Bordeaux mixture (Bm) is a copper (Cu)-based pesticide that has been widely used for controlling citrus scab and citrus canker. However, frequent spraying of Bm is toxic to citrus. To our knowledge, few studies are available that discuss how the photosynthetic characteristics and chloroplast ultrastructure of citrus leaves are affected by Cu toxicity induced by excessive Bm. In the study, two-year-old seedlings of Citrus grandis (C. grandis) and Citrus sinensis (C. sinensis), which were precultured in pots, were foliar-sprayed with deionized water (as control) or Bm diluted 500-fold at intervals of 7 days for 6 times (4 times as recommended by the manufacturer) to investigate the leaf Cu absorption, photosynthesis, chloroplast ultrastructure and antioxidant enzymatic activities. Bm foliar-sprayed 6 times on citrus seedlings increased the leaf Cu content, decreased the photosynthetic pigments content and destroyed the chloroplast ultrastructure, which induced leaf chlorosis and photosynthetic inhibition. A lower Cu absorption, a higher light photon-electron transfer efficiency, a relative integrity of chloroplast ultrastructure and a promoted antioxidant protection contributed to a higher photosynthetic activity of C. grandis than C. sinensis under excessive spraying of Bm. The present study provides crucial references for screening and selecting citrus species with a higher tolerance to Cu toxicity induced by excessive Bm.
Over-applied copper (Cu)-based agrochemicals are toxic to citrus trees. However, less information is available discussing the ultrastructural alterations in Cu-stressed citrus species. In the present study, seedlings of Citrus sinensis and Citrus grandis that differed in Cu-tolerance were sandy-cultured with nutrient solution containing 0.5 µM Cu (as control) or 300 µM Cu (as Cu toxicity) for 18 weeks. At the end of the treatments, the physiological parameters and ultrastructural features of the citrus leaves and roots were analyzed. The results indicate that Cu toxicity significantly decreased the ratio of shoot biomass to dry weight, the Cu translocation factor and the total chlorophyll of two citrus species. The anatomical and ultrastructural alterations verified that excessive Cu resulted in starch granules accumulated in the leaves and roots of the two citrus species. Under Cu toxicity, increased root flocculent precipitate and thickened root cell wall might reduce the Cu translocation from citrus roots to the shoots. Compared with C. sinensis, C. grandis maintained a relatively integral root cellular structure under Cu toxicity, which provided a structural basis for a higher Cu tolerance than C. sinensis. The present results increase our understanding of the physiological and ultrastructural responses to Cu toxicity in citrus species.
Excessive copper (Cu) of rhizosphere inhibited the growth and development of citrus seedlings. Lignin deposition on the cell wall promotes plant Cu tolerance. However, the lignin biosynthesis in citrus leaves and roots that respond to Cu toxicity is not fully understood. In this study, young seedlings of 'Xuegan' [Citrus sinensis (L.) Osbeck, a less Cu-tolerant species] and 'Shatian pomelo' [Citrus grandis (L.) Osbeck, a more Cu-tolerant species] were treated with nutrient solution containing 0.5 (as Control), 100, 300 or 500 µM Cu for 15 weeks in sandy culture. By the end of treatments, citrus leaves and roots were sampled to investigate the biomass allocation, Cu distribution, the lignin biosynthesis and deposition. The results indicated that Cu stress from 100 to 500 µM increased the root/shoot biomass ratio, promoting Cu and lignin accumulation in the leaves and roots of the tested citrus species. Besides, 300 µM Cu stress increased the accumulation of three lignin monomers of citrus species. The metabolomic profile indicated that Cu toxicity altered the lignin components of citrus species. The citrus roots are more prominent in the lignin precursor biosynthesis under Cu toxicity than citrus leaves. The histochemical staining supported that Cu stress improved the deposition of both guaiacy and syringy lignin units in citrus roots. The enzyme activity and gene expression revealed that activating lignin-biosynthetic enzymes, such as L-phenylalanine ammonia-lyase, peroxidase and laccase, played an essential role in lignin biosynthesis. Our results demonstrated that excessive Cu induced lignin biosynthesis in citrus leaves and roots to different extents. The findings from the present study increased our understanding of lignin biosynthesis in Cu-stressed citrus species, which would provide a theoretical basis for the citrus Cu-tolerant mechanisms.
Excessive copper (Cu) has become a common physiological disorder restricting the sustainable production of citrus. Coumarin (COU) is a hydroxycinnamic acid that can protect plants from heavy metal toxicity. No data to date are available on the ameliorative effect of COU on plant Cu toxicity. ‘Xuegan’ (Citrus sinensis (L.) Osbeck) seedlings were treated for 24 weeks with nutrient solution containing two Cu levels (0.5 (Cu0.5) and 400 (Cu400) μM CuCl2) × four COU levels (0 (COU0), 10 (COU10), 50 (COU50), and 100 (COU100) μM COU). There were eight treatments in total. COU supply alleviated Cu400-induced increase in Cu absorption and oxidative injury in roots and leaves, decrease in growth, nutrient uptake, and leaf pigment concentrations and CO2 assimilation (ACO2), and photo-inhibitory impairment to the whole photosynthetic electron transport chain (PETC) in leaves, as revealed by chlorophyll a fluorescence (OJIP) transient. Further analysis suggested that the COU-mediated improvement of nutrient status (decreased competition of Cu2+ with Mg2+ and Fe2+, increased uptake of nutrients, and elevated ability to maintain nutrient balance) and mitigation of oxidative damage (decreased formation of reactive oxygen species and efficient detoxification system in leaves and roots) might lower the damage of Cu400 to roots and leaves (chloroplast ultrastructure and PETC), thereby improving the leaf pigment levels, ACO2, and growth of Cu400-treated seedlings.
Citrus species are prone to suffer from copper (Cu) toxicity because of improper application of Cu-based agrochemicals. Copper immobilization mediated by pectin methylesterase (PME) in the root cell wall (CW) is effective for Cu detoxification. However, the underlying mechanisms of the structural modification and stress responses of citrus root CW pectin to Cu toxicity have been less discussed. In the present study, seedlings of 'Shatian pummelo' (Citrus grandis L. Osbeck) and 'Xuegan' (Citrus sinensis L. Osbeck), which differ in Cu tolerance, were irrigated with nutrient solution containing 0.5 (as control), 100, 300 or 500 μM Cu for 18 weeks in sandy culture or 24 h in hydroponics. At the end of treatments in the 18-week sandy culture, Cu toxicity on CW pectin content, Cu distribution, degree of pectin methylesterification (DPM) and the PME enzyme activity were discussed. At the genome-wide level, PME gene family was identified from the two citrus species, and qRT-PCR array of citrus PMEs under control and 300 μM Cu stress for 18 weeks were performed to screen the Cu-responsive PME genes. Moreover, the candidate genes that responded to Cu toxicity were further examined within 24 h. The results showed that Cu toxicity increased the root CW pectin content. The root CW pectin under Cu toxicity was remodeled by upregulation of the expression of the Cu-responsive PME genes followed by increasing PME activity, which mainly promoted low methylesterased pectin level and the Cu content on root CW pectin. Compared with C. sinensis, C. grandis root CW had a lower DPM and higher Cu content on the Cu-stressed root CW pectin, contributing to its higher Cu tolerance. Our present study provided theoretical evidence for root CW pectin remodeling in response to Cu toxicity of citrus species.
Most commercial citrus fruits are grown in acidic soils with high copper (Cu) and low organic matter levels in China. Sweet orange (Citrus sinensis (L.) Osbeck cv. Xuegan) seedlings were treated with 0 (HA0), 0.1 (HA0.1), or 0.5 (HA0.5) mM humic acid (HA) and 0.5 (Cu0.5) or 400 (Cu400 or Cu excess) μM CuCl2 for 24 weeks. The purpose was to validate the hypothesis that HA reduces the oxidative injury caused by Cu400 in roots and leaves via the coordination of strengthened antioxidant defense and glyoxalase systems. Copper excess increased the superoxide anion production rate by 27.0% and 14.2% in leaves and by 47.9% and 33.9% in roots, the malonaldehyde concentration by 199.6% and 27.8% in leaves and by 369.4% and 77.4% in roots, and the methylglyoxal concentration by 18.2% and 6.6% in leaves and by 381.8% and 153.3% in roots, as well as the H2O2 production rate (HPR) by 70.5% and 16.5% in roots, respectively, at HA0 and HA0.5. Also, Cu400 increased the leaf HPR at HA0, but not at HA0.5. The addition of HA reduced the Cu400-induced production and accumulation of reactive oxygen species and methylglyoxal and alleviated the impairment of Cu400 to the antioxidant defense system (ascorbate-glutathione cycle, antioxidant enzymes, sulfur-containing compounds, and sulfur-metabolizing enzymes) and glyoxalase system in roots and leaves. The HA-mediated amelioration of Cu toxicity involved reduced oxidative injury due to the coordination of strengthened antioxidant defense and glyoxalase systems. These findings highlight the promise of HA for sustainable citrus cultivation in heavy metal (Cu)-polluted soils.
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) μM CuCl2 and 2.5 (B2.5) or 25 (B25) μM 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 μM 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 μM 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.
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