a better capacity to keep cell wall and cytoskeleton integrity than C. grandis roots in response to B-toxicity, which might be responsible for the higher B-tolerance of C. sinensis. In addition, proteins involved in nucleic acid metabolism, biological regulation and signal transduction might play a role in the higher B-tolerance of C. sinensis.

Root iTRAQ protein profile analysis of two Citrus species differing in aluminum-tolerance in response to long-term aluminum-toxicity.

BACKGROUND: Limited information is available on aluminum (Al)-toxicity-responsive proteins in woody plant roots. Seedlings of 'Xuegan' (Citrus sinensis) and 'Sour pummelo' (Citrus grandis) were treated for 18 weeks with nutrient solution containing 0 (control) or 1.2 mM AlCl3 · 6H2O (+Al). Thereafter, we investigated Citrus root protein profiles using isobaric tags for relative and absolute quantification (iTRAQ). The aims of this work were to determine the molecular mechanisms of plants to deal with Al-toxicity and to identify differentially expressed proteins involved in Al-tolerance. RESULTS: C. sinensis was more tolerant to Al-toxicity than C. grandis. We isolated 347 differentially expressed proteins from + Al Citrus roots. Among these proteins, 202 (96) proteins only presented in C. sinensis (C. grandis), and 49 proteins were shared by the two species. Of the 49 overlapping proteins, 45 proteins were regulated in the same direction upon Al exposure in the both species. These proteins were classified into following categories: sulfur metabolism, stress and defense response, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, cell transport, biological regulation and signal transduction, cell wall and cytoskeleton metabolism, and jasmonic acid (JA) biosynthesis. The higher Al-tolerance of C. sinensis may be related to several factors, including: (a) activation of sulfur metabolism; (b) greatly improving the total ability of antioxidation and detoxification; (c) up-regulation of carbohydrate and energy metabolism; (d) enhancing cell transport; (e) decreased (increased) abundances of proteins involved in protein synthesis (proteiolysis); (f) keeping a better balance between protein phosphorylation and dephosphorylation; and (g) increasing JA biosynthesis. CONCLUSIONS: Our results demonstrated that metabolic flexibility was more remarkable in C. sinenis than in C. grandis roots, thus improving the Al-tolerance of C. sinensis. This provided the most integrated view of the adaptive responses occurring in Al-toxicity roots.