In China, high copper (Cu) and low organic matter often occur in some citrus orchard soils. However, the underlying mechanisms by which humic acid (HA) stimulates growth and mitigates Cu toxicity of citrus seedlings are unclear. After being treated with 0, 0.1, or 0.5 mM sodium humate and 0.5 or 400 mu M CuCl2 (Cu excess) for 24 weeks, sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] seedlings were used to examine the impacts of HA-Cu interactions on seedling growth, nutrient uptake, leaf pigments, and photosynthetic performance that was revealed by chlorophyll a fluorescence transient. Copper excess reduced root, stem, and leaf dry weight (DW) by 42.4%, 65.4%, and 61.6%, respectively at 0 mM HA, and by 17.3%, 25.4%, and 31.4%, respectively at 0.5 mM HA; and that the levels of Cu in leaves, stems, and roots declined with elevating HA supply. Copper excess caused some rotten and dead fibrous roots at 0 mM HA, but not at 0.5 mM HA. Adding HA lowered Cu uptake per root DW (UPR), the levels of Cu in leaves, stems, and roots, and the competition of Cu2+ with Mg2+ and Fe2+, and therefore mitigated root impairment caused by Cu excess. The HA-mediated alleviation of root damage caused by Cu excess increased the uptake per plant and UPR of nitrogen, potassium, magnesium, phosphorus, calcium, sulfur, boron, and manganese, and therefore alleviated Cu excess-induced decline in seedling growth, impairment to leaf photosynthetic electron transport chains, and decrease in leaf pigments. For 0.5 mu M Cu-treated seedlings, adding HA promoted seedling growth by improving root nutrient uptake and leaf photosynthetic performance. Cu excess aggravated the impacts of HA supplementation on seedling growth, leaf photosynthetic performance, and root nutrient uptake.

Both copper (Cu) excess and boron (B) deficiency are often observed in some citrus orchard soils. The molecular mechanisms by which B alleviates excessive Cu in citrus are poorly understood. Seedlings of sweet orange (Citrus sinensis (L.) Osbeck cv. Xuegan) were treated with 0.5 (Cu0.5) or 350 (Cu350 or Cu excess) mu M CuCl2 and 2.5 (B2.5) or 25 (B25) mu M HBO3 for 24 wk. Thereafter, this study examined the effects of Cu and B treatments on gene expression levels revealed by RNA-Seq, metabolite profiles revealed by a widely targeted metabolome, and related physiological parameters in leaves. Cu350 upregulated 564 genes and 170 metabolites, and downregulated 598 genes and 58 metabolites in leaves of 2.5 mu M B-treated seedlings (LB2.5), but it only upregulated 281 genes and 100 metabolites, and downregulated 136 genes and 40 metabolites in leaves of 25 mu M B-treated seedlings (LB25). Cu350 decreased the concentrations of sucrose and total soluble sugars and increased the concentrations of starch, glucose, fructose and total nonstructural carbohydrates in LB2.5, but it only increased the glucose concentration in LB25. Further analysis demonstrated that B addition reduced the oxidative damage and alterations in primary and secondary metabolisms caused by Cu350, and alleviated the impairment of Cu350 to photosynthesis and cell wall metabolism, thus improving leaf growth. LB2.5 exhibited some adaptive responses to Cu350 to meet the increasing need for the dissipation of excessive excitation energy (EEE) and the detoxification of reactive oxygen species (reactive aldehydes) and Cu. Cu350 increased photorespiration, xanthophyll cycle-dependent thermal dissipation, nonstructural carbohydrate accumulation, and secondary metabolite biosynthesis and abundances; and upregulated tryptophan metabolism and related metabolite abundances, some antioxidant-related gene expression, and some antioxidant abundances. Additionally, this study identified some metabolic pathways, metabolites and genes that might lead to Cu tolerance in leaves.

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 mu M Cu (Cu excess or Cu exposure) and 2.5, 10, or 25 mu M 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 mu M B-treated seedlings, while it only increased the malondialdehyde level by 48.5% (97.8%) in leaves (roots) of 25 mu M 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.

Nitrogen-deficiency (ND) usually occurs in some citrus orchard soils in China. The roles of reactive oxygen species (ROS) and methylglyoxal (MG) generation and their detoxification systems in ND tolerance of horticultural woody plants still need to be revealed. For the first time, we examined the effects of ND on ROS and MG generation and their detoxification systems in leaves and roots of Citrus sinensis seedlings. The objectives are to test the hypotheses that N-deficient leaves and roots can keep high abilities to scavenge ROS and MG, thereby protecting them from oxidative damage, and that ND-induced alterations of ROS and MG formation and their detoxification systems in leaves and roots are different. ND augmented superoxide anion production rate and MG concentrations, but it decreased malondialdehyde (MDA) concentrations and electrolyte leakage in leaves and roots. ND increased the activities of most enzymes involved in ROS (ascorbate-glutathione cycle-related enzymes, antioxidant enzymes, and sulfur metabolism-related enzymes) and MG (glyoxalases) detoxification expressed on a protein basis with a few exceptions, and the concentrations of ascorbate, phytochelatins, and total non-protein thiols in leaves and roots. These results suggested that nitrogen-deficient leaves and roots could keep high abilities to detoxify ROS and MG, and protect them from oxidative damage. Generally viewed, ND affected the production and removal of ROS and MG more in roots than in leaves.