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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 CuCl

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.

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.

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.

Gamma radiation has been widely applied in citrus breeding to create seedless cultivar. However, little is yet known regarding the effect of γ-radiation mutagenesis on seed development in genus Citrus. In this report, a seedless mutant created from polyembryonic ‘Meiguicheng’ orange (Citrus sinensis) with γ-rays and its wild type were used to investigate the relationship between γ-radiation and the mechanism of seedlessness. Results indicated that both the seedless mutant and its wild type were diploid, producing highly similar flowers. Radiation-induced chromosomal aberrations significantly reduced pollen quantity and viability in the seedless mutant. However, cytological observation on pollen mother cells showed that there was no significant difference in irregular chromosomal behaviors at each given meiotic stage between the mutant and its wild type, indicating that the reduction of pollen viability in the seedless mutant might not be caused by meiotic disturbances. Also, γ-radiation-induced chromosomal aberrations caused a significantly higher abortion rate (86.21%) of megagametophyte during megasporogenesis and early megagametogenesis, resulting in female sterility in the seedless mutant. Intercrossing between the seedless mutant and its wild type revealed that pollen grains from the mutant efficiently fertilised the wild type, suggesting that the mutant is male fertile thereby could be used as male parent in further hybridization breeding programs. However, fruits developed from the flowers of cross-pollinated with wild-type pollen still produced very few seeds, most of which were much smaller in size and contained only nucellar embryos inside. Thus, the seedlessness of the mutant was caused by strong female sterility. Nucellar embryos could develop independently to produce seeds in this cultivar, though with low incidence.

In south China, Magnesium (Mg) deficiency is commonly found in Citrus orchard due to soil acidification. However, less information is available discussing the global metabolic profile of Mg-deficient leaves in Citrus species. In the study, seedlings of ‘Xuegan’ [Citrus sinensis (L.) Osbeck] were treated with Mg-plus (2 mM, as Control) or Mg-deficiency (0 mM) for 16 weeks by sandy culture, respectively. The untargeted metabolomics based on UHPLC-Q-TOF/MS were investigated to reveal the metabolic responses to Mg-deficiency in leaves of C. sinensis. The results indicated severe growth inhibition and obvious leaf chlorosis in Mg-deficient seedlings of C. sinensis compared to control. The principal component analysis (PCA) and orthogonal partial least squares-discriminate analysis (OPLS-DA) on the LC/GC data demonstrated a clear discrimination between the profiles of metabolites in control leaves and Mg-deficient leaves of C.sinensis. Besides, the identification and classification of those differential expressed metabolites (DEMs) revealed Mg-deficiency upregulated relative abundances of 78.42 % lipids and lipid-like molecules. In contrast, Mg-deficiency downregulated 89.42 % phenylpropanoids and polyketides in leaves of C.sinensis. Remarkably, relative abundances of most monosaccharides and amino acids increased while relative abundances of most leaf pigment compounds and organic acids decreased in response to Mg-deficiency in C. sinensis leaves. Those results support both primary and secondary metabolic modifications in C. sinensis leaves exposed to Mg-deficiency. Overall, the findings of present study contribute to a better understanding on the metabolic changes of Mg-deficient Citrus species, which will provide references for nutrient management and quality control in Citrus production.

Nitrogen (N) is an essential mineral nutrient for plant growth. Appropriate N fertilization may enhance the efficiency of N use and improve crop yield. However, the effects of N form and level on citrus plants are not well understood. We hypothesized that citrus plants are ammonium (as NH4+-N or AN) partial or insensitive based on the characteristics of preferential growth in the acidic red soil of southern China, where AN is the dominant N form in the soil. Citrus (Citrus reticulata Blanco cv. Ponkan) seedlings were cultivated in sand in the presence of three N levels (1, 4 and 8 mmol L−1) supplied in two N forms (AN; nitrate, as NO3--N or NN). Biomass, root morphology, leaf characteristics, N uptake, and assimilation were determined. Compared with NN treatment, plant growth characteristics, including biomass, growth rate, root and leaf characteristics were all markedly inhibited under AN conditions, resulting in malondialdehyde (MDA) accumulation throughout the plants, except the specific leaf area. Under AN conditions, higher N concentration and free amino acid (FAA) content were observed, while N accumulation, soluble protein content, and the activities of nitrate reductase (NR), glutamine synthetase (GS) and glutamate synthase (GOGAT) in root and leaf were reduced, except for GOGAT in root. Moreover, the trend in these changes was amplified by increasing N supply, and significant interactions between N form and level were also found. Taken together, our results demonstrate that citrus plants are highly ammonium sensitive, the findings provide a theoretical basis for the optimized N management in the high-quality production of citrus.

Little information is available on the molecular mechanisms of boron (B)-induced alleviation of aluminum (Al)-toxicity. 'Sour pummelo' (Citrus grandis) seedlings were irrigated for 18 weeks with nutrient solution containing different concentrations of B (2.5 or 20μM H3BO3) and Al (0 or 1.2mM AlCl3·6H2O). B alleviated Al-induced inhibition in plant growth accompanied by lower leaf Al. We used cDNA-AFLP to isolate 127 differentially expressed genes from leaves subjected to B and Al interactions. These genes were related to signal transduction, transport, cell wall modification, carbohydrate and energy metabolism, nucleic acid metabolism, amino acid and protein metabolism, lipid metabolism and stress responses. The ameliorative mechanisms of B on Al-toxicity might be related to: (a) triggering multiple signal transduction pathways; (b) improving the expression levels of genes related to transport; (c) activating genes involved in energy production; and (d) increasing amino acid accumulation and protein degradation. Also, genes involved in nucleic acid metabolism, cell wall modification and stress responses might play a role in B-induced alleviation of Al-toxicity. To conclude, our findings reveal some novel mechanisms on B-induced alleviation of Al-toxicity at the transcriptional level in C. grandis leaves.

Limited data are available on boron (B)-toxicity-responsive proteins in plants. We first applied 2-dimensional electrophoresis (2-DE) to compare the effects of B-toxicity on leaf protein profiles in B-tolerant Citrus sinensis and B-intolerant Citrus grandis seedlings, and identified 27 (20) protein species with increased abundances and 23 (25) protein species with decreased abundances from the former (latter). Generally speaking, B-toxicity increased the abundances of protein species involved in antioxidation and detoxification, proteolysis, cell transport, and decreased the abundances of protein species involved in protein biosynthesis in the two citrus species. The higher B-tolerance of C. sinensis might include following several aspects: (a) protein species related to photosynthesis and energy metabolism in C. sinensis leaves were more adaptive to B-toxicity than in C. grandis ones, which was responsible for the higher photosynthesis and for the better maintenance of energy homeostasis in the former; and (b) the increased requirement for detoxification of reactive oxygen species and cytotoxic compounds due to decreased photosynthesis was less in B-toxic C. sinensis leaves than in B-toxic C. grandis ones. B-toxicity-responsive protein species involved in coenzyme biosynthesis differed between the two species, which might also contribute to the higher B-tolerance of C. sinensis. Biological significance B-toxicity occurs in many regions all over the world, especially in arid and semiarid regions due to the raising of B-rich water tables with high B accumulated in topsoil. In China, B-toxicity often occurs in some citrus orchards. However, the mechanisms of citrus B-tolerance are still not fully understood. Here, we first used 2-DE to identify some new B-toxicity-responsive-proteins involved in carbohydrate and energy metabolism, antioxidation and detoxification, signal transduction and nucleotide metabolism. Our results showed that proteins involved in photosynthesis and energy metabolism displayed more adaptive to B-toxicity in B-tolerant C. sinensis than in B-intolerant C. grandis, which might play a key role in citrus B-tolerance. Therefore, our results reveal some new mechanisms on plant B-response and tolerance.

Here, we provide the data from a comparative proteomics approach used to investigate the response of boron (B)-tolerant ‘Xuegan’ (Citrus sinensis) and B-intolerant ‘Sour pummelo’ (Citrus grandis) leaves to B-toxicity. Using two-dimensional gel electrophoresis (2-DE) technique, we identified 50 and 45 protein species with a fold change of more than 1.5 and a P-value of less than 0.05 from B-toxic C. sinensis and C. grandis leaves. These B-toxicity-responsive protein species were mainly involved in carbohydrate and energy metabolism, antioxidation and detoxification, stress responses, coenzyme biosynthesis, protein and amino acid metabolism, signal transduction, cell transport, cytoskeleton, nucleotide metabolism, and cell cycle and DNA processing. A detailed analysis of this data may be obtained from Sang et al. (J. Proteomics 114 (2015))[1].

Seedlings of Citrus sinensis were fertilized with boron (B)-deficient (0 μM H 3 BO 3 ) or -sufficient (10 μM H 3 BO 3 ) nutrient solution for 15 weeks. Thereafter, iTRAQ analysis was employed to compare the abundances of proteins from B-deficient and -sufficient roots. In B-deficient roots, 164 up-regulated and 225 down-regulated proteins were identified. These proteins were grouped into the following functional categories: protein metabolism, nucleic acid metabolism, stress responses, carbohydrate and energy metabolism, cell transport, cell wall and cytoskeleton metabolism, biological regulation and signal transduction, and lipid metabolism. The adaptive responses of roots to B-deficiency might include following several aspects: ( a ) decreasing root respiration; ( b ) improving the total ability to scavenge reactive oxygen species (ROS); and ( c ) enhancing cell transport. The differentially expressed proteins identified by iTRAQ are much larger than those detected using 2D gel electrophoresis, and many novel B-deficiency-responsive proteins involved in cell transport, biological regulation and signal transduction, stress responses and other metabolic processes were identified in this work. Our results indicate remarkable metabolic flexibility of citrus roots, which may contribute to the survival of B-deficient plants. This represents the most comprehensive analysis of protein profiles in response to B-deficiency. Biological significance In this study, we identified many new proteins involved in cell transport, biological regulation and signal transduction, stress responses and other metabolic processes that were not previously known to be associated with root B-deficiency responses. Therefore, our manuscript represents the most comprehensive analysis of protein profiles in response to B-deficiency and provides new information about the plant response to B-deficiency. This article is part of a Special Issue entitled: Translational Plant Proteomics.

‘Sour pummelo’ (Citrus grandis) seedlings were irrigated for 18 weeks with nutrient solution containing 0 (−Al) and 1.2 mM (+Al) AlCl3·6H2O × 0, 10 and 500 μM sodium nitroprusside (SNP), a nitric oxide (NO) donor. Leaf malate content did not significantly change in response to SNP with or without aluminum (Al) except for an increase under 1.2 mM Al + 10 μM SNP, while leaf citrate content decreased with increasing SNP supply. Root malate content kept unchanging with or without Al except for an increase under 500 μM SNP in the absence of Al, while SNP-treated roots had a higher or similar citrate content. Al decreased or did not affect malate content in roots and leaves, and citrate content in roots, but increased leaf citrate content. Al-treated roots and leaves displayed lower or similar activities of acid-metabolizing enzymes [phosphoenolpyruvate carboxylase (PEPC), NAD-malate dehydrogenase (NAD-MDH), NADP-malic enzyme (NADP-ME), citrate synthase (CS), aconitase (ACO), NADP-isocitrate dehydrogenase (NADP-IDH), phosphoenolpyruvate phosphatase (PEPP) and pyruvate kinase (PK)] except that they had higher or similar activities of NADP-ME and PK and Al-treated leaves had a higher or similar activity of PEPP. In conclusion, the OA metabolism in leaves and roots is differently affected by NO and Al interactions.

Ethychlozate (ethyl 5-chloro-1H-3-indazolylacetate, Figaron) was applied to 12-year-old ‘Jiefangzhong’ (a high-acid cultivar) loquat (Eriobotrya japonica) trees at 0 (control), 75, 150 and 250 mg L−1 on March 19th, 90 days after flowering. To determine pulp titratable acidity (TA), organic acid concentration and acid-metabolizing enzyme activity, fruits were collected at 0, 13, 25, 42 and 49 days after treatments (DAT), when fruits were ripe. Malate was the major organic acid determining loquat pulp acidity. Ethychlozate was very effective in reducing pulp malate concentration, thus decreasing pulp acidity. The best result for reducing acidity (malate) was obtained when ethychlozate was applied at 250 mg L−1. The ethychlozate-treated pulp showed lower phosphoenolpyruvate carboxylase (PEPC) and NAD-malate dehydrogenase (NAD-MDH) activities through the experimental period, but higher or similar NADP-malic enzyme (NADP-ME) from 25 to 49 DAT compared to non-sprayed control. The reduction in malate concentration in the ethychlozate-treated pulp was probably caused by decreased malate biosynthesis and increased malate degradation.

Changes in organic acid concentration and related enzyme activities in loquat ( Eriobotrya japonica Lindl.) pulp were studied, using low-acid ‘Changhong 3’ and high-acid ‘Jiefangzhong’ cultivars. Both titratable acidity (TA) and malic acid concentration increased during the early stages of fruit development and decreased at the later stages. The difference in TA between the two cultivars could be explained by the difference in malic acid concentration, which could result from a difference in NAD-malate dehydrogenase (NAD-MDH) and NADP-malic enzyme (NADP-ME) activities. Although the difference in malic acid concentration between the two cultivars could not result from a difference in phosphoenolpyruvate carboxylase (PEPC) activity, malic acid concentration in both ‘Changhong 3’ throughout fruit development and ‘Jiefangzhong’ at the early stages increased linearly and curvilinearly with increasing PEPC activity, respectively. Therefore, NAD-MDH, NADP-ME and PEPC activities may play significant roles in malic acid biosynthesis and degradation.

The thermotolerance of the sun-exposed peel and the shaded peel of ‘Fuji’ apple ( Malus domestica Borkh.) fruit was evaluated by measuring pigments, chlorophyll a fluorescence transients and O 2 evolution or uptake after exposure to 25, 35, 40, 42, 44, 46 or 48 °C for 30 min in the dark. A major effect of heat stress at 46–48 °C on the chlorophyll a fluorescence transients was the appearance of a very clear K step at 200–300 μs for both peel types. The K step was slightly more pronounced in the sun-exposed peel than in the shaded peel, suggesting that the resistance of oxygen-evolving complex to heat stress is slightly lower in the sun-exposed peel than in the shaded peel. Minimal fluorescence ( F O ), relative to the value at 25 °C, increased to a greater extent in the shaded peel than in the sun-exposed peel after exposure to 46–48 °C, but the temperature dependencies of F O changes were similar for both peel types. Maximum quantum yield of PSII ( F V / F M ) decreased to a similar extent in the sun-exposed peel and the shaded peel as temperature rose from 25 to 44 °C, but the sun-exposed peel reached slightly lower values at 46–48 °C. Correspondingly, gross O 2 evolution rate, relative to that at 25 °C, was also slightly lower in the sun-exposed peel than in the shaded peel at 46–48 °C. In response to heat stress, the ratio of Q A -reducing reaction centers (RCs) to total RCs and the ratio of Q B -reducing RCs to Q A -reducing RCs decreased, but both of them decreased to lower values in the sun-exposed peel than in the shaded peel at 46–48 °C, indicating that the capacity of electron transfer between P 680 + and Q B via Q A was damaged to a greater extent in the sun-exposed peel than in the shaded peel. At each given temperature, dark respiration was similar between the two peel types. Overall, it appears that the exposure to higher surface temperature under high light does not make the sun-exposed peel more tolerant of heat stress than the shaded peel of apple fruit.

Seedlings of Citrus grandis were fertilized every other days for 15 weeks with nutrient solution containing 0 (deficiency), 10 μM (control) or 500 μM (excess) H3BO3. CO2 assimilation and chlorophyll (Chl) content decreased to a greater degree in B-deficient than in B-excess leaves, but photosynthetic enzyme activities were similarly decreased. Starch accumulated in B-deficient leaves, but not in B-excess ones. Chlorophyll a fluorescence transient showed that the positive L- and K-steps were more pronounced in B-excess than in B-deficient leaves. Maximum quantum yield of primary photochemistry (Fv/Fm), maximum variable fluorescence (Fv), oxygen-evolving complex (OEC) were less decreased in B-deficient than in B-excess leaves, whereas minimum fluorescence (F0) was less increased in B-deficient leaves. Boron-deficient leaves displayed higher or similar antioxidant enzyme activities and higher ascorbate (AsA) and reduced glutathione (GSH) contents compared to B-excess leaves. Content of thiobarbituric acid (TBA) reactive compounds was less increased by B-deficiency than by B-excess. We conclude that B-deficient leaves are less damaged by oxidative stress than B-excess leaves due to their higher ability to scavenge reactive oxygen species. Both the donor (i.e. the OEC) and the acceptor sides of photosystem II were less photoinhibited by B-deficiency than by B-excess. The greater decrease in CO2 assimilation and Chl content in B-deficient leaves may be caused by the excessive accumulation of starch. The reduction of CO2 assimilation by B-excess is probably caused by a combination of factors such as oxidative damage, reduced photosynthetic enzyme activities and impaired electron transport capacity.

Seedlings of sweet orange (Citrus sinensis) were fertilized for 14 weeks with boron (B)-free or B-sufficient (2.5 or 10 microM H(3)BO(3)) nutrient solution every other day. Boron deficiency resulted in an overall inhibition of plant growth, with a reduction in root, stem and leaf dry weight (DW). Boron-starved leaves showed decreased CO(2) assimilation and stomatal conductance, but increased intercellular CO(2) concentrations. Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) and stromal fructose-1,6-bisphosphatase (FBPase) were lower in B-deficient leaves than in controls. Contents of glucose, fructose and starch were increased in B-deficient leaves while sucrose was decreased. Boron-deficient leaves displayed higher or similar superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) activities, while dehydroascorbate reductase (DHAR) and catalase (CAT) activities were lower. Expressed on a leaf area or protein basis, B-deficient leaves showed a higher ascorbate (AsA) concentration, but a similar AsA concentration on a DW basis. For reduced glutathione (GSH), we found a similar GSH concentration on a leaf area or protein basis and an even lower content on a DW basis. Superoxide anion (O(2)(-)) generation, malondialdehyde (MDA) concentration and electrolyte leakage were higher in B-deficient than in control leaves. In conclusion, CO(2) assimilation may be feedback-regulated by the excessive accumulation of starch and hexoses in B-deficient leaves via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes. Although B-deficient leaves remain high in activity of antioxidant enzymes, their antioxidant system as a whole does not provide sufficient protection from oxidative damage.