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.

Stress Physiology and Molecular Biology of Fruit Crops.

Fruit crops provide various kinds of fruit commodities that are of significant nutritional benefit and economic value to humans [...].

Citrus sinensis manganese tolerance: Insight from manganese-stimulated secretion of root exudates and rhizosphere alkalization.

We used manganese (Mn)-tolerant 'Xuegan' (Citrus sinensis) seedlings as materials and examined the characterization of Mn uptake and Mn-activated-release of root exudates under hydroponic conditions. We observed that root and shoot Mn bioaccumulation factor (BCF) reduced with the increase of Mn supply, and that Mn transfer factor (Tf) reduced greatly as Mn supply increased from 0 to 500 µM, beyond which Tf slightly increased with increasing Mn supply, suggesting that Mn supply reduced the ability to absorb and accumulate Mn in roots and shoots, as well as root-to-shoot Mn translocation. Without Mn, roots alkalized the solution pH from 5.0 to above 6.2, while Mn supply reduced root-induced alkalization. As Mn supply increased from 0 to 2000 µM, the secretion of root total phenolics (TPs) increased, while the solution pH decreased. Mn supply did not alter the secretion of root total free amino acids, total soluble sugars, malate, and citrate. Mn-activated-release of TPs was inhibited by low temperature and anion channel inhibitors, but not by protein biosynthesis inhibitor. Using widely targeted metabolome, we detected 48 upregulated [35 upregulated phenolic compounds + 13 other secondary metabolites (SMs)] and three downregulated SMs, and 39 upregulated and eight downregulated primary metabolites (PMs). These findings suggested that reduced ability to absorb and accumulate Mn in roots and shoots and less root-to-shoot Mn translocation in Mn-toxic seedlings, rhizosphere alkalization, and Mn-activated-release of root exudates (especially phenolic compounds) contributed to the high Mn tolerance of C. sinensis seedlings.

Regulation of magnesium and calcium homeostasis in citrus seedlings under varying magnesium supply.

Magnesium (Mg) and calcium (Ca) are two essential macronutrients in plants; however, the characteristics of Mg and Ca concentrations in organ, subcellular and chemical forms and their relationships in citrus plants, especially under varying Mg supply, are not well understood. In this study, Citrus sinensis seedlings (cv. Xuegan) were cultivated in conditions of Mg deficiency (0 mmol Mg2+ L-1) and Mg sufficiency (2 mmol Mg2+ L-1) to investigate the responses of Mg and Ca homeostasis in different organs and fractions. Compared with Mg sufficiency, Mg deficiency significantly decreased root and shoot growth, with the shoot biomass reduction of branch organs was greater than that of parent organs. In addition to increasing the Ca concentration in the parent stem and lateral root organs, Mg deficiency significantly decreased the concentrations and accumulations of Mg and Ca in citrus seedlings, further altering their distribution in different organs. More than 50% of Ca and Mg were sequestrated in the cell wall and soluble fractions, respectively, with Mg concentration decreasing by 15.4% in roots and 46.9% in leaves under Mg deficiency, while Ca concentration decreased by 27.6% in roots and increased by 23.6% in parent leaves. Approximately 90% of Mg exists in inorganic, water-soluble, and pectate and protein-bound forms, and nearly 90% of Ca exists in water-soluble, pectate and protein-bound, phosphate and oxalate acid forms. Except for the decreased inorganic Mg in roots and water-soluble Mg and Ca in leaves, Mg deficiency increased the proportions of Mg and Ca in all chemical forms. However, Mg deficiency generally increased the Ca/Mg ratio in various organs, subcellular and chemical forms, with negative relationships between Mg concentration and Ca/Mg ratio, and the variations of Mg and Ca were highly separated between Mg supply and organs. In conclusion, our results provide insights into the effects of Mg supply on Mg and Ca homeostasis in citrus plants.

Regulation on copper-tolerance in Citrus sinensis seedlings by boron addition: Insights from root exudates, related metabolism, and gene expression.

Boron (B) can alleviate Citrus copper (Cu)-toxicity. However, the underlying mechanism by which B mitigates Cu-toxicity is unclear. 'Xuegan' (Citrus sinensis) seedlings were exposed to 0.5 (control) or 350 (Cu-toxicity) µM Cu and 2.5 or 25 µM B for 24 weeks. Thereafter, we investigated the secretion of low molecular weight compounds [LMWCs; citrate, malate, total soluble sugars (TSS), total phenolics (TP), and total free amino acids (TFAA)] by excised roots and their concentrations in roots and leaves, as well as related enzyme gene expression and activities in roots and leaves. Cu-stress stimulated root release of malate and TFAA, which might contribute to citrus Cu-tolerance. However, B-mediated-mitigation of Cu-stress could not be explained in this way, since B addition failed to further stimulate malate and TFAA secretion. Indeed, B addition decreased Cu-stimulated-secretion of malate. Further analysis suggested that Cu-induced-exudation of malate and TFAA was not regulated by their levels in roots. By contrast, B addition increased malate, citrate, and TFAA concentrations in Cu-toxic roots. Cu-toxicity increased TP concentration in 25 µM B-treated leaves, but not in 2.5 µM B-treated leaves. Our findings suggested that the internal detoxification of Cu by LMWCs played a role in B-mediated-alleviation of Cu-toxicity.

An Integrated Analysis of Metabolome, Transcriptome, and Physiology Revealed the Molecular and Physiological Response of Citrus sinensis Roots to Prolonged Nitrogen Deficiency.

Citrus sinensis seedlings were supplied with a nutrient solution containing 15 (control) or 0 (nitrogen (N) deficiency) mM N for 10 weeks. Extensive metabolic and gene reprogramming occurred in 0 mM N-treated roots (RN0) to cope with N deficiency, including: (a) enhancing the ability to keep phosphate homeostasis by elevating the abundances of metabolites containing phosphorus and the compartmentation of phosphate in plastids, and/or downregulating low-phosphate-inducible genes; (b) improving the ability to keep N homeostasis by lowering the levels of metabolites containing N but not phosphorus, upregulating N compound degradation, the root/shoot ratio, and the expression of genes involved in N uptake, and resulting in transitions from N-rich alkaloids to carbon (C)-rich phenylpropanoids and phenolic compounds (excluding indole alkaloids) and from N-rich amino acids to C-rich carbohydrates and organic acids; (c) upregulating the ability to maintain energy homeostasis by increasing energy production (tricarboxylic acid cycle, glycolysis/gluconeogenesis, oxidative phosphorylation, and ATP biosynthetic process) and decreasing energy utilization for amino acid and protein biosynthesis and new root building; (d) elevating the transmembrane transport of metabolites, thus enhancing the remobilization and recycling of useful compounds; and (e) activating protein processing in the endoplasmic reticulum. RN0 had a higher ability to detoxify reactive oxygen species and aldehydes, thus protecting RN0 against oxidative injury and delaying root senescence.

Roles of Hormones in Elevated pH-Mediated Mitigation of Copper Toxicity in Citrus sinensis Revealed by Targeted Metabolome.

The effects of copper (Cu)-pH interactions on the levels of hormones and related metabolites (HRMs) in Citrus sinensis leaves and roots were investigated. Our findings indicated that increased pH mitigated Cu toxicity-induced alterations of HRMs, and Cu toxicity increased low-pH-induced alterations of HRMs. Increased pH-mediated decreases in ABA, jasmonates, gibberellins, and cytokinins, increases in (±)strigol and 1-aminocyclopropanecarboxylic acid, and efficient maintenance of salicylates and auxins homeostasis in 300 µM Cu-treated roots (RCu300); as well as efficient maintenance of hormone homeostasis in 300 µM Cu-treated leaves (LCu300) might contribute to improved leaf and root growth. The upregulation of auxins (IAA), cytokinins, gibberellins, ABA, and salicylates in pH 3.0 + 300 µM Cu-treated leaves (P3CL) vs. pH 3.0 + 0.5 µM Cu-treated leaves (P3L) and pH 3.0 + 300 µM Cu-treated roots (P3CR) vs. pH 3.0 + 0.5 µM Cu-treated roots (P3R) might be an adaptive response to Cu toxicity, so as to cope with the increased need for reactive oxygen species and Cu detoxification in LCu300 and RCu300. Increased accumulation of stress-related hormones (jasmonates and ABA) in P3CL vs. P3L and P3CR vs. P3R might reduce photosynthesis and accumulation of dry matter, and trigger leaf and root senescence, thereby inhibiting their growth.

Effects of Nitrogen Deficiency on the Metabolism of Organic Acids and Amino Acids in Oryza sativa.

Organic acids metabolism and nitrogen (N) metabolism in rice seedlings and the relationship between them are not fully understood. In this study, rice (Oryza sativa L. ssp. Indica) variety "Huanghuazhan" was used as the experimental material, and three N levels (5 mM, 1 mM, and 0 mM NH4NO3) were set by the hydroponic method for different levels of N treatment. Our results showed that the increased content of malate in rice leaves caused by reducing N level was related to the increased synthesis of malate (the activity of leaf PEPC increased)and the decreased degradation of malate (the activity of leaf NADP-ME decreased), while the increased contents of citrate and isocitrate in rice leaves caused by reducing N level might not be caused by the increased biosynthesis, but due to the decrease in degradation of citrate and isocitrate (the activities of leaf CS, ACO, and NADP-IDH decreased). The increased content of malate in rice roots caused by reducing N level might be related to the increased biosynthesis and the decreased degradation of root malate (the activities of root NAD-MDH and PEPC increased, while the activity of NADP-ME decreased). Compared to the control (5 mM NH4NO3), the increased content of citrate in rice roots caused by reducing N level might be related to the increased biosynthesis rather than the decreased degradation of citrate, due to the higher activities of CS and ACO in rice roots under 0 mM N and 1mM N treatment when compared to that of the control ones. At the same time, the increased content of isocitrate in roots was related to the increased isomerization of isocitrate (the activity of root ACO increased) and the decreased degradation of isocitrate (the activity of root NADP-IDH decreased). With the reducing N level, the activities of N metabolism-related enzymes, such as nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT), decreased in rice leaves and roots, resulting in the decreased contents of total free amino acids (TFAAs) and soluble proteins in rice seedlings, and finally led to the growth inhibition. Our results showed that the dynamics of organic acids metabolism caused by reducing N level were different in rice leaves and roots. In conclusion, there was a close correlation between organic acids metabolism and N metabolism in rice leaves and roots under N-limited conditions; furthermore, such a correlation was more obvious in rice leaves than that of roots.

Characterization of copper-induced-release of exudates by Citrus sinensis roots and their possible roles in copper-tolerance.

Copper (Cu) excess is often observed in old Citrus orchards. Little information is available on the characterization of Cu-induced-release of root exudates and their possible roles in plant Cu-tolerance. Using sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] seedlings as materials, we investigated the impacts of 0, 0.5, 25, 150, 350, 550, 1000, 2000 or 5000 µM CuCl2 (pH 4.8) on Cu uptake, root exudates [malate, citrate, total phenolics (TP), total soluble sugars (TSS) and total free amino acids (TFAA)], electrolyte leakage and malondialdehyde, and solution pH under hydroponic conditions; the time-course of root exudates and solution pH in response to Cu; and the impacts of protein synthesis and anion-channel inhibitors, and temperature on Cu-induced-secretion of root exudates and solution pH. About 70% of Cu was accumulated in 0 and 0.5 µM Cu-exposed roots, while over 97% of Cu was accumulated in ≥25 µM Cu-exposed roots. Without Cu, the seedlings could alkalize the solution pH from 4.8 to above 6.0. Cu-stimulated-secretion of root exudates elevated with the increment of Cu concentration from 0 to 1000 µM, then decreased or remained unchanged with the further increment of Cu concentration, while root electrolyte leakage and malondialdehyde (root-induced alkalization) increased (lessened) with the increment of Cu concentration from 0 to 5000 µM. Further analysis indicated that Cu-stimulated-secretion of root exudates was an energy-dependent process and could repressed by inhibitors, and that there was no discernible delay between the onset of exudate release and the addition of Cu. To conclude, both root-induced alkalization and Cu-stimulated-release of root exudates played a key role in sweet orange Cu-tolerance via increasing root Cu accumulation and reducing Cu uptake and phytotoxicity.

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.

Adaptive responses of carbon and nitrogen metabolisms to nitrogen-deficiency in Citrus sinensis seedlings.

BACKGROUND: In China, nitrogen (N)-deficiency often occurs in Citrus orchards, which is one of the main causes of yield loss and fruit quality decline. Little information is known about the adaptive responses of Citrus carbon (C) and N metabolisms to N-deficiency. Seedlings of 'Xuegan' (Citrus sinensis (L.) Osbeck) were supplied with nutrient solution at an N concentration of 0 (N-deficiency), 5, 10, 15 or 20 mM for 10 weeks. Thereafter, we examined the effects of N supply on the levels of C and N in roots, stems and leaves, and the levels of organic acids, nonstructural carbohydrates, NH4+-N, NO3--N, total soluble proteins, free amino acids (FAAs) and derivatives (FAADs), and the activities of key enzymes related to N assimilation and organic acid metabolism in roots and leaves. RESULTS: N-deficiency elevated sucrose export from leaves to roots, C and N distributions in roots and C/N ratio in roots, stems and leaves, thus enhancing root dry weight/shoot dry weight ratio and N use efficiency. N-deficient leaves displayed decreased accumulation of starch and total nonstructural carbohydrates (TNC) and increased sucrose/starch ratio as well as a partitioning trend of assimilated C toward to sucrose, but N-deficient roots displayed elevated accumulation of starch and TNC and reduced sucrose/starch ratio as well as a partitioning trend of assimilated C toward to starch. N-deficiency reduced the concentrations of most FAADs and the ratios of total FAADs (TFAADs)/N in leaves and roots. N-deficiency reduced the demand for C skeleton precursors for amino acid biosynthesis, thus lowering TFAADs/C ratio in leaves and roots. N-deficiency increased (decreased) the relative amounts of C-rich (N-rich) FAADs, thus increasing the molar ratio of C/N in TFAADs in leaves and roots. CONCLUSIONS: Our findings corroborated our hypothesis that C and N metabolisms displayed adaptive responses to N-deficiency in C. sinensis seedlings, and that some differences existed between roots and leaves in N-deficiency-induced alterations of and C and N metabolisms.

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.

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.

Citrus Physiological and Molecular Response to Boron Stresses.

Since the essentiality of boron (B) to plant growth was reported nearly one century ago, the implication of B in physiological performance, productivity and quality of agricultural products, and the morphogenesis of apical meristem in plants has widely been studied. B stresses (B deficiency and toxicity), which lead to atrophy of canopy and deterioration of Citrus fruits, have long been discovered in citrus orchards. This paper reviews the research progress of B stresses on Citrus growth, photosynthesis, light use efficiency, nutrient absorption, organic acid metabolism, sugar metabolism and relocation, and antioxidant system. Moreover, the beneficial effects of B on plant stress tolerance and further research in this area were also discussed.

Molecular mechanisms for magnesium-deficiency-induced leaf vein lignification, enlargement and cracking in Citrus sinensis revealed by RNA-Seq.

Citrus sinensis (L.) Osbeck seedlings were fertigated with nutrient solution containing 2 [magnesium (Mg)-sufficiency] or 0 mM (Mg-deficiency) Mg(NO3)2 for 16 weeks. Thereafter, RNA-Seq was used to investigate Mg-deficiency-responsive genes in the veins of upper and lower leaves in order to understand the molecular mechanisms for Mg-deficiency-induced vein lignification, enlargement and cracking, which appeared only in the lower leaves. In this study, 3065 upregulated and 1220 downregulated, and 1390 upregulated and 375 downregulated genes were identified in Mg-deficiency veins of lower leaves (MDVLL) vs Mg-sufficiency veins of lower leaves (MSVLL) and Mg-deficiency veins of upper leaves (MDVUL) vs Mg-sufficiency veins of upper leaves (MSVUL), respectively. There were 1473 common differentially expressed genes (DEGs) between MDVLL vs MSVLL and MDVUL vs MSVUL, 1463 of which displayed the same expression trend. Magnesium-deficiency-induced lignification, enlargement and cracking in veins of lower leaves might be related to the following factors: (i) numerous transciption factors and genes involved in lignin biosynthesis pathways, regulation of cell cycle and cell wall metabolism were upregulated; and (ii) reactive oxygen species, phytohormone and cell wall integrity signalings were activated. Conjoint analysis of proteome and transcriptome indicated that there were 287 and 56 common elements between DEGs and differentially abundant proteins (DAPs) identified in MDVLL vs MSVLL and MDVUL vs MSVUL, respectively, and that among these common elements, the abundances of 198 and 55 DAPs matched well with the transcript levels of the corresponding DEGs in MDVLL vs MSVLL and MDVUL vs MSVUL, respectively, indicating the existence of concordances between protein and transcript levels.

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.