Search

Over-application of copper (Cu) is prevalent in citrus orchards of South China. Therefore, the evaluation of citrus Cu tolerance is of significance for better nutrient management and in the breeding of Cu-tolerant citrus species. In this study, seedlings of ‘Xuegan’ [Citrus sinensis (L.) Osbeck] and ‘Shatian pummelo’ [Citrus grandis (L.) Osbeck] were treated with 0.5 µM Cu (Control) or 300 µM Cu (Cu toxicity) for 18 weeks in sandy culture medium. The results demonstrated that excess Cu decreased seedling biomass, plant height, and root length and significantly increased Cu accumulation of C. sinensis and C. grandis. The Cu distribution in citrus tissues was following the order: lateral roots > primary roots > stems > leaves. Compared to C. sinensis, C. grandis had a 22.4% higher Cu concentration in the lateral roots under Cu toxicity. According to the stomata limiting theory, the inhibition of the net photosynthetic rate of C. sinensis leaves by Cu toxicity was mainly attributed to non-stomata limitation. The chlorophyll a transient indicated much more severe damage to the structure and function of leaf photosynthetic system II (PS II) in C. sinensis than C. grandis by Cu toxicity. Conclusively, a higher Cu retention in the lateral roots, the maintenance of a stable PS II structure, and higher electron transferring rate from PS II to PS I implied the relatively higher Cu tolerance of C. grandis seedlings. These results provide essential information for further investigation on the Cu-tolerant mechanisms of citrus species and the future breeding of Cu-tolerant citrus species.

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.

Copper (Cu)-toxic effects on Citrus grandis growth and Cu uptake, as well as gene expression and physiological parameters in leaves were investigated. Using RNA-Seq, 715 upregulated and 573 downregulated genes were identified in leaves of C. grandis seedlings exposed to Cu-toxicity (LCGSEC). Cu-toxicity altered the expression of 52 genes related to cell wall metabolism, thus impairing cell wall metabolism and lowering leaf growth. Cu-toxicity downregulated the expression of photosynthetic electron transport-related genes, thus reducing CO2 assimilation. Some genes involved in thermal energy dissipation, photorespiration, reactive oxygen species scavenging and cell redox homeostasis and some antioxidants (reduced glutathione, phytochelatins, metallothioneins, l-tryptophan and total phenolics) were upregulated in LCGSEC, but they could not protect LCGSEC from oxidative damage. Several adaptive responses might occur in LCGSEC. LCGSEC displayed both enhanced capacities to maintain homeostasis of Cu via reducing Cu uptake by leaves and preventing release of vacuolar Cu into the cytoplasm, and to improve internal detoxification of Cu by accumulating Cu chelators (lignin, reduced glutathione, phytochelatins, metallothioneins, l-tryptophan and total phenolics). The capacities to maintain both energy homeostasis and Ca homeostasis might be upregulated in LCGSEC. Cu-toxicity increased abscisates (auxins) level, thus stimulating stomatal closure and lowering water loss (enhancing water use efficiency and photosynthesis).

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.

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.

Background In this study, we aimed to test the hypothesis that magnesium (Mg) remobilization in citrus plants is regulated by Mg supply and contributes to differences in the growth of the parent and branch organs. Citrus seedlings were grown in sand under Mg deficient (0 mmol Mg2+ L−1, -Mg) and Mg sufficient (2 mmol Mg2+ L−1, + Mg) conditions. The effects on biomass, Mg uptake and transport, gas exchange and chlorophyll fluorescence, as well as related morphological and physiological parameters were evaluated in different organs. Results Mg deficiency significantly decreased plant biomass, with a decrease in total plant biomass of 39.6%, and a greater than twofold decrease in the branch organs compared with that of the parent organs. Reduced photosynthesis capacity was caused by a decreased in pigment levels and photosynthetic electron transport chain disruption, thus affecting non-structural carbohydrate accumulation and plant growth. However, the adaptive responses of branch leaves to Mg deficiency were greater than those in parent leaves. Mg deficiency inhibited plant Mg uptake but enhanced Mg remobilization from parent to branch organs, thus changing related growth variables and physiological parameters, including protein synthesis and antioxidant enzyme activity. Moreover, in the principal components analysis, these variations were highly clustered in both the upper and lower parent leaves, but highly separated in branch leaves under the different Mg conditions. Conclusions Mg deficiency inhibits the growth of the parent and branch organs of citrus plants, with high Mg mobility contributing to differences in physiological metabolism. These findings suggest that Mg management should be optimized for sustainable citrus production.

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 &mu, M 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.

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.

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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12870-019-1683-4) contains supplementary material, which is available to authorized users.

Figure S1. Mg-deficiency effects on Citrus sinensis seedling growth (a) and Mg-deficient symptoms in the upper and lower leaves (b). Table S1. PCA for physiological parameters of upper leaves. Table S2. PCA for physiological parameters of lower leaves. Table S3. PCA for physiological parameters of roots. (DOCX 447 kb)

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.

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.

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.

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.

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].

Two novel DREB (dehydration-responsive element-binding protein) genes, designated as CiDREB1A and CiDREB1B, were cloned from chicory (Cichorium intybus). Both of these genes contained a conserved EREBP/AP2 domain and were classified into the A-1 subgroup of the DREB subfamily based on phylogenetic analysis. Quantitative real-time PCR analysis revealed that low temperature, but not ABA, greatly induced the expression of both CiDREB1 genes, suggesting that these genes are involved in ABA-independent stress signaling pathways. A subcellular localization assay revealed that both CiDREBs localized to the nucleus. In addition, we showed by yeast one-hybrid analysis that these two CiDREB proteins bind to the DRE motif of RD19A. These results suggest that CiDREB1A and CiDREB1B are important regulators of stress-responsive signaling in chicory.

In order to evaluate differential growth, photosynthesis and H + -ATPase activity responses to salt-induced stress, two Jerusalem artichoke ( Helianthus tuberosus L.) genotypes (Nanyu No. 1 and Qingyu No. 2) were used in sand-culture experiment with different concentrations of NaCl (0, 30, 60, 90, 120 and 150 mM). After 20 days of growth, the NaCl stress resulted in a decrease of biomass accumulation, relative leaf expansion rate and photosynthetic rate, but an increase of proline content in both genotypes. Compared with Qingyu No. 2, Nanyu No. 1 had lower biomass, photosynthetic rate, gas exchange and transpiration rate, but higher proline content, activities of plasma membrane H + -ATPase (PM H + -ATPase) and vacuolar membrane H + -ATPase (VM H + -ATPase). Hence, the NaCl adaptation strategy in Nanyu No. 1 was by lowering photosynthetic rate, stomatal conductance and transpiration rate while maintaining high H + -ATPase activities, whereas the adjustment of Qingyu No. 2 was by keeping much higher rate of proline accumulation and concentration of chlorophyll. The differences in salt tolerance showed that different adaptation mechanisms existed between cultivars of Jerusalem artichoke. The findings offered the possibility of selecting salt-tolerant genotypes of Jerusalem artichoke.

To explore genetic variability for two Jerusalem artichoke ( Helianthus tuberosus ) cultivars, N1 (the sixth-generation cultivated with 75% seawater irrigation for six years) and N7 (a general variety), a experiment was conducted to study the changes in physiological attributes under different concentrations (0%, 10% and 25% of seawater concentration in greenhouse and 0%, 30% and 50% of seawater concentration in the field) of seawater salinity stress. In the greenhouse experiment, decreases of dry growth rate, but increases of dry matter percentage and membrane injury occurred in both the genotypes at 10% and 25% seawater treatments, although lesser cell membrane damage was observed in N1 than N7. N1 accumulated greater contents of Na + , Cl − , soluble sugar and proline in leaves compared with N7. In the field experiment, the yields of shoot, root and tuber, and the contents of total-sugar and inulin in tubers of N1 were higher than those of N7. Lesser degree of salt injury in N1 indicated that the relatively salt-tolerant cultivar had higher K + /Na + ratio, lower Na + /Ca 2+ ratio, and the salt-induced enhancement of osmotic adjustment.

Two Jerusalem artichoke (Helianthus tuberosus L.) genotypes, NY-1 and NY-7, were subjected to different seawater concentrations (0, 10, 20, 30, 40, and 50%) for various periods of time to determine the effects on seedling growth, ion content, and photosynthetic productivity in a greenhouse. Under different seawater concentrations, sprouting rates varied greatly among the genotypes. The differences in relative growth rate (RGR), leaf chlorophyll content, total leaf area (TLA), plant dry weight (PDW), photosynthetic rate (A), stomatal conductance (gs), and efficiency of the light harvesting of photosystem II (Fv′/Fm′) were significant between NY-1 and NY-7 after 12 days of stress at 40 and 50% seawater. Seawater treatments resulted in the reduction of almost all the growth parameters and coincident increases of Na+ and Ca2+ concentrations in plant tissues. Our results indicate that there is great variability for seawater tolerance among H. tuberosus varieties, and that greater photosynthesis capacity, higher RGR, and relatively higher tissue Na+ accumulation at high seawater concentrations appears to be associated with seawater tolerance in H. tuberosus varieties.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2133-9) contains supplementary material, which is available to authorized users.

Effects of Al-toxicity on plant growth and root Al concentration in Citrus sinensis and C. grandis seedlings. (DOC 184 kb)

List of differentially expressed proteins in Al-toxicity Citrus sinensis (CS) and C. grandis (CG) roots. (DOC 418 kb)

Specific primer pairs used for qRT-PCR expression analysis. (DOC 40 kb)

OBJECTIVE: It is known that the vacuolating cytotoxin (VacA) could induce apoptosis. However, the mechanism remained to be elucidated. The aim of this study is to investigate the role of Bcl family of proteins (Bcl-2 and Bax) and the mitochondrial voltage-dependent anion channel (VDAC) in VacA-induced apoptosis of AGS cells. METHODS: Plasmid pGBKT7-VacA p58 was constructed and transfected into the AGS cells. RT-PCR and Western blotting were used to determine the expressions of cytochrome c, caspase-3, Bax, Bcl-2 and VDAC1 mRNA and proteins. RESULTS: VacA p58 can induce cytochrome c release and activate caspase-3 in AGS cells. It up-regulated the expressions of Bax and VDAC1 mRNA and proteins, and decreased the expression of Bcl-2 in AGS cells. CONCLUSION: VacA p58 induces apoptosis in AGS cells. This apoptotic process is associated with the up-regulation of Bax/VDAC1 and downregulation of Bcl-2. These findings suggest that the release of cytochrome c by VacA p58 is mainly through VDAC-dependent and Bcl-2 family-dependent pathways.

Background Boron (B)-deficiency is a widespread problem in many crops, including Citrus. MicroRNAs (miRNAs) play important roles in nutrient deficiencies. However, little is known on B-deficiency-responsive miRNAs in plants. In this study, we first identified miRNAs and their expression pattern in B-deficient Citrus sinensis roots by Illumina sequencing in order to identify miRNAs that might be involved in the tolerance of plants to B-deficiency. Results We isolated 52 (40 known and 12 novel) up-regulated and 82 (72 known and 10 novel) down-regulated miRNAs from B-deficient roots, demonstrating remarkable metabolic flexibility of roots, which might contribute to the tolerance of plants to B-deficiency. A model for the possible roles of miRNAs in the tolerance of roots to B-deficiency was proposed. miRNAs might regulate the adaptations of roots to B-deficiency through following several aspects: (a) inactivating reactive oxygen species (ROS) signaling and scavenging through up-regulating miR474 and down-regulating miR782 and miR843; (b) increasing lateral root number by lowering miR5023 expression and maintaining a certain phenotype favorable for B-deficiency-tolerance by increasing miR394 expression; (c) enhancing cell transport by decreasing the transcripts of miR830, miR5266 and miR3465; (d) improving osmoprotection (miR474) and regulating other metabolic reactions (miR5023 and miR821). Other miRNAs such as miR472 and miR2118 in roots increased in response to B-deficiency, thus decreasing the expression of their target genes, which are involved in disease resistance, and hence, the disease resistance of roots. Conclusions Our work demonstrates the possible roles of miRNAs and related mechanisms in the response of plant roots to B-deficiency.

Proline accumulation is an important mechanism for osmotic regulation under salt stress. In this study, we evaluated proline accumulation profiles in roots, stems and leaves of Jerusalem artichoke (Helianthus tuberosus L.) plantlets under NaCl stress. We also examined HtP5CS, HtOAT and HtPDH enzyme activities and gene expression patterns of putative HtP5CS1, HtP5CS2, HtOAT, HtPDH1, and HtPDH2 genes. The objective of our study was to characterize the proline regulation mechanisms of Jerusalem artichoke, a moderately salt tolerant species, under NaCl stress. Jerusalem artichoke plantlets were observed to accumulate proline in roots, stems and leaves during salt stress. HtP5CS enzyme activities were increased under NaCl stress, while HtOAT and HtPDH activities generally repressed. Transcript levels of HtP5CS2 increased while transcript levels of HtOAT, HtPDH1 and HtPDH2 generally decreased in response to NaCl stress. Our results supports that for Jerusalem artichoke, proline synthesis under salt stress is mainly through the Glu pathway, and HtP5CS2 is predominant in this process while HtOAT plays a less important role. Both HtPDH genes may function in proline degradation.