TY - JOUR
AU1 - Wan,, Huixue
AU2 - Du,, Jiayi
AU3 - He,, Jiali
AU4 - Lyu,, Deguo
AU5 - Li,, Huifeng
AB - Abstract To unravel the physiological and molecular regulation mechanisms underlying the variation in copper (Cu)accumulation, translocation and tolerance among five apple rootstocks, seedlings were exposed to either basal or excess Cu. Excess Cu suppressed plant biomass and root architecture, which was less pronounced in Malus prunifolia Borkh., indicating its relatively higher Cu tolerance. Among the five apple rootstocks, M. prunifolia exhibited the highest Cu concentration and bio-concentration factor in roots but the lowest translocation factor, indicating its greater ability to immobilize Cu and restrict translocation to the aerial parts. Higher Cu concentration in cell wall fraction but lower Cu proportion in membrane-containing and organelle-rich fractions were found in M. prunifolia. Compared with the other four apple rootstocks under excess Cu conditions, M. prunifolia had a lower increment of hydrogen peroxide in roots and leaves and malondialdehyde in roots, but higher concentrations of carbohydrates and enhanced antioxidants. Transcript levels of genes involved in Cu uptake, transport and detoxification revealed species-specific differences that are probably related to alterations in Cu tolerance. M. prunifolia had relatively higher gene transcript levels including copper transporters 2 (COPT2), COPT6 and zinc/iron-regulated transporter-related protein 2 (ZIP2), which probably took part in Cu uptake, and C-type ATP-binding cassette transporter 2 (ABCC2), copper chaperone for Cu/Zn superoxide dismutase (CCS), Cu/Zn superoxide dismutase 1 (CSD1) and metallothionein 2 (MT2) probably implicated in Cu detoxification, and relatively lower mRNA levels of yellow stripe-like transporter 3 (YSL3) and heavy metal ATPase 5 (HMA5) involved in transport of Cu to aerial parts. These results suggest that M. prunifolia is more tolerant to excess Cu than the other four apple rootstocks under the current experimental conditions, which is probably attributed to more Cu retention in roots, subcellular partitioning, well-coordinated antioxidant defense mechanisms and transcriptional expression of genes involved in Cu uptake, translocation and detoxification. Introduction Long-term use of urban composts, copper (Cu)-based fungicides, ripening agents and wastewater irrigation have resulted in increasing Cu accumulation in orchard soils (Komarek et al. 2010, Liu et al. 2011, Girotto et al. 2014). Li et al. (2005) found that Cu content in apple orchard soil increased substantially due to the application of more than 16 kg ha−1 Cu per year, ranging from 2.5 to 9 mg Cu kg−1 soil. In Liaodong Peninsula of China, 5.56% of the soil samples from 48 investigated apple orchards exceeded the allowable levels of Cu (150 mg kg−1, dry weight), with the maximum concentration of 229 mg kg−1 (Wang et al. 2015a). Copper contamination is not only a serious environmental problem in many parts of the world but also is responsible for reductions in agricultural productivity (Komarek et al. 2010, Fan et al. 2011). Excessive amounts of Cu in orchard soils can impair the growth of roots and shoots, restrain photosystem II activity, inhibit protein synthesis and enzyme activities, reduce carbohydrate concentrations and induce plant senescence and even death, resulting in lower fruit productivity (Liu et al. 2011, Chen et al. 2013, Cambrolle et al. 2015, Wang et al. 2015b, Brunetto et al. 2016, Hippler et al. 2016). Previous studies have demonstrated that Cu uptake, compartmentalization and tolerance characteristics are highly variable among plant species or even among cultivars within a species (Hamed et al. 2017, Cao et al. 2018, Lange et al. 2018), which makes it possible and feasible to select crops with relatively high Cu tolerance. Rootstocks of horticultural crops regulate plant growth and development and affect Cu uptake, accumulation and toxicity through ion exclusion or retention (Liu et al. 2011, Hippler et al. 2016). Progress has been made in demonstrating the physiological mechanisms of excess Cu uptake, accumulation and tolerance in rootstocks of grapevine (Cambrolle et al. 2015, Marastoni et al. 2019) and citrus (Hippler et al. 2016). To our knowledge, however, relatively little literature is currently available on Cu accumulation and tolerance in apple rootstocks. To survive in Cu-polluted soil, plants have evolved physiological and transcriptional regulatory mechanisms to avoid Cu toxicity, which include the regulation of Cu uptake, chelation, efflux and sequestration (Printz et al. 2016, Wang et al. 2016). In recent years, studies have shown that subcellular distribution in plants is extremely important for Cu accumulation, migration and detoxification (Mwamba et al. 2016). Previous studies have explored the subcellular distribution of Cu in various cultivated plants such as maize (Niu et al. 2012), tomato (Dong et al. 2013), castor (Kang et al. 2015) and oilseed rape (Mwamba et al. 2016). However, the results have not been consistent. For example, cell wall immobilization played an important role in Cu detoxification in apple trees (Wang et al. 2016) and castor beans (Huang et al. 2016), while the alleviation of Cu toxicity in tomatoes (Dong et al. 2013) was mainly associated with vacuolar sequestration. These inconsistent results may be ascribed to species-dependent processes, thus driving the interest to determine the subcellular distribution of Cu in different apple rootstocks, which is crucial for the selection of high Cu tolerance species. In plants, one of the important effects of excess Cu toxicity is the induction of reactive oxygen species (ROS) such as superoxide (O2·−) and hydrogen peroxide (H2O2) (Thounaojam et al. 2012, Shahid et al. 2014). The increased production of ROS disturbs the redox balance in cells, leading to lipid peroxidation, biological macromolecule deterioration, ion leakage and DNA strand cleavage (He et al. 2011, Farmer and Mueller 2013, Shahid et al. 2014). To prevent Cu-induced ROS injuries, plants have evolved several protective mechanisms such as inducing carbohydrates and enhancing non-enzymatic and enzymatic systems (Mellado et al. 2012, Mostofa et al. 2015, Cao et al. 2018). Total soluble sugar participates in plant osmotic conditioning, membrane lipid biosynthesis and oxidative detoxification under heavy metal (HM) stress (He et al. 2013a, Cao et al. 2018). The non-enzymatic metabolites including free proline, soluble phenols, ascorbate (ASC), total thiols (T-SH) and glutathione (GSH) are important for scavenging ROS (Yadav et al. 2017, Zhou et al. 2017). In addition, antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), also play important roles in scavenging ROS and reducing its deleterious effects (Thounaojam et al. 2012, Shahid et al. 2014). However, to date, few studies have been carried out to investigate the physiological response of different apple rootstocks to excess Cu stress in terms of variation in Cu toxicity and detoxification. Copper uptake, translocation and detoxification are controlled by many genes that encode proteins involved in these processes (Yruela 2009, Branzini et al. 2012). Copper exists as the divalent cation (Cu2+) and cuprous copper (Cu+) in its natural condition (Yruela 2009). Before being absorbed by plants, Cu2+ in solution can be reduced to Cu+ by two ferric reductase oxidases (FRO4 and FRO5), and then Cu+ would be transported across the plasma membrane by the high-affinity copper transporters, COPT1, COPT2 and COPT6 (Jung et al. 2012, Li et al. 2018, Sanz et al. 2018). Alternatively, Cu2+ in solution may directly enter root cells through low-affinity divalent cation transporters with low specificity, such as zinc/iron-regulated transporter-related protein 2 (ZIP2) and ZIP4 (Henri et al. 2003, Puig et al. 2010). Once it enters the root cell, Cu can be sequestrated in vacuoles in the form of complexes of phytochelatins (PCs) through C-type ATP-binding cassette transporter 2 (ABCC2) (Won-Yong et al. 2010, Sharma et al. 2016). Copper ions stored in the vacuoles can be exported to the cytoplasm by tonoplast-localized transporters such as copper transporter 5 (COPT5) (Sharma et al. 2016). In the cytoplasm, the copper chaperone for copper/zinc (Cu/Zn) superoxide dismutase (CCS) located in cytosol and plastids (Chu et al. 2005) can deliver Cu to cytosolic Cu/Zn SOD enzymes (CSD) (Yruela 2009), which are important for scavenging ROS. Moreover, Cu ions in the cytosol are often complexed with chelators including metallothioneins (MTs) and nicotianamine (NA) to alleviate Cu toxicity in plants (Hall 2002, Yruela 2009, Hossain et al. 2012, Benyo et al. 2016). The yellow stripe-like transporter (YSL) is located in the plasma membrane, where it transports the Cu–NA complex from the roots to the aerial parts of plants (Waters et al. 2006, Chen et al. 2011, Migocka and Malas 2018). Alternatively, cytosolic HM ions can be excluded from the apoplast by plasma membrane-localized transporters such as P-type heavy metal ATPase 5 (HMA5), which is critical for xylem loading of Cu+ in rice (Deng et al. 2013). The differential transcriptional regulation of the key genes involved in Cu uptake, translocation and detoxification may lead to different Cu accumulation and tolerance in plants (Migocka and Malas 2018). However, no information is currently available on the transcriptional regulation of genes involved in variation in Cu accumulation and tolerance in apple rootstocks. To obtain insight into the physiological and transcriptional regulatory mechanisms underlying the variation in Cu accumulation, transport and tolerance in apple rootstocks, seedlings of five different species were exposed to either basal or excess Cu levels for 12 days. We hypothesized that (i) there would be variations in Cu accumulation and tolerance among different apple rootstocks under excess Cu-exposed conditions and (ii) these variations are associated with Cu subcellular distribution and physiological and transcriptional regulation. To examine these hypotheses, we analyzed growth characteristics; Cu concentration, bio-concentration factor (BCF) and translocation factor (Tf); subcellular distribution of Cu; ROS and malondialdehyde (MDA); soluble sugars and starch; antioxidants; and transcriptional regulation of several genes involved in Cu uptake, translocation and detoxification. Characterization of physiological and molecular regulation mechanisms involved in Cu accumulation and detoxification in apple rootstocks will provide a basis for further screening or engineering Cu-tolerant rootstocks. Materials and methods Plant material, Cu exposure and harvesting Seeds of Malus asiatica Nakai. (Ma), Malus baccata Borkh. (Mb), Malus hupehensis Rehd. (Mh), Malus micromalus Makino. (Mm) and Malus prunifolia Borkh. (Mp) were stratified at 0–4 °C in sand for 60 days. After germination, seeds were planted in nursery seedling plates filled with seedling substrate. After cultivation for 40 days in a greenhouse under natural light and temperature conditions (day/night temperature, 26/18 °C; relative air humidity, 50–60%), seedlings of similar size (six to seven leaves, ~ 8 cm tall) were selected and transferred to aerated quarter Hoagland nutrient solution (with basal dose of Cu: 0.125 μM CuSO4·5H2O). The Hoagland solution was adjusted to pH 5.5 and refreshed every 3 days. After cultivation in the hydroponic system for 10 weeks, 24 plants with similar height and growth performance from each species were divided into two groups (12 plants in each group). Subsequently, plants in the two groups from each apple rootstock were exposed to either basal or excess (20 μM) Cu by adding CuSO4·5H2O to the quarter Hoagland nutrient solution for 12 days. Before harvest, roots were washed with 0.1 mM EDTA for 5 min and rinsed three times for 5 min in deionized water to desorb Cu2+ from the root surface (Feng et al. 2018). Next, root, stem and leaf tissues were separated, and fresh weight of each tissue was recorded. The harvested samples were wrapped with tinfoil and immediately frozen in liquid nitrogen. The frozen samples were then milled to fine powder with a ball mill (Retsch, Haan, Germany). An equal amount of fine powder from two plants in each treatment was combined and thoroughly mixed for their further analysis and stored at −80 °C. Subsamples of roots were harvested for root scanning analysis. Analysis of biomass, root characteristics and photosynthetic pigments The fresh powder (∼50 mg) from each tissue per plant was dried at 60 °C for 72 h to determine the fresh-to-dry mass ratio. Biomass of each tissue of plants was calculated based on fresh-to-dry mass ratio and the fresh weight of each part. For the analysis of root architecture, subsamples of roots were scanned and analyzed by a WinRHIZO Root Analyzer System (WinRHIZO 2012b; Regent Instruments Canada Inc., Montreal, Canada) according to Ma et al. (2014). To determine chlorophyll and carotenoid contents in leaves, the fresh leaf was extracted in 5 ml of 80% acetone in darkness for 24 h. Then the absorbance of the mixture was recorded spectrophotometrically at 663, 646 and 470 nm (He et al. 2011). Copper concentration, BCF and Tf Copper concentration in the roots, stems and leaves were determined by graphite furnace atomic absorption spectrophotometry (Hitachi 180-80, Hitachi Ltd, Tokyo, Japan) after digestion with a mixture containing 7 ml concentrated HNO3 and 1 ml concentrated HClO4 at 170 °C as described by Zhou et al. (2016). The BCF was defined as the ratio of metal concentration (μg g−1) in plant roots or aerial tissues to that in the solution (μg g−1) (Zacchini et al. 2009). The Tf was calculated as metal concentration (μg g−1) in the aerial tissues of a plant divided by that in roots multiplied by 100% (Zacchini et al. 2009). Analysis of Cu subcellular distribution Root samples were separated into four subcellular fractions (cell wall fraction, organelle-rich fraction, membrane-containing fraction and soluble fraction) by the gradient centrifugation technique as suggested by Fu et al. (2011). Frozen roots (∼200 mg) were homogenized in 10 ml of pre-cold extraction buffer containing 250 mM sucrose, 50 mM Tris-HCl (pH 7.5) and 1 mM dithioerythritol at 4 °C. The homogenate was filtered through a nylon cloth (100 μm), and the residue that mainly contained cell walls and cell wall debris was designated as the cell wall fraction. The filtrate was subsequently centrifuged at 10,000g for 30 min, and the resultant deposition containing organelle-rich fraction was collected. Then the supernatant continued centrifugation at 100,000g for 30 min, and the pellet was considered as the membrane-containing fraction, while the supernatant was taken as the soluble fraction (Mwamba et al. 2016). All steps were performed at 4 °C. The fractions were oven-dried at 65 °C to dryness, and digested with a mixture containing 7 ml concentrated HNO3 and 1 ml concentrated HClO4 at 170 °C according to Lai (2015) with some modifications. Copper concentration in the digests was measured by graphite furnace atomic absorption spectrophotometry (Hitachi 180-80, Hitachi Ltd). Determination of O2·−, H2O2 and MDA The concentrations of O2·− and H2O2 in the root, stem and leaf tissues were determined spectrophotometrically at 530 and 410 nm, respectively, as suggested by Zhang et al. (2010) and He et al. (2011). The concentrations of MDA in samples were measured using a spectrophotometer at 450, 532 and 600 nm, according to the method of Lei et al. (2007). Analysis of total soluble sugars and starch Total soluble sugar and starch in plant tissues were measured by the anthrone method as suggested (Yemm and Willis 1954). The fresh plant tissues (∼100 mg) were extracted in 3 ml of 80% ethanol at 80 °C for 30 min. After centrifugation at 6000g for 10 min, the first supernatant was collected. The pellet was extracted again with the same extraction procedures, and the supernatant was combined with the previous one. After adding anthrone reagent to the supernatant and heating in boiling water, the absorbance of the mixture was recorded spectrophotometrically at 620 nm. The standard curve was established by a series of diluted glucose solutions. The pellet retained after the extraction of the soluble sugars was further extracted by HClO4 to measure starch in plant tissues. Starch (expressed as glucose equivalent) in the supernatant was determined using the same methods as above. Analysis of antioxidants and antioxidative enzyme activities The concentration of free proline, soluble phenolics, ASC and T-SH was determined spectrophotometrically, according to He et al. (2013b). Reduced GSH was analyzed as suggested by Wang et al. (2013). The soluble proteins in plant tissues were extracted and quantified as suggested by Luo et al. (2008). The enzyme activities of SOD, POD, CAT and APX were determined as suggested by He et al. (2011), and GR according to the method of Wang et al. (2013). Analysis of the levels of gene transcripts Total RNA extraction and purification and quantitative reverse transcription (RT)-PCR were carried out according to the method of Zhou et al. (2016). Total RNA of roots was isolated and purified by using a plant RNA extraction kit (R6827, Omega Bio-Tek, Norcross, GA, USA). The concentrations and quality of extracted RNA were measured by spectrophotometer analysis (NanoDrop 2000, Thermo Fisher Scientific Ltd, New York, USA) and agarose gel electrophoresis, respectively. The first strand cDNA was generated by 1 μg total RNA in a total volume of 20 μl using PrimeScript RT Reagent Kit with gDNA Eraser (DRR037A, Takara, Dalian, China). Quantitative PCR for each gene was performed using 10 μl 2× SYBR Green Premix Ex Taq II (DRR820A, Takara), 0.5 μl cDNA and 0.2 μl of 20 μm primer, which had been designed specifically for each plant gene (Table S1 available as Supplementary Data at Tree Physiology Online) with a CFX96 Real Time system to test (CFX96, Bio-Rad, Hercules, CA, USA). The β-Actin gene served as a reference gene. The homogeneity of PCR products was confirmed by a melting curve program. PCR products were sequenced and aligned with homologs in other model plants to ensure the specificity (Figure S1 available as Supplementary Data at Tree Physiology Online). The relative mRNA expression was calculated using the 2—∆∆CT method (Livak and Schmittgen 2001). The expression level was set to 1 for each gene in roots of M. asiatica exposed to basal Cu, and fold changes of transcripts were subsequently calculated in roots of other apple rootstocks under different Cu conditions. The gene expression heatmap was generated on the log base 2 average expression fold values using the command heatmap.2 in the package ‘gplots’ in R (http://www.r-project.org/) according to Luo et al. (2015). Table 1 Biomass of roots, stems and leaves, root architecture and photosynthetic pigments of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters following the values in the same column indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh.; Chl, chlorophyll. Rootstocks Cu treatments Root (g DW) Stem (g DW) Leaf (g DW) Total biomass (g DW) Total root length (m) Total root surface area (cm2) Total root volume (cm3) Chl (a + b) (mg g−1 DW) Carotenoid (mg g−1 DW) Ma Basal Cu 3.12 ± 0.13 a 3.08 ± 0.16 cde 4.19 ± 0.09 ab 10.39 ± 0.20 b 73.26 ± 1.22 a 947.38 ± 83.36 a 9.90 ± 1.68 a 4.27 ± 0.02 b 0.77 ± 0.01 ab Excess Cu 1.90 ± 0.04 b 2.84 ± 0.13 ef 3.34 ± 0.22 de 8.08 ± 0.21 c 35.32 ± 1.41 e 341.69 ± 17.60 cd 3.55 ± 0.56 b 4.00 ± 0.13 de 0.71 ± 0.02 de Mb Basal Cu 2.97 ± 0.04 a 3.79 ± 0.19 b 3.75 ± 0.10 bcd 10.51 ± 0.25 b 67.27 ± 5.53 ab 869.30 ± 38.77 a 8.98 ± 0.06 a 4.44 ± 0.03 a 0.79 ± 0.01 a Excess Cu 1.63 ± 0.00 bc 1.86 ± 0.04 g 3.16 ± 0.16 de 6.65 ± 0.19 d 32.72 ± 0.39 e 341.69 ± 17.60 cd 2.85 ± 0.26 b 3.98 ± 0.02 de 0.71 ± 0.00 de Mh Basal Cu 2.90 ± 0.01 a 3.30 ± 0.01 cd 4.45 ± 0.20 a 10.65 ± 0.26 b 51.48 ± 2.55 cd 774.96 ± 31.92 a 9.34 ± 0.79 a 4.11 ± 0.02 cd 0.73 ± 0.01 cd Excess Cu 1.52 ± 0.08 c 1.44 ± 0.05 h 2.87 ± 0.27 e 5.83 ± 0.33 e 18.84 ± 1.27 f 256.93 ± 28.21 d 2.80 ± 0.45 b 3.67 ± 0.03 f 0.65 ± 0.01 f Mm Basal Cu 2.87 ± 0.07 a 3.41 ± 0.03 c 3.97 ± 0.22 abc 10.25 ± 0.21 b 70.42 ± 0.60 a 835.07 ± 104.11 a 8.12 ± 1.94 a 4.27 ± 0.00 b 0.75 ± 0.01 bc Excess Cu 1.89 ± 0.05 b 2.58 ± 0.06 f 3.36 ± 0.15 de 7.83 ± 0.15 c 41.17 ± 2.50 de 447.84 ± 28.74 bc 3.88 ± 0.27 b 3.88 ± 0.05 e 0.69 ± 0.01 e Mp Basal Cu 2.87 ± 0.28 a 4.43 ± 0.52 a 4.41 ± 0.33 a 11.71 ± 0.47 a 63.87 ± 10.05 ab 908.02 ± 117.35 a 10.93 ± 1.28 a 4.21 ± 0.03 bc 0.75 ± 0.01 bc Excess Cu 1.97 ± 0.09 b 2.93 ± 0.18 def 3.52 ± 0.18 cd 8.42 ± 0.14 c 55.98 ± 1.78 bc 583.39 ± 3.05 b 4.85 ± 0.10 b 4.30 ± 0.03 ab 0.73 ± 0.01 cd P value Cu **** **** **** **** **** **** **** **** **** S ns **** ns **** **** ns ns **** ** Cu × S ns **** ns ** * ns ns *** ns Rootstocks Cu treatments Root (g DW) Stem (g DW) Leaf (g DW) Total biomass (g DW) Total root length (m) Total root surface area (cm2) Total root volume (cm3) Chl (a + b) (mg g−1 DW) Carotenoid (mg g−1 DW) Ma Basal Cu 3.12 ± 0.13 a 3.08 ± 0.16 cde 4.19 ± 0.09 ab 10.39 ± 0.20 b 73.26 ± 1.22 a 947.38 ± 83.36 a 9.90 ± 1.68 a 4.27 ± 0.02 b 0.77 ± 0.01 ab Excess Cu 1.90 ± 0.04 b 2.84 ± 0.13 ef 3.34 ± 0.22 de 8.08 ± 0.21 c 35.32 ± 1.41 e 341.69 ± 17.60 cd 3.55 ± 0.56 b 4.00 ± 0.13 de 0.71 ± 0.02 de Mb Basal Cu 2.97 ± 0.04 a 3.79 ± 0.19 b 3.75 ± 0.10 bcd 10.51 ± 0.25 b 67.27 ± 5.53 ab 869.30 ± 38.77 a 8.98 ± 0.06 a 4.44 ± 0.03 a 0.79 ± 0.01 a Excess Cu 1.63 ± 0.00 bc 1.86 ± 0.04 g 3.16 ± 0.16 de 6.65 ± 0.19 d 32.72 ± 0.39 e 341.69 ± 17.60 cd 2.85 ± 0.26 b 3.98 ± 0.02 de 0.71 ± 0.00 de Mh Basal Cu 2.90 ± 0.01 a 3.30 ± 0.01 cd 4.45 ± 0.20 a 10.65 ± 0.26 b 51.48 ± 2.55 cd 774.96 ± 31.92 a 9.34 ± 0.79 a 4.11 ± 0.02 cd 0.73 ± 0.01 cd Excess Cu 1.52 ± 0.08 c 1.44 ± 0.05 h 2.87 ± 0.27 e 5.83 ± 0.33 e 18.84 ± 1.27 f 256.93 ± 28.21 d 2.80 ± 0.45 b 3.67 ± 0.03 f 0.65 ± 0.01 f Mm Basal Cu 2.87 ± 0.07 a 3.41 ± 0.03 c 3.97 ± 0.22 abc 10.25 ± 0.21 b 70.42 ± 0.60 a 835.07 ± 104.11 a 8.12 ± 1.94 a 4.27 ± 0.00 b 0.75 ± 0.01 bc Excess Cu 1.89 ± 0.05 b 2.58 ± 0.06 f 3.36 ± 0.15 de 7.83 ± 0.15 c 41.17 ± 2.50 de 447.84 ± 28.74 bc 3.88 ± 0.27 b 3.88 ± 0.05 e 0.69 ± 0.01 e Mp Basal Cu 2.87 ± 0.28 a 4.43 ± 0.52 a 4.41 ± 0.33 a 11.71 ± 0.47 a 63.87 ± 10.05 ab 908.02 ± 117.35 a 10.93 ± 1.28 a 4.21 ± 0.03 bc 0.75 ± 0.01 bc Excess Cu 1.97 ± 0.09 b 2.93 ± 0.18 def 3.52 ± 0.18 cd 8.42 ± 0.14 c 55.98 ± 1.78 bc 583.39 ± 3.05 b 4.85 ± 0.10 b 4.30 ± 0.03 ab 0.73 ± 0.01 cd P value Cu **** **** **** **** **** **** **** **** **** S ns **** ns **** **** ns ns **** ** Cu × S ns **** ns ** * ns ns *** ns View Large Table 1 Biomass of roots, stems and leaves, root architecture and photosynthetic pigments of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters following the values in the same column indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh.; Chl, chlorophyll. Rootstocks Cu treatments Root (g DW) Stem (g DW) Leaf (g DW) Total biomass (g DW) Total root length (m) Total root surface area (cm2) Total root volume (cm3) Chl (a + b) (mg g−1 DW) Carotenoid (mg g−1 DW) Ma Basal Cu 3.12 ± 0.13 a 3.08 ± 0.16 cde 4.19 ± 0.09 ab 10.39 ± 0.20 b 73.26 ± 1.22 a 947.38 ± 83.36 a 9.90 ± 1.68 a 4.27 ± 0.02 b 0.77 ± 0.01 ab Excess Cu 1.90 ± 0.04 b 2.84 ± 0.13 ef 3.34 ± 0.22 de 8.08 ± 0.21 c 35.32 ± 1.41 e 341.69 ± 17.60 cd 3.55 ± 0.56 b 4.00 ± 0.13 de 0.71 ± 0.02 de Mb Basal Cu 2.97 ± 0.04 a 3.79 ± 0.19 b 3.75 ± 0.10 bcd 10.51 ± 0.25 b 67.27 ± 5.53 ab 869.30 ± 38.77 a 8.98 ± 0.06 a 4.44 ± 0.03 a 0.79 ± 0.01 a Excess Cu 1.63 ± 0.00 bc 1.86 ± 0.04 g 3.16 ± 0.16 de 6.65 ± 0.19 d 32.72 ± 0.39 e 341.69 ± 17.60 cd 2.85 ± 0.26 b 3.98 ± 0.02 de 0.71 ± 0.00 de Mh Basal Cu 2.90 ± 0.01 a 3.30 ± 0.01 cd 4.45 ± 0.20 a 10.65 ± 0.26 b 51.48 ± 2.55 cd 774.96 ± 31.92 a 9.34 ± 0.79 a 4.11 ± 0.02 cd 0.73 ± 0.01 cd Excess Cu 1.52 ± 0.08 c 1.44 ± 0.05 h 2.87 ± 0.27 e 5.83 ± 0.33 e 18.84 ± 1.27 f 256.93 ± 28.21 d 2.80 ± 0.45 b 3.67 ± 0.03 f 0.65 ± 0.01 f Mm Basal Cu 2.87 ± 0.07 a 3.41 ± 0.03 c 3.97 ± 0.22 abc 10.25 ± 0.21 b 70.42 ± 0.60 a 835.07 ± 104.11 a 8.12 ± 1.94 a 4.27 ± 0.00 b 0.75 ± 0.01 bc Excess Cu 1.89 ± 0.05 b 2.58 ± 0.06 f 3.36 ± 0.15 de 7.83 ± 0.15 c 41.17 ± 2.50 de 447.84 ± 28.74 bc 3.88 ± 0.27 b 3.88 ± 0.05 e 0.69 ± 0.01 e Mp Basal Cu 2.87 ± 0.28 a 4.43 ± 0.52 a 4.41 ± 0.33 a 11.71 ± 0.47 a 63.87 ± 10.05 ab 908.02 ± 117.35 a 10.93 ± 1.28 a 4.21 ± 0.03 bc 0.75 ± 0.01 bc Excess Cu 1.97 ± 0.09 b 2.93 ± 0.18 def 3.52 ± 0.18 cd 8.42 ± 0.14 c 55.98 ± 1.78 bc 583.39 ± 3.05 b 4.85 ± 0.10 b 4.30 ± 0.03 ab 0.73 ± 0.01 cd P value Cu **** **** **** **** **** **** **** **** **** S ns **** ns **** **** ns ns **** ** Cu × S ns **** ns ** * ns ns *** ns Rootstocks Cu treatments Root (g DW) Stem (g DW) Leaf (g DW) Total biomass (g DW) Total root length (m) Total root surface area (cm2) Total root volume (cm3) Chl (a + b) (mg g−1 DW) Carotenoid (mg g−1 DW) Ma Basal Cu 3.12 ± 0.13 a 3.08 ± 0.16 cde 4.19 ± 0.09 ab 10.39 ± 0.20 b 73.26 ± 1.22 a 947.38 ± 83.36 a 9.90 ± 1.68 a 4.27 ± 0.02 b 0.77 ± 0.01 ab Excess Cu 1.90 ± 0.04 b 2.84 ± 0.13 ef 3.34 ± 0.22 de 8.08 ± 0.21 c 35.32 ± 1.41 e 341.69 ± 17.60 cd 3.55 ± 0.56 b 4.00 ± 0.13 de 0.71 ± 0.02 de Mb Basal Cu 2.97 ± 0.04 a 3.79 ± 0.19 b 3.75 ± 0.10 bcd 10.51 ± 0.25 b 67.27 ± 5.53 ab 869.30 ± 38.77 a 8.98 ± 0.06 a 4.44 ± 0.03 a 0.79 ± 0.01 a Excess Cu 1.63 ± 0.00 bc 1.86 ± 0.04 g 3.16 ± 0.16 de 6.65 ± 0.19 d 32.72 ± 0.39 e 341.69 ± 17.60 cd 2.85 ± 0.26 b 3.98 ± 0.02 de 0.71 ± 0.00 de Mh Basal Cu 2.90 ± 0.01 a 3.30 ± 0.01 cd 4.45 ± 0.20 a 10.65 ± 0.26 b 51.48 ± 2.55 cd 774.96 ± 31.92 a 9.34 ± 0.79 a 4.11 ± 0.02 cd 0.73 ± 0.01 cd Excess Cu 1.52 ± 0.08 c 1.44 ± 0.05 h 2.87 ± 0.27 e 5.83 ± 0.33 e 18.84 ± 1.27 f 256.93 ± 28.21 d 2.80 ± 0.45 b 3.67 ± 0.03 f 0.65 ± 0.01 f Mm Basal Cu 2.87 ± 0.07 a 3.41 ± 0.03 c 3.97 ± 0.22 abc 10.25 ± 0.21 b 70.42 ± 0.60 a 835.07 ± 104.11 a 8.12 ± 1.94 a 4.27 ± 0.00 b 0.75 ± 0.01 bc Excess Cu 1.89 ± 0.05 b 2.58 ± 0.06 f 3.36 ± 0.15 de 7.83 ± 0.15 c 41.17 ± 2.50 de 447.84 ± 28.74 bc 3.88 ± 0.27 b 3.88 ± 0.05 e 0.69 ± 0.01 e Mp Basal Cu 2.87 ± 0.28 a 4.43 ± 0.52 a 4.41 ± 0.33 a 11.71 ± 0.47 a 63.87 ± 10.05 ab 908.02 ± 117.35 a 10.93 ± 1.28 a 4.21 ± 0.03 bc 0.75 ± 0.01 bc Excess Cu 1.97 ± 0.09 b 2.93 ± 0.18 def 3.52 ± 0.18 cd 8.42 ± 0.14 c 55.98 ± 1.78 bc 583.39 ± 3.05 b 4.85 ± 0.10 b 4.30 ± 0.03 ab 0.73 ± 0.01 cd P value Cu **** **** **** **** **** **** **** **** **** S ns **** ns **** **** ns ns **** ** Cu × S ns **** ns ** * ns ns *** ns View Large Statistical analysis All statistical tests were performed with Statgraphics (STN, St Louis, MO, USA). All data were checked for normality before statistical analysis. Two-way analyses of variance (ANOVAs) were employed with CuSO4·5H2O (Cu) and species (S) as the two factors to test all parameters for significant changes. If interactions were significant, a posteriori comparisons of means were made. All P values of these multicomparisons were corrected by the Tukey-HSD method to reduce the chance of type I errors. Differences between means were considered significant when the P value of the ANOVA F test was <0.05. Results Growth characteristics To analyze the Cu-induced phytotoxic effects, biomass, root characteristics and photosynthetic pigments were determined (Table 1). The plant biomass of each tissue varied greatly among the apple rootstocks. Among the five apple rootstocks, the largest total biomass was found in M. prunifolia, regardless of Cu treatment. After exposure to elevated Cu, root biomass was negatively affected in all apple rootstocks, but this was less pronounced in M. prunifolia. The negative effects of excess Cu on the biomass of stems and leaves were also found in apple rootstocks. The reduction in total biomass was the lowest in M. asiatica (22.3%), followed by M. micromalus (23.7%), M. prunifolia (28.1%), M. baccata (36.7%) and M. hupehensis (45.3%) (Table 1). Marked differences in root characteristics were observed among the different apple rootstocks. Similarly, elevated Cu exposure led to significant declines in total root length, root surface area and root volume of the five apple rootstocks, but this effect was less pronounced in M. prunifolia and more pronounced in M. hupehensis (Table 1). Treatment with excess Cu markedly reduced the photosynthetic pigments of different apple rootstocks, except for M. prunifolia, relative to their respective controls (Table 1). Copper concentration, BCF and Tf The Cu concentrations in roots, stems and leaves are shown in Figure 1. Comparatively, there were remarkable differences in tissue Cu concentrations among the different apple rootstocks. Excess Cu caused marked Cu accumulation in roots, stems and leaves of all plants compared with plants undergoing non-toxic Cu treatment. In general, most Cu was accumulated in the roots followed by the stems and leaves. Among all the apple rootstocks, M. prunifolia accumulated the maximum amount of Cu in roots, with the lowest found in the roots of M. hupehensis. However, the highest Cu concentrations in stems and leaves were found in M. hupehensis and M. baccata, respectively (Figure 1b and c). Figure 1. View largeDownload slide Copper concentrations in roots (a), stems (b) and leaves (c) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. ****P ≤ 0.0001. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Figure 1. View largeDownload slide Copper concentrations in roots (a), stems (b) and leaves (c) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. ****P ≤ 0.0001. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. In the present study, BCFs of all tissues in the different apple rootstocks were calculated to assess the ability of plants to accumulate Cu (Figure 2a). In general, BCFs were highest in the roots of all apple rootstocks, followed by the stems and leaves. In the roots, the highest BCF was found in M. prunifolia, whereas BCFs of the aerial parts were higher in M. baccata and M. hupehensis (Figure 2a). The Tf could reveal the ability of plants to transport HMs from roots to aerial tissues (Zacchini et al. 2009). Higher Tfs in the aerial tissues were observed in M. baccata and M. hupehensis, whereas Tfs in the aerial parts of M. micromalus and M. prunifolia were lower than those of the other apple rootstocks (Figure 2b). Figure 2. View largeDownload slide BCF (a) in roots, stems and leaves and Tf (b) in stems and leaves of five apple rootstocks exposed to excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters on the bars for the same tissue indicate significant differences between the treatments. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Figure 2. View largeDownload slide BCF (a) in roots, stems and leaves and Tf (b) in stems and leaves of five apple rootstocks exposed to excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters on the bars for the same tissue indicate significant differences between the treatments. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Subcellular distribution of Cu Compared with non-elevated Cu supply conditions, excess Cu in the growth medium increased Cu concentration in all subcellular fractions of the roots, irrespective of apple rootstock species (Table 2). However, significant differences in the subcellular distribution of Cu were observed among the five apple rootstocks. In general, Cu was mainly stored in the cell wall fraction, followed by the soluble fraction, the membrane-containing fraction and the organelle-rich fraction (Table 2). Among the five apple rootstocks, no significant differences in Cu concentration in subcellular fractions were observed in all plants under non-toxic Cu conditions. Nevertheless, the highest Cu concentration in the cell wall fraction, soluble fraction and organelle-rich fraction were found in M. prunifolia at 38.9%, 27.8% and 29.1%, respectively, higher on average than those in the other investigated rootstocks under elevated Cu conditions. Inversely, the Cu concentration of the membrane-containing fraction was the highest in M. baccata and M. hupehensis (Table 2). Table 2 Subcellular distribution of Cu and its proportion in roots of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters following the values in the same column indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. ****P ≤ 0.0001. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Rootstocks Cu treatments Cu concentration (μg g−1 DW) Cu distribution ratio (%) Membrane-containing fraction Cell wall fraction Soluble fraction Organelles fraction Membrane-containing fraction Cell wall fraction Soluble fraction Organelles fraction Ma Basal Cu 8.24 ± 0.32 d 10.73 ± 0.23 f 18.52 ± 0.04 e 1.31 ± 0.09 c 21.23 ± 0.78 a 27.66 ± 0.65 f 47.73 ± 0.24 a 3.38 ± 0.18 ab Excess Cu 114.68 ± 1.76 b 544.51 ± 15.12 c 545.61 ± 26.20 a 28.92 ± 0.47 b 9.31 ± 0.18 e 44.16 ± 0.30 c 44.18 ± 0.60 bc 2.35 ± 0.12 cd Mb Basal Cu 6.32 ± 0.28 d 15.61 ± 1.76 f 18.46 ± 0.71 e 0.57 ± 0.04 c 15.59 ± 1.50 cd 37.87 ± 2.31 e 45.13 ± 0.64 ab 1.41 ± 0.17 e Excess Cu 190.42 ± 31.17 a 471.75 ± 22.02 d 410.19 ± 12.34 b 32.21 ± 0.20 a 17.12 ± 2.30 bcd 42.76 ± 2.16 cd 37.21 ± 1.68 f 2.92 ± 0.09 abc Mh Basal Cu 3.53 ± 0.54 d 7.23 ± 0.06 f 7.18 ± 0.21 e 0.23 ± 0.01 c 19.24 ± 2.25 ab 39.96 ± 1.89 cde 39.54 ± 0.62 def 1.26 ± 0.01 e Excess Cu 161.40 ± 1.49 a 388.75 ± 4.58 e 350.08 ± 6.10 c 13.52 ± 2.21 b 17.29 ± 0.15 bc 41.66 ± 0.54 cde 37.51 ± 0.62 ef 3.54 ± 0.01 a Mm Basal Cu 8.85 ± 0.27 d 21.08 ± 0.38 f 22.92 ± 1.00 e 1.74 ± 0.01 c 16.21 ± 0.22 bcd 38.66 ± 1.33 de 41.94 ± 1.14 cd 3.19 ± 0.03 ab Excess Cu 67.99 ± 11.31 c 687.18 ± 9.70 b 285.77 ± 8.72 d 28.79 ± 2.58 b 6.30 ± 0.87 e 64.40 ± 1.04 a 26.71 ± 0.01 h 2.68 ± 0.16 bc Mp Basal Cu 5.79 ± 0.73 d 17.82 ± 0.39 f 17.17 ± 2.00 e 1.48 ± 0.20 c 13.60 ± 0.80 d 42.44 ± 2.03 cd 40.35 ± 1.95 de 3.60 ± 0.72 a Excess Cu 112.44 ± 1.70 b 934.28 ± 3.97 a 541.77 ± 6.31 a 38.57 ± 2.01 a 6.95 ± 0.10 e 57.76 ± 0.15 b 33.49 ± 0.23 g 1.80 ± 0.02 de P values Cu **** **** **** **** **** **** **** **** S **** **** **** **** **** **** **** **** Cu × S **** **** **** **** **** **** **** **** Rootstocks Cu treatments Cu concentration (μg g−1 DW) Cu distribution ratio (%) Membrane-containing fraction Cell wall fraction Soluble fraction Organelles fraction Membrane-containing fraction Cell wall fraction Soluble fraction Organelles fraction Ma Basal Cu 8.24 ± 0.32 d 10.73 ± 0.23 f 18.52 ± 0.04 e 1.31 ± 0.09 c 21.23 ± 0.78 a 27.66 ± 0.65 f 47.73 ± 0.24 a 3.38 ± 0.18 ab Excess Cu 114.68 ± 1.76 b 544.51 ± 15.12 c 545.61 ± 26.20 a 28.92 ± 0.47 b 9.31 ± 0.18 e 44.16 ± 0.30 c 44.18 ± 0.60 bc 2.35 ± 0.12 cd Mb Basal Cu 6.32 ± 0.28 d 15.61 ± 1.76 f 18.46 ± 0.71 e 0.57 ± 0.04 c 15.59 ± 1.50 cd 37.87 ± 2.31 e 45.13 ± 0.64 ab 1.41 ± 0.17 e Excess Cu 190.42 ± 31.17 a 471.75 ± 22.02 d 410.19 ± 12.34 b 32.21 ± 0.20 a 17.12 ± 2.30 bcd 42.76 ± 2.16 cd 37.21 ± 1.68 f 2.92 ± 0.09 abc Mh Basal Cu 3.53 ± 0.54 d 7.23 ± 0.06 f 7.18 ± 0.21 e 0.23 ± 0.01 c 19.24 ± 2.25 ab 39.96 ± 1.89 cde 39.54 ± 0.62 def 1.26 ± 0.01 e Excess Cu 161.40 ± 1.49 a 388.75 ± 4.58 e 350.08 ± 6.10 c 13.52 ± 2.21 b 17.29 ± 0.15 bc 41.66 ± 0.54 cde 37.51 ± 0.62 ef 3.54 ± 0.01 a Mm Basal Cu 8.85 ± 0.27 d 21.08 ± 0.38 f 22.92 ± 1.00 e 1.74 ± 0.01 c 16.21 ± 0.22 bcd 38.66 ± 1.33 de 41.94 ± 1.14 cd 3.19 ± 0.03 ab Excess Cu 67.99 ± 11.31 c 687.18 ± 9.70 b 285.77 ± 8.72 d 28.79 ± 2.58 b 6.30 ± 0.87 e 64.40 ± 1.04 a 26.71 ± 0.01 h 2.68 ± 0.16 bc Mp Basal Cu 5.79 ± 0.73 d 17.82 ± 0.39 f 17.17 ± 2.00 e 1.48 ± 0.20 c 13.60 ± 0.80 d 42.44 ± 2.03 cd 40.35 ± 1.95 de 3.60 ± 0.72 a Excess Cu 112.44 ± 1.70 b 934.28 ± 3.97 a 541.77 ± 6.31 a 38.57 ± 2.01 a 6.95 ± 0.10 e 57.76 ± 0.15 b 33.49 ± 0.23 g 1.80 ± 0.02 de P values Cu **** **** **** **** **** **** **** **** S **** **** **** **** **** **** **** **** Cu × S **** **** **** **** **** **** **** **** View Large Table 2 Subcellular distribution of Cu and its proportion in roots of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters following the values in the same column indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. ****P ≤ 0.0001. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Rootstocks Cu treatments Cu concentration (μg g−1 DW) Cu distribution ratio (%) Membrane-containing fraction Cell wall fraction Soluble fraction Organelles fraction Membrane-containing fraction Cell wall fraction Soluble fraction Organelles fraction Ma Basal Cu 8.24 ± 0.32 d 10.73 ± 0.23 f 18.52 ± 0.04 e 1.31 ± 0.09 c 21.23 ± 0.78 a 27.66 ± 0.65 f 47.73 ± 0.24 a 3.38 ± 0.18 ab Excess Cu 114.68 ± 1.76 b 544.51 ± 15.12 c 545.61 ± 26.20 a 28.92 ± 0.47 b 9.31 ± 0.18 e 44.16 ± 0.30 c 44.18 ± 0.60 bc 2.35 ± 0.12 cd Mb Basal Cu 6.32 ± 0.28 d 15.61 ± 1.76 f 18.46 ± 0.71 e 0.57 ± 0.04 c 15.59 ± 1.50 cd 37.87 ± 2.31 e 45.13 ± 0.64 ab 1.41 ± 0.17 e Excess Cu 190.42 ± 31.17 a 471.75 ± 22.02 d 410.19 ± 12.34 b 32.21 ± 0.20 a 17.12 ± 2.30 bcd 42.76 ± 2.16 cd 37.21 ± 1.68 f 2.92 ± 0.09 abc Mh Basal Cu 3.53 ± 0.54 d 7.23 ± 0.06 f 7.18 ± 0.21 e 0.23 ± 0.01 c 19.24 ± 2.25 ab 39.96 ± 1.89 cde 39.54 ± 0.62 def 1.26 ± 0.01 e Excess Cu 161.40 ± 1.49 a 388.75 ± 4.58 e 350.08 ± 6.10 c 13.52 ± 2.21 b 17.29 ± 0.15 bc 41.66 ± 0.54 cde 37.51 ± 0.62 ef 3.54 ± 0.01 a Mm Basal Cu 8.85 ± 0.27 d 21.08 ± 0.38 f 22.92 ± 1.00 e 1.74 ± 0.01 c 16.21 ± 0.22 bcd 38.66 ± 1.33 de 41.94 ± 1.14 cd 3.19 ± 0.03 ab Excess Cu 67.99 ± 11.31 c 687.18 ± 9.70 b 285.77 ± 8.72 d 28.79 ± 2.58 b 6.30 ± 0.87 e 64.40 ± 1.04 a 26.71 ± 0.01 h 2.68 ± 0.16 bc Mp Basal Cu 5.79 ± 0.73 d 17.82 ± 0.39 f 17.17 ± 2.00 e 1.48 ± 0.20 c 13.60 ± 0.80 d 42.44 ± 2.03 cd 40.35 ± 1.95 de 3.60 ± 0.72 a Excess Cu 112.44 ± 1.70 b 934.28 ± 3.97 a 541.77 ± 6.31 a 38.57 ± 2.01 a 6.95 ± 0.10 e 57.76 ± 0.15 b 33.49 ± 0.23 g 1.80 ± 0.02 de P values Cu **** **** **** **** **** **** **** **** S **** **** **** **** **** **** **** **** Cu × S **** **** **** **** **** **** **** **** Rootstocks Cu treatments Cu concentration (μg g−1 DW) Cu distribution ratio (%) Membrane-containing fraction Cell wall fraction Soluble fraction Organelles fraction Membrane-containing fraction Cell wall fraction Soluble fraction Organelles fraction Ma Basal Cu 8.24 ± 0.32 d 10.73 ± 0.23 f 18.52 ± 0.04 e 1.31 ± 0.09 c 21.23 ± 0.78 a 27.66 ± 0.65 f 47.73 ± 0.24 a 3.38 ± 0.18 ab Excess Cu 114.68 ± 1.76 b 544.51 ± 15.12 c 545.61 ± 26.20 a 28.92 ± 0.47 b 9.31 ± 0.18 e 44.16 ± 0.30 c 44.18 ± 0.60 bc 2.35 ± 0.12 cd Mb Basal Cu 6.32 ± 0.28 d 15.61 ± 1.76 f 18.46 ± 0.71 e 0.57 ± 0.04 c 15.59 ± 1.50 cd 37.87 ± 2.31 e 45.13 ± 0.64 ab 1.41 ± 0.17 e Excess Cu 190.42 ± 31.17 a 471.75 ± 22.02 d 410.19 ± 12.34 b 32.21 ± 0.20 a 17.12 ± 2.30 bcd 42.76 ± 2.16 cd 37.21 ± 1.68 f 2.92 ± 0.09 abc Mh Basal Cu 3.53 ± 0.54 d 7.23 ± 0.06 f 7.18 ± 0.21 e 0.23 ± 0.01 c 19.24 ± 2.25 ab 39.96 ± 1.89 cde 39.54 ± 0.62 def 1.26 ± 0.01 e Excess Cu 161.40 ± 1.49 a 388.75 ± 4.58 e 350.08 ± 6.10 c 13.52 ± 2.21 b 17.29 ± 0.15 bc 41.66 ± 0.54 cde 37.51 ± 0.62 ef 3.54 ± 0.01 a Mm Basal Cu 8.85 ± 0.27 d 21.08 ± 0.38 f 22.92 ± 1.00 e 1.74 ± 0.01 c 16.21 ± 0.22 bcd 38.66 ± 1.33 de 41.94 ± 1.14 cd 3.19 ± 0.03 ab Excess Cu 67.99 ± 11.31 c 687.18 ± 9.70 b 285.77 ± 8.72 d 28.79 ± 2.58 b 6.30 ± 0.87 e 64.40 ± 1.04 a 26.71 ± 0.01 h 2.68 ± 0.16 bc Mp Basal Cu 5.79 ± 0.73 d 17.82 ± 0.39 f 17.17 ± 2.00 e 1.48 ± 0.20 c 13.60 ± 0.80 d 42.44 ± 2.03 cd 40.35 ± 1.95 de 3.60 ± 0.72 a Excess Cu 112.44 ± 1.70 b 934.28 ± 3.97 a 541.77 ± 6.31 a 38.57 ± 2.01 a 6.95 ± 0.10 e 57.76 ± 0.15 b 33.49 ± 0.23 g 1.80 ± 0.02 de P values Cu **** **** **** **** **** **** **** **** S **** **** **** **** **** **** **** **** Cu × S **** **** **** **** **** **** **** **** View Large The Cu distribution ratio in different subcellular fractions differed markedly in the five apple rootstocks (Table 2). Copper treatment decreased the Cu proportion in the membrane-containing fraction in roots of all plants except for M. baccata and M. hupehensis, while it increased the Cu proportion in the cell wall fraction, except for M. hupehensis, compared with the controls under non-toxic Cu conditions. In most cases, excess Cu significantly decreased Cu distribution ratio of soluble fraction (Table 2). The Cu distribution ratios of the organelle-rich fraction declined by 30.5%, 16.0% and 50.0% in the roots of M. asiatica, M. micromalus and M. prunifolia, respectively, under excess Cu conditions compared with those under basal Cu conditions. The opposite pattern was observed in M. baccata and M. hupehensis. Among the five apple rootstocks, the Cu proportion of the membrane-containing fraction was lower in M. asiatica, M. micromalus and M. prunifolia than in the other two apple rootstocks in the presence of elevated Cu (Table 2). O2·−, H2O2 and MDA O2·− and H2O2, which are types of ROS that often accumulate in plants exposed to HMs, were measured in the present study to assess excess Cu toxicity in apple rootstocks (Figure 3). O2·− concentration was greatly induced in all the tissues of the apple rootstocks in the presence of elevated Cu compared with those under basal Cu conditions, with the highest accumulation found in roots of M. baccata, stems of M. micromalus and leaves of M. baccata and M. hupehensis (Figure 3a1–a3). With excess Cu exposure, O2·− concentration was significantly lower in the leaves of M. micromalus and M. prunifolia than that in the other three apple rootstocks. The concentration of H2O2 in the roots of M. baccata and M. hupehensis was higher than that in the other three apple rootstocks (Figure 3b1). High Cu levels resulted in significant increases in H2O2 accumulation in roots of all apple rootstocks. In stems, the H2O2 concentration was significantly enhanced by 89.3% and 77.0% in M. baccata and M. hupehensis, respectively, when exposed to 20 μM Cu, compared with their respective controls under non-elevated Cu conditions (Figure 3b2). However, H2O2 concentration remained unaffected in stems of M. asiatica, M. micromalus and M. prunifolia. In leaves, excess Cu led to significant increases in H2O2 accumulation in all apple rootstocks, but this effect was less pronounced in M. asiatica and M. prunifolia (Figure 3b3). Figure 3. View largeDownload slide O2·− (a1–a3), H2O2 (b1–b3) and MDA (c1–c3) in roots (a1–c1), stems (a2–c2) and leaves (a3–c3) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; M,: M. prunifolia Borkh. Figure 3. View largeDownload slide O2·− (a1–a3), H2O2 (b1–b3) and MDA (c1–c3) in roots (a1–c1), stems (a2–c2) and leaves (a3–c3) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; M,: M. prunifolia Borkh. Malondialdehyde (MDA), an indicator of cell membrane lipid oxidation, was measured in the apple rootstocks to evaluate oxidative stress (Figure 3c1–c3). Changes in MDA concentration were species specific. After 20 μM Cu exposure for 12 days, MDA concentration significantly increased in all plants compared with plants under non-toxic Cu conditions. M. hupehensis displayed the highest, but M. micromalus and M. prunifolia showed lowest MDA accumulation in roots of plants exposed to excess Cu relative to their respective controls (Figure 3c1). Significant effects of excess Cu on MDA concentration in stems and leaves were also found in all apple rootstocks. Remarkably higher MDA concentration was found in stems of M. hupehensis (82.9%) and in leaves of M. baccata (40.1%) with elevated Cu exposure than those with basal Cu supply. However, upon exposure to high levels of Cu, MDA concentration increased by only 34.7% and 27.8% in stems and leaves of M. prunifolia, respectively, compared with that of the control plants (Figure 3c2–c3). Total soluble sugars and starch Changes in the concentrations of total soluble sugars and starch under Cu stress were species specific (Figure 4). Total soluble sugar concentration declined by 11.6–17.5% in the roots of M. micromalus and M. prunifolia when exposed to 20 μM Cu compared with the controls, but it remained unaffected in the roots of the other three apple rootstocks exposed to elevated Cu (Figure 4a1). In stems, the concentration of total soluble sugars was remarkably enhanced in M. asiatica, M. baccata, M. micromalus and M. prunifolia by 33.8%, 52.4%, 28.8% and 70.6%, respectively, under the excess Cu treatment compared with those with non-toxic Cu supply (Figure 4a2). Excess Cu only led to significant increases in total soluble sugars in the leaves of M. micromalus and M. prunifolia (Figure 4a3). The highest concentration of starch was found in the roots of M. asiatica and M. prunifolia, the stems of M. asiatica and the leaves of M. prunifolia under excess Cu conditions (Figure 4b1–b3). Figure 4. View largeDownload slide Total soluble sugars (a1–a3) and starch (b1–b3) in roots (a1 and b1), stems (a2 and b2) and leaves (a3 and b3) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; ****P ≤ 0.0001; ns, not significant. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Figure 4. View largeDownload slide Total soluble sugars (a1–a3) and starch (b1–b3) in roots (a1 and b1), stems (a2 and b2) and leaves (a3 and b3) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; ****P ≤ 0.0001; ns, not significant. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Non-enzymatic and enzymatic antioxidants The highest concentration of free proline was found in the roots of M. prunifolia (Figure 5a1). Generally, the concentration of free proline increased in all tissues of most apple rootstocks, especially in the leaves, when exposed to elevated Cu (Figure 5a1–a3). Conversely, excess Cu decreased soluble phenolics in roots, stems and leaves of all plants except for the stems of M. baccata, M. hupehensis and M. prunifolia, and the leaves of M. prunifolia, relative to plants under non-toxic Cu conditions (Figure 5b1–b3). The concentration of ASC was enhanced by excess Cu exposure in roots, stems and leaves of all apple rootstocks, except for leaves of M. hupehensis (Figure 5c1–c3). After exposure to excess Cu, the highest concentration of ASC was observed in the roots of M. asiatica, the stems of M. micromalus and the leaves of M. prunifolia. Total thiols (T-SH) in the roots of all plants showed a similar Cu response to that of the soluble phenolics in roots (Figure 5d1). By contrast, the concentration of T-SH was significantly higher in the leaves of all apple rootstocks, except for M. hupehensis, exposed to high Cu compared with the controls. The T-SH in the stems of different apple rootstocks displayed distinct responses to Cu stress (Figure 5d2). Upon excessive Cu exposure, the concentration of GSH was only significantly induced in the stems of M. baccata and M. hupehensis and the leaves of M. prunifolia (Figure 5e1–e3). Figure 5. View largeDownload slide Free proline (a1–a3), soluble phenolics (b1–b3), ASC (c1–c3), T-SH (d1–d3) and GSH (e1–e3) in roots (a1–e1), stems (a2–e2) and leaves (a3–e3) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Figure 5. View largeDownload slide Free proline (a1–a3), soluble phenolics (b1–b3), ASC (c1–c3), T-SH (d1–d3) and GSH (e1–e3) in roots (a1–e1), stems (a2–e2) and leaves (a3–e3) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. The species-specific differences in antioxidant enzymes were significant in all tissues of the different apple rootstocks (Figure 6). Superoxide dismutase (SOD) activities were significantly enhanced by 170.6% and 165.3% in the roots of M. micromalus and M. prunifolia after the imposition of elevated Cu (Figure 6a1). In contrast, excess Cu markedly suppressed the activities of SOD in the leaves of all plants compared with those under non-elevated Cu conditions, but this effect was less pronounced in M. prunifolia (Figure 6a3). Generally, POD activities were higher in roots, stems and leaves of analyzed apple rootstocks with the exception of the stems of M. baccata, M. hupehensis and M. micromalus exposed to elevated Cu compared with those under non-toxic Cu conditions. Peroxidase (POD) activities were always higher in the leaves of M. micromalus and M. prunifolia than in the leaves of the other three apple rootstocks, irrespective of whether Cu supply was basal or in excess (Figure 6b3). Upon elevated Cu exposure, CAT activities were increased by 37.5%, 26.8% and 47.0% in the roots of M. asiatica, M. hupehensis and M. prunifolia, respectively, but were suppressed in the stems and leaves of all rootstocks except for the stems of M. prunifolia (Figure 6c1–c3). The activities of APX were elevated in the roots of all rootstocks especially in M. prunifolia exposed 20 μM Cu compared with the controls. By contrast, APX activities were only enhanced in the stems of M. prunifolia, and the leaves of M. baccata and M. prunifolia (Figure 6d2–d3). Excess Cu resulted in higher GR activities in the roots of M. baccata, M. micromalus and M. prunifolia, and the stems of M. baccata and M. prunifolia, while repressed GR activities in the leaves of all plants with the lowest reduction found in M. prunifolia (Figure 6 e1–e3). Figure 6. View largeDownload slide Superoxide dismutase (SOD) (a1–a3), POD (b1–b3), CAT (c1–c3), APX (d1–d3) and GR (e1-e3) in roots (a1–e1), stems (a2–e2) and leaves (a3–e3) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001; ns, not significant. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Figure 6. View largeDownload slide Superoxide dismutase (SOD) (a1–a3), POD (b1–b3), CAT (c1–c3), APX (d1–d3) and GR (e1-e3) in roots (a1–e1), stems (a2–e2) and leaves (a3–e3) of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). Different letters on the bars for the same tissue indicate significant differences between the treatments. P values of the ANOVAs of Cu, species (S) and their interaction (Cu × S) are indicated. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001; ns, not significant. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Transcriptional regulation of genes involved in Cu uptake, transport and detoxification To obtain information on the molecular responses to Cu in the investigated rootstocks, transcript levels of 13 genes involved in Cu accumulation, transport and detoxification were tested (Figure 7). In the roots, COPT1, COPT2, COPT6, ZIP2 and ZIP4 are probably involved in Cu+/Cu2+ uptake. The transcript levels of these genes displayed marked differences among the different rootstocks (Figure 7). Excess Cu led to a downregulation of COPT1, COPT2 and ZIP2 in the roots of all investigated rootstocks compared with the plants under non-toxic Cu conditions (Figure 7). However, the transcript levels of COPT6 and ZIP4 increased in the roots of all rootstocks due to elevated Cu exposure, especially COPT6 in M. prunifolia (Figure 7). Under excessive Cu exposure, the transcript levels of COPT2, COPT6 and ZIP2 were always higher in the roots of M. prunifolia than those in the roots of the other rootstocks. Figure 7. View largeDownload slide Heatmap of genes encoding proteins involved in Cu uptake, transport and detoxification in fine roots of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). The expression level was set to 1 for each gene in roots of M. asiatica exposed to basal Cu. The gene expression heatmap was generated on the log base 2 average expression fold values. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. Figure 7. View largeDownload slide Heatmap of genes encoding proteins involved in Cu uptake, transport and detoxification in fine roots of five apple rootstocks exposed to either basal or excess Cu for 12 days. Data indicate means ± SE (n = 6). The expression level was set to 1 for each gene in roots of M. asiatica exposed to basal Cu. The gene expression heatmap was generated on the log base 2 average expression fold values. Ma, M. asiatica Nakai.; Mb, M. baccata Borkh.; Mh, M. hupehensis Rehd.; Mm, M. micromalus Makino.; Mp, M. prunifolia Borkh. After HMs are bound by chelators, the HM-containing complexes can be transported from the cytosol to vacuoles by ABCC2 (Won-Yong et al. 2010, Sharma et al. 2016). The mRNA levels of ABCC2, a homolog of AtABCC2, were elevated in the roots of all apple rootstocks treated by elevated Cu compared with the controls under basal Cu conditions (Figure 7). The transcript levels of ABCC2 were the highest in the roots of M. prunifolia and were the lowest in the roots of M. hupehensis in the presence of Cu (Figure 7). COPT5, which participates in Cu exportation from the vacuole to the cytosol, decreased in the roots of those rootstocks exposed to excess Cu, with the most reduction found in M. prunifolia (Figure 7). The genes of CCS and CSD play a pivotal role in the regulation of oxidative stress protection. The transcript levels of the homologous gene AtCCS in M. micromalus and M. prunifolia were 6.1–22.5- and 1.9–6.8- folds higher, respectively, than those in the other rootstocks after the imposition of excess Cu (Figure 7). High Cu levels led to the downregulation of CCS in the roots of M. asiatica, M. baccata and M. hupehensis, and the repression was more pronounced in M. hupehensis than in the other rootstocks (Figure 7). Similar to CCS, excess Cu led to a drastic reduction in the transcription of CSD1 in M. asiatica and M. hupehensis, while the transcript levels of this gene were significantly enhanced in the other apple rootstocks, especially M. prunifolia (Figure 7). The highest mRNA levels of CSD1 were found in the roots of M. prunifolia under elevated Cu conditions, on average 4.3-fold higher than in the roots of the other rootstocks (Figure 7). Metallothioneins (MTs) are involved in HM chelation and effective in metal detoxification (Benavides et al. 2005). In roots, the mRNA levels of the orthologous gene of AtMT2 varied significantly among the different rootstocks (Figure 7). Enhanced mRNA levels of MT2 were detected in the roots of M. prunifolia compared with the other rootstocks, irrespective of the Cu treatment applied. Excess Cu led to the downregulation of MT2 in all rootstocks, but a lower decrease was observed in the roots of M. prunifolia (Figure 7). Nicotianamine synthase (NAS), which catalyzes NA synthesis, confers plants tolerance to metals including Cu (Lin and Aarts 2012). High levels of variation were observed in the roots for mRNA levels of NAS1 among the different rootstocks (Figure 7). The transcript levels of NAS1 in M. prunifolia were 1.0–9.6-fold higher than those in the other four rootstocks under basal Cu conditions. The transcript levels of NAS1 were enhanced by excess Cu exposure in M. baccata and M. micromalus, but the opposite effect was observed in the other three rootstocks (Figure 7). Both genes of YSL3 and HMA5 are involved in Cu translocation from the roots to the aerial parts. After elevated Cu exposure, root YSL3 expression was downregulated in M. asiatica and M. prunifolia, while the opposite trend was observed in the other three rootstocks compared with their respective controls (Figure 7). The transcript levels of YSL3 were the highest in the roots of M. hupehensis but were the lowest in the roots of M. prunifolia under excess Cu conditions (Figure 7). The transcript levels of HMA5 in M. baccata, M. hupehensis and M. micromalus were 1.57-, 1.83- and 1.37- fold higher under high Cu level conditions, while they declined in the other two rootstocks (Figure 7). Discussion Copper tolerance, accumulation and subcellular distribution varied greatly among different apple rootstocks In plants, many indicators such as photosynthesis, chlorophyll, tissue biomass and root configuration have been used as indicators to evaluate metal toxicity and tolerance (He et al. 2015, Zhou et al. 2017). Growth inhibition is frequently found in plants exposed to excess levels of Cu (Yadav et al. 2018). For many crops, the critical toxicity level of Cu is believed to be ~20 μg g−1 of leaf dry matter (Kabata and Pendias 1992). After elevated Cu exposure, leaf Cu concentration of the five apple rootstocks ranged from 5.2 to 11.3 μg g−1 dry weight (DW) (Figure 1c). However, tissue biomass, total root length, total root surface area and total root volume of all rootstocks were severely affected by excess Cu (Table 1), indicating that these rootstocks are relatively sensitive to excess Cu under hydroponic conditions. Hydroponics is an easy and key experimental method to evaluate metal accumulation and tolerance in plants (Dos Santos Utmazian et al. 2007, Yang et al. 2015). Although hydroponic systems will change root structure and cannot reflect the absorption abilities under field conditions, they provide the basic information on metal uptake and tolerance mechanisms (Drzewiecka et al. 2012). Previous study has demonstrated that results from hydroponic and field experiments were well correlated for metal accumulation and tolerance in plants (Watson et al. 2003). The growth inhibition caused by excess Cu may be ascribed to interference with photosynthesis and nutrient uptake (Borghi et al. 2008). Among the investigated apple rootstocks, the adverse effects of elevated Cu on biomass and root architecture varied significantly, which suggest that this is broad variation in Cu tolerance among species (cultivars). Similar findings were reported in willow (Cao et al. 2018), rice (Cao et al. 2014) and grapevine (Cambrolle et al. 2015). When exposed to excess Cu, M. prunifolia exhibited the lowest repression in root configuration and reduction in photosynthetic pigments, which are most suitable to compare Cu tolerance of apple rootstocks in the present study (Table 1). Therefore, M. prunifolia appears to have a greater Cu tolerance than the other four rootstocks under the experimental conditions of the current study. Heavy metals confined to the roots could protect the susceptible aerial parts from metal toxicity (Cao et al. 2018). In the present study, the higher Cu concentration and BCF in the roots than in the aerial parts indicated that the investigated apple rootstocks may have an exclusion strategy, a common resistance trait involving holding and detoxifying metals in the roots and limiting translocation to the aerial parts (Figures 1 and 2), which would be in accordance with previous studies (Cao et al. 2018, Feng et al. 2018). Moreover, retaining the majority of metals in the roots would be beneficial to avoid leaching and toxic metal transfer into the food chain, which would ultimately affect human health (Chen et al. 2018). In the aerial parts, Cu was mainly accumulated in the stems of apple rootstocks (Figure 1b), likely protecting the photosynthetic organs in the leaves from Cu toxicity. It has long been recognized that Cu accumulation and tolerance abilities differ greatly among different species or varieties (Cao et al. 2018), which makes it possible and feasible to seek crops with relatively low Cu accumulation in the aerial parts and high Cu tolerance. In this study, M. prunifolia exhibited the greatest Cu concentration in the roots but the lowest Tf when compared with the other rootstocks (Figures 1 and 2), suggesting that it may have developed defense strategies to restrict HM translocation to its aerial parts, and consequently a relatively higher Cu tolerance. On the contrary, the lower Cu tolerance in M. hupehensis than that of the other rootstocks may be attributed to its higher Cu accumulation in the aerial parts. Previous studies have provided evidence that agrees with this theory, as they indicated that the tolerance of plants to HMs was explained by low metal uptake and translocation (Zhou et al. 2017). The subcellular distribution of Cu is extremely crucial for the accumulation, migration and detoxification of Cu in plants (Wang et al. 2016). Cell walls, which act as the first barrier preventing the entrance of metals into cells, is a major site for apoplastic sequestration of HMs to limit their transport into the cytoplasm (Fu et al. 2011). Likewise, vacuoles represent key sites for the storage of excessive metals after being chelated by cytosolic ligands such as sulfur-rich peptides and organic acids (Clemens et al. 2002). In the current study, most Cu retention in the roots was an important strategy to lower its toxicity to the aerial parts, which may play a key role in Cu tolerance of apple rootstocks. Compared with the other rootstocks, M. prunifolia exhibited the highest Cu accumulation in the roots but suffered less Cu injuries, thus we expected roots of this species may adopt well strategy in Cu immobilization and detoxification at the subcellular level. Based on the preliminary experiments, our analysis focused on the subcellular distribution of Cu in the roots (Table 2). After exposure to elevated levels of Cu, most Cu in the roots of the different rootstocks was stored in the cell walls and in the soluble fraction mostly containing vacuoles (Mwamba et al. 2016, Wang et al. 2016). Only a small proportion of Cu was detected in the membrane and organelles, suggesting that Cu compartmentalization in the cell wall is effective and potentially the most important mechanism for Cu tolerance in apple rootstocks, which was consistent with previous study (Xu et al. 2013). However, the opposite results were observed in tomato seedlings, which stored more Cu in vacuoles than in cell walls (Dong et al. 2013). These contrasting results may be suggestive of species-specific processes. Overall, Cu compartmentalization in the cell walls of roots could protect sensitive membranes and organelles from Cu toxicity and further limit Cu transport from the roots to the aerial parts. This may be confirmed by the lower Cu concentration observed in the aerial parts of the apple rootstocks (Figures 1 and 2). The subcellular distribution of Cu in different fractions of roots exhibited great differences among the different rootstocks, which may be attributed to the different levels of Cu tolerance in the different plants (Wang et al. 2016). Although cell walls appeared instrumental in counteracting Cu toxicity, the percentage of Cu retained in this site was significantly different in the roots of the different rootstocks, with <50% in M. asiatica, M. baccata and M. hupehensis (Table 2). Previous studies have reported that >50% of Cu was retained in the cell walls in Hydrilla verticillata (Xu et al. 2013), while the opposite results were found in Brassica napus (Mwamba et al. 2016). These results appear to indicate that variation in cell wall binding capacity could partially account for the variable level of metal tolerance within and among plant species. Compared with the other four rootstocks, higher Cu concentration or distribution ratio of the cell wall fraction and lower Cu distribution ratio of the membrane-containing fraction and the organelle fraction was found in M. prunifolia (Table 2). This indicates that this species may have a higher Cu tolerance and adopt more efficient strategies to prevent Cu toxicity and restrict Cu translocation. Moreover, it is interesting to note that a large percentage of Cu was distributed in the membrane-containing fraction in plants under basal Cu conditions. However, excess Cu decreased this ratio and the Cu distribution ratio of the organelles fraction in most apple rootstocks except for M. baccata and M. hupehensis (Table 2), suggesting that these species have a relatively weaker ability to prevent Cu interfering with the organelles and a lower Cu tolerance, which is in line with the more severe growth inhibition observed (Table 1). Carbohydrate status and antioxidant defense play key roles in Cu tolerance of apple rootstocks It has been well documented that excess Cu exposure can cause the accumulation of ROS, leading to oxidative stress (Thounaojam et al. 2012). In herbaceous plants, Cu-induced oxidative stress can cause programmed cell death leading to decreased growth (Marzilli et al. 2018). In the current study, overproduction of O2·− and H2O2 and the accumulation of MDA were observed in the tissues of the plants exposed to elevated Cu (Figure 3). Therefore, Cu-induced decline in growth and reduction in photosynthetic pigments of the five apple rootstocks were associated with Cu-induced oxidative stress (Table 1 and Figure 3). More Cu was accumulated in the roots of M. prunifolia (Figure 1), but lower concentrations of H2O2 and MDA in roots and O2·− in leaves were observed in M. prunifolia (Figure 3), when compared with the other rootstocks in the presence of elevated Cu. These results indicate that M. prunifolia is more robust, and it may experience less oxidative stress under high levels of Cu exposure, which is probably related to its well-coordinated physiological regulatory mechanisms. A comparative analysis of carbohydrate changes in response to high stress levels in plants differing in metal tolerance could be another way to detect possible tolerance traits (He et al. 2013a, Zhou et al. 2017). Total soluble sugars and starch, which can be immediately hydrolyzed to soluble sugars if needed, will provide energy to counteract metal-induced oxidative stress (Ding et al. 2017, Shi et al. 2015). In accordance with the lower accumulation of ROS and MDA, the increases in total soluble sugars in the stems and leaves and starch in the stems of M. prunifolia exposed to elevated Cu (Figure 4) may have contributed to osmotic regulation and oxidant detoxification as observed for willow species (Cao et al. 2018). In the present study, the antioxidant levels of the rootstocks greatly varied (Figures 5 and 6), indicating that these plants have different antioxidant responses to excess Cu. The stimulation of free proline and ASC in all tissues and T-SH in the leaves of most of the analyzed rootstocks was proposed to be part of a strategy for alleviating Cu-induced oxidative stress, as observed in other plants such as willow and Brassica juncea (Yadav et al. 2017, Cao et al. 2018). However, changes in soluble phenolics in the rootstocks were modest (Figure 5), which does not support the theory of an important role in apple rootstocks for scavenging excess Cu-induced ROS. Glutathione (GSH) is a low molecular weight thiol compound, which can chelate directly with metal and scavenge overproduced ROS in metal-stressed plants (Seth et al. 2012). Moreover, GSH can be utilized as a precursor for the biosynthesis of phytochelatins (PCs), which are necessary for chelating metal in the cytosol and vacuoles of plant cells (Luo et al. 2016, Szalai et al. 2009). The application of exogenous GSH reduced cadmium (Cd)-induced H2O2 production and alleviated Cd toxicity in poplar trees (Ding et al. 2017). A higher accumulation of free proline in roots and stems, soluble phenolics in leaves and ASC and GSH in leaves of M. prunifolia suggests higher Cu tolerance in this species than the other four rootstocks, which corresponds well to the lower ROS and MDA accumulation (Figures 3 and 5). In general, excess Cu enhanced the activities of antioxidant enzymes in the roots of most rootstocks, but the trend in SOD, CAT and APX was the opposite in the leaves of all plants (Figure 6). These results suggest that the response of antioxidant enzyme activities to excess Cu in leaves is different from that of the roots of apple rootstocks. This is in line with previous results from maize (Merlos et al. 2016). Compared with antioxidant enzymes, the strong increase in the concentrations of free proline, ASC, T-SH and GSH suggest that non-enzymatic metabolites may be more important as ROS scavengers in leaves of apple rootstocks (Figure 5). Compared with the other four rootstocks, higher activities of all analyzed enzymes in roots and SOD, APX and GR in leaves were found in M. prunifolia under excess Cu conditions (Figure 6), indicating that this species has a higher free radical-scavenging capacity. This was corroborated by the lower ROS concentration and lipid peroxidation that resulted in a higher Cu tolerance. Different gene expression patterns underlying the variation in Cu accumulation and tolerance in apple rootstocks Alongside the excess Cu-induced distinct physiological responses including Cu accumulation and tolerance, different transcriptional regulation of genes involved in Cu uptake, detoxification and transport was also observed in the roots of the analyzed apple rootstocks (Figure 7). The differential transcriptional expression of COPT1, COPT2, COPT6, ZIP2 and ZIP4 in the roots of the rootstocks in response to excess Cu (Figure 7) suggests that these genes play key roles in Cu uptake. In the roots of Populus trichocarpa, the transcript levels of PtCOPT1 and PtCOPT2 were negatively regulated by Cu, while PtCOPT6 expression was enhanced under excess Cu conditions (Zhang et al. 2015). Furthermore, COPT1 antisense transgenic plants displayed a decrease in Cu uptake (Sancenón et al. 2004). In agreement with these results, excess Cu led to decreased mRNA levels of COPT1 and COPT2 but increased mRNA levels of COPT6 in apple rootstocks (Figure 7). In addition, enhanced expression of ZIP2 resulted in increased Cu accumulation in Arabidopsis thaliana (Wintz et al. 2003). In the present study, relatively higher mRNA levels of COPT2, COPT6, ZIP2 and ZIP4 were found in the roots of M. prunifolia during exposure to excess Cu compared with the other rootstocks, which was consistent with the highest Cu accumulation in roots of this species (Figure 1a). Although M. prunifolia accumulated the highest Cu in its roots, the negative effects of Cu on plant growth were less pronounced in this species, indicating that it may adopt a more efficient strategy to deal with Cu toxicity. It has been shown that the ABC transporter gene PxABCC2 was more induced by excess Cu in Populus deltoides than in Populus canadensis, thereby resulting in less cellular oxidative damage in P. deltoides (Benyo et al. 2016). Similarly, the transcript levels of this gene were induced in the roots of the apple rootstocks in response to excess Cu. Higher expression levels of ABCC2 were found in M. prunifolia (Figure 7), corresponding with its higher Cu tolerance. In A. thaliana, AtCOPT5 is implicated in vacuolar remobilization of Cu to different organs (Shahid et al. 2014, Sharma et al. 2016). Evidence from a previous study demonstrated that the copt5 T-DNA insertion mutants of A. thaliana accumulated more Cu in vacuoles than the wild-type plants with a simultaneous decline in Cu content in the siliques and seeds (Klaumann et al. 2011). Consistently, excess Cu led to the downregulation of COPT5 in all apple rootstocks (Figure 7), indicating that apple rootstocks may adopt a strategy to restrict Cu remobilization and transport to other tissues. Malus prunifolia exhibited higher Cu accumulation in roots but lower Cu translocation to the above-ground tissues (Figures 1 and 2), which may be partially attributed to the lower transcript levels of COPT5. Under HM exposure conditions, plants can regulate the mRNA levels of several genes involved in the detoxification of HMs, including Cu (Guo et al. 2008, Lin and Aarts 2012). The upregulation of AtCCS and AtCSD1 were found in both the roots and shoots of A. thaliana in response to excess Cu to reduce Cu toxicity (del Pozo et al. 2010). Similarly, transgenic tobacco with overexpressed grapevine VvCSD1 and VvCSD2 had lower Cu sensitivity and higher Cu tolerance compared with the wild type (Leng et al. 2017). In the present study, the species-specific differences in the transcriptional regulation of these two genes are probably related to variations in Cu tolerance. Upregulated CCS and CSD1 mRNA levels were detected in the roots of M. prunifolia more so than in those of the other apple rootstocks (Figure 7). This resulted in higher levels of SOD activity (Figure 6), which may lead to a greater capacity for excess Cu detoxification. In plants, HM tolerance is also associated with the ability to complex the metal by ion ligands or chelators in the cytosol (Shahid et al. 2014). Greater Cu and Cd tolerance were observed in those A. thaliana plants that overexpressed the Brassica juncea gene encoding metallothionein 2 (MT2) in comparison with wild-type seedlings (An et al. 2006). In the present study, Cu decreased the mRNA levels of MT2 in all apple rootstocks, which does not infer an important role in apple rootstocks for Cu detoxification. However, the higher expression of MT2 in M. prunifolia compared with the other apple rootstocks irrespective of Cu treatment may lead to a relatively greater capacity for Cu complexation and detoxification (Figure 7). Figure 8. View largeDownload slide A schematic model for tolerant apple rootstocks with enhanced Cu tolerance compared with sensitive species. Cu+/Cu2+ uptake and accumulation in tolerant rootstocks (top left) and sensitive rootstocks (top right) and the molecular mechanisms of excess Cu transport and detoxification in tolerant rootstocks (bottom). Tolerant rootstocks exhibited higher Cu accumulation in roots but lower root-to-shoot translocation capacities and enhanced antioxidant defense system, resulting in lower growth reduction than sensitive rootstocks. FRO4/5: ferric reductase oxidase 4/5; COPT1, COPT2, COPT5 and COPT6: copper transporters 1, 2, 5 and 6; ZIP2 and ZIP4: zinc/iron-regulated transporter-related proteins 2 and 4; ABCC2: ATP-binding cassette transporter 2; CCS: Cu/Zn superoxide dismutase; CSD1: Cu/Zn SOD enzyme 1; MT2: metallothionein 2; NAS1: nicotianamine synthase 1; YSL3: yellow stripe-like transporter 3; HMA5: P-type heavy metal ATPase 5. Figure 8. View largeDownload slide A schematic model for tolerant apple rootstocks with enhanced Cu tolerance compared with sensitive species. Cu+/Cu2+ uptake and accumulation in tolerant rootstocks (top left) and sensitive rootstocks (top right) and the molecular mechanisms of excess Cu transport and detoxification in tolerant rootstocks (bottom). Tolerant rootstocks exhibited higher Cu accumulation in roots but lower root-to-shoot translocation capacities and enhanced antioxidant defense system, resulting in lower growth reduction than sensitive rootstocks. FRO4/5: ferric reductase oxidase 4/5; COPT1, COPT2, COPT5 and COPT6: copper transporters 1, 2, 5 and 6; ZIP2 and ZIP4: zinc/iron-regulated transporter-related proteins 2 and 4; ABCC2: ATP-binding cassette transporter 2; CCS: Cu/Zn superoxide dismutase; CSD1: Cu/Zn SOD enzyme 1; MT2: metallothionein 2; NAS1: nicotianamine synthase 1; YSL3: yellow stripe-like transporter 3; HMA5: P-type heavy metal ATPase 5. The HM–NA complexes can be translocated across membrane systems by YSL proteins localized in the plasma membrane for long-distance translocation from roots to shoots (Waters et al. 2006). Greater Zn concentration in young leaves and seeds and Cu concentration in sepals were observed in transgenic tobacco overexpressing NAS compared with the wild-type plants (Takahashi et al. 2003). Similarly, the expression of AtYSL3 was lower in wild-type plants than it was in the small ubiquitin-like modifier E3 ligase (siz1) mutant under excess Cu stress. This led to a lower shoot-to-root ratio of Cu concentration in the wild-type plants compared with the mutants (Chen et al. 2011). These results indicate that increased NAS and YSL expression are important for metal translocation from roots to shoots in plants. In the present study, the lowest expression levels of both genes were found in M. prunifolia (Figure 7), which was consistent with the observation of the lowest Cu root-to-shoot translocation in M. prunifolia than in the other apple rootstocks (Figure 2). Excess Cu led to the downregulation of NAS1 and YSL3 in M. asiatica and M. prunifolia (Figure 7), indicating that these two species could be more sufficient at preventing excessive translocation of Cu from their roots to their aerial parts, reducing its toxicity in the aerial parts. Alternatively, cytosolic HM ions, including those of Cu, can also be excluded from the apoplast by HMA5, and this process is important for HM uptake and xylem loading in the root steles in plants (Deng et al. 2013). Evidence from previous research has demonstrated that increased Cu concentration in the roots coupled with a decreased Cu concentration of xylem sap were observed in OsHMA5 mutants compared with the wild-type rice (Deng et al. 2013). In agreement with the higher Cu accumulation in roots and lower Cu translocation from roots to the aerial parts in M. prunifolia compared with other apple rootstocks, the transcript levels of HMA5 were the lowest in the roots of M. prunifolia under Cu exposure conditions (Figures 1, 2 and 7). Taken together, our results suggest that the lower Cu accumulation in the aerial parts and relatively high Cu tolerance in M. prunifolia may be ascribed to the higher repression of the transcripts of YSL3 and HMA5 (Figures 1, 2 and 7), which are important and effective ways to restrict Cu translocation and reduce its toxicity in the aerial parts. As summarized in Figure 8, large variations in Cu accumulation and tolerance existed among the investigated apple rootstocks. Excess Cu was mainly accumulated in the roots and partially translocated to the aerial parts, thereby resulting in decreases in biomass, root architecture and photosynthetic pigments, which were less pronounced in M. prunifolia, but more pronounced in M. baccata and M. hupehensis. Compared with the other four apple rootstocks, M. prunifolia had the largest concentrations and BCF in the roots, but only moderate Cu concentration in the stems and leaves, and the lowest Tf, indicating that this species may have a lower capability to transport Cu to its aerial parts, which may contribute to its relatively higher Cu tolerance. Copper subcellular distribution is relevant to species differences in terms of Cu tolerance. Copper sequestration in the cell walls played a key role in Cu tolerance and detoxification in apple rootstocks. The large accumulation of Cu led to a burst of ROS and lipid peroxidation in the tissues of apple rootstocks, which were more pronounced in M. baccata and M. hupehensis. After exposure to excess Cu, M. prunifolia had a relatively lower increment of H2O2 in roots and leaves and MDA in roots, but enhanced concentrations of total soluble sugars in stems and leaves, and starch in roots and stems, and enhanced antioxidants. In agreement with more Cu accumulation in roots, lower Cu translocation capacity and higher Cu tolerance, the transcript levels of several genes involved in Cu uptake and detoxification, including COPT2, COPT6, ZIP2, ZIP4, ABCC2, COPT5 and MT2, were higher, whereas the mRNA levels of genes tha participated in Cu transport to the aboveground parts, such as YSL3 and HMA5, were lower in M. prunifolia compared with other species, indicating this species has a relatively higher Cu tolerance than the other four apple rootstocks under the current experimental conditions. Overall, our results indicate that variations in Cu accumulation and tolerance among different apple rootstocks are associated with physiological and molecular regulation mechanisms, including Cu retention in roots, subcellular partitioning, antioxidant defense mechanisms and transcriptional regulation of genes involved in Cu uptake, translocation and detoxification. Conflict of interest None declared. Acknowledgments Special thanks are given to Xiaolei Zhuang, Xu Zhang and Fan Bu for their help in harvesting the plants. Funding This work was funded by the National Natural Science Foundation of China (31501712), the National Key Research and Development Program of China (2016YFD0201115), the Program for Liaoning Excellent Talents in University (LJQ2015098), the China Agriculture Research System (CARS-27) and the Scientific Research Foundation of Talent Introduction of Shenyang Agricultural University (20153007). handling Editor Heinz Rennenberg References An ZG , Li CJ , Zu YG , Du YJ , Wachter A , Gromes R , Rausch T ( 2006 ) Expression of BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana seedlings . J Exp Bot 57 : 3575 – 3582 . Google Scholar Crossref Search ADS PubMed WorldCat Benavides MP , Gallego SM , Tomaro ML ( 2005 ) Cadmium toxicity in plants . Braz J Plant Physiol 17 : 21 – 34 . Google Scholar Crossref Search ADS WorldCat Benyo DE , Horvath E , Nemeth T et al. ( 2016 ) Physiological and molecular responses to heavy metal stresses suggest different detoxification mechanism of Populus deltoides and P. × canadensis . J Plant Physiol 201 : 62 – 70 . Google Scholar Crossref Search ADS PubMed WorldCat Borghi M , Tognetti R , Monteforti G , Sebastiani L ( 2008 ) Responses of two poplar species (Populus alba and Populus × canadensis) to high copper concentrations . Environ Exp Bot 62 : 290 – 299 . Google Scholar Crossref Search ADS WorldCat Branzini A , Gonzalez RS , Zubillaga M ( 2012 ) Absorption and translocation of copper, zinc and chromium by Sesbania virgata . J Environ Manage 102 : 50 – 54 . Google Scholar Crossref Search ADS PubMed WorldCat Brunetto G , Bastos de Melo GW , Terzano R , Del D , Astolfi S , Tomasi N , Pii Y , Mimmo T , Cesco S ( 2016 ) Copper accumulation in vineyard soils: rhizosphere processes and agronomic practices to limit its toxicity . Chemosphere 162 : 293 – 307 . Google Scholar Crossref Search ADS PubMed WorldCat Cambrolle J , Garcia JL , Figueroa ME , Cantos M ( 2015 ) Evaluating wild grapevine tolerance to copper toxicity . Chemosphere 120 : 171 – 178 . Google Scholar Crossref Search ADS PubMed WorldCat Cao F , Wang R , Cheng W , Zeng F , Ahmed IM , Hu X , Zhang G , Wu F ( 2014 ) Genotypic and environmental variation in cadmium, chromium, lead and copper in rice and approaches for reducing the accumulation . Sci Total Environ 496 : 275 – 281 . Google Scholar Crossref Search ADS PubMed WorldCat Cao Y , Zhang Y , Ma C , Li H , Zhang J , Chen G ( 2018 ) Growth, physiological responses, and copper accumulation in seven willow species exposed to Cu-a hydroponic experiment . Environ Sci Pollut Res Int 25 : 19875 – 19886 . Google Scholar Crossref Search ADS PubMed WorldCat Chen CC , Chen YY , Tang IC , liang HM , Lai CC , Chiou JM , Yeh KC ( 2011 ) Arabidopsis SUMO E3 ligase SIZ1 is involved in excess copper tolerance . Plant Physiol 156 : 2225 – 2234 . Google Scholar Crossref Search ADS PubMed WorldCat Chen J , Liu YQ , Yan XW , Wei GH , Zhang JH , Fang LC ( 2018 ) Rhizobium inoculation enhances copper tolerance by affecting copper uptake and regulating the ascorbate-glutathione cycle and phytochelatin biosynthesis-related gene expression in Medicago sativa seedlings . Ecotox Environ Safe 162 : 312 – 323 . Google Scholar Crossref Search ADS WorldCat Chen PY , Lee YI , Chen BC , Juang KW ( 2013 ) Effects of calcium on rhizotoxicity and the accumulation and translocation of copper by grapevines . Plant Physiol Bioch 73 : 375 – 382 . Google Scholar Crossref Search ADS WorldCat Chu CC , Lee WC , Guo WY , Pan SM , Chen LJ , Li HM , Jinn TL ( 2005 ) A copper chaperone for superoxide dismutase that confers three types of copper/zinc superoxide dismutase activity in Arabidopsis . Plant Physiol 139 : 425 – 436 . Google Scholar Crossref Search ADS PubMed WorldCat Clemens S , Palmgren MG , Kramer U ( 2002 ) A long way ahead: understanding and engineering plant metal accumulation . Trends Plant Sci 7 : 309 – 315 . Google Scholar Crossref Search ADS PubMed WorldCat del T , Cambiazo V , Gonzalez M ( 2010 ) Gene expression profiling analysis of copper homeostasis in Arabidopsis thaliana . Biochem Bioph Res Co 393 : 248 – 252 . Google Scholar Crossref Search ADS WorldCat Deng F , Yamaji N , Xia J , Ma JF ( 2013 ) A member of the heavy metal P-type ATPase OsHMA5 is involved in xylem loading of copper in rice . Plant Physiol 163 : 1353 – 1362 . Google Scholar Crossref Search ADS PubMed WorldCat Ding S , Ma CF , Shi WG , Liu W , Lu Y , Liu QF , Luo ZB ( 2017 ) Exogenous glutathione enhances cadmium accumulation and alleviates its toxicity in Populus × canescens . Tree Physiol 37 : 1697 – 1712 . Google Scholar Crossref Search ADS PubMed WorldCat Dong YX , Wang XF , Cui XM ( 2013 ) Exogenous nitric oxide involved in subcellular distribution and chemical forms of Cu2+ under copper stress in tomato seedlings . J Integr Agr 12 : 1783 – 1790 . Google Scholar Crossref Search ADS WorldCat Dos Santos Utmazian MN , Wieshammer G , Vega R , Wenzel WW ( 2007 ) Hydroponic screening for metal resistance and accumulation of cadmium and zinc in twenty clones of willows and poplars . Environ Pollut 148 : 155 – 165 . Google Scholar Crossref Search ADS PubMed WorldCat Drzewiecka K , Mleczek M , Gasecka M , Magdziak Z , Golinski P ( 2012 ) Changes in Salix viminalis L. cv. ‘Cannabina’ morphology and physiology in response to nickel ions-hydroponic investigations . J Hazard Mater 217-218 : 429 – 438 . Google Scholar Crossref Search ADS PubMed WorldCat Fan J , He Z , Ma LQ , Stoffella PJ ( 2011 ) Accumulation and availability of copper in citrus grove soils as affected by fungicide application . J Soil Sediment 11 : 639 – 648 . Google Scholar Crossref Search ADS WorldCat Farmer EE , Mueller MJ ( 2013 ) ROS-mediated lipid peroxidation and RES-activated signaling . Annu Rev Plant Biol 64 : 429 . Google Scholar Crossref Search ADS PubMed WorldCat Feng J , Lin Y , Yang Y , Shen Q , Huang J , Wang S , Zhu X , Li Z ( 2018 ) Tolerance and bioaccumulation of Cd and Cu in Sesuvium portulacastrum . Ecotox Environ Safe 147 : 306 – 312 . Google Scholar Crossref Search ADS WorldCat Fu XP , Dou CM , Chen YX , Chen XC , Shi JY , Yu MG , Xu J ( 2011 ) Subcellular distribution and chemical forms of cadmium in Phytolacca americana L . J Hazard Mater 186 : 103 – 107 . Google Scholar Crossref Search ADS PubMed WorldCat Girotto E , Ceretta CA , Brunetto G et al. ( 2014 ) Copper availability assessment of cu-contaminated vineyard soils using black oat cultivation and chemical extractants . Environ Monit Assess 186 : 9051 – 9063 . Google Scholar Crossref Search ADS PubMed WorldCat Guo WJ , Meetam M , Goldsbrough PB ( 2008 ) Examining the specific contributions of individual Araidopsis metallothioneins to copper distribution and metal tolerance . Plant Physiol 146 : 1697 – 1706 . Google Scholar Crossref Search ADS PubMed WorldCat Hall JL ( 2002 ) Cellular mechanisms for heavy metal detoxification and tolerance . J Exp Bot 53 : 1 – 11 . Google Scholar Crossref Search ADS PubMed WorldCat Hamed SM , Selim S , Klöck G , Abdelgawad H ( 2017 ) Sensitivity of two green microalgae to copper stress: growth, oxidative and antioxidants analyses . Ecotox Environ Safe 144 : 19 – 25 . Google Scholar Crossref Search ADS WorldCat He J , Qin J , Long L et al. ( 2011 ) Net cadmium flux and accumulation reveal tissue-specific oxidative stress and detoxification in Populus × canescens . Physiol Plant 143 : 50 – 63 . Google Scholar Crossref Search ADS PubMed WorldCat He J , Ma C , Ma Y , Li H , Kang J , Liu T , Polle A , Peng C , Luo ZB ( 2013a) Cadmium tolerance in six poplar species . Environ Sci Pollut R 20 : 163 – 174 . Google Scholar Crossref Search ADS WorldCat He J , Li H , Luo J et al. ( 2013b) A transcriptomic network underlies microstructural and physiological responses to cadmium in Populus × canescens . Plant Physiol 162 : 424 – 439 . Google Scholar Crossref Search ADS PubMed WorldCat He J , Li H , Ma C , Zhang Y , Polle A , Rennenberg H , Cheng X , Luo ZB ( 2015 ) Overexpression of bacterial gamma-glutamylcysteine synthetase mediates changes in cadmium influx, allocation and detoxification in poplar . New Phytol 205 : 240 – 254 . Google Scholar Crossref Search ADS PubMed WorldCat Henri W , Tama F , Wu YY , Victoria F , Chen W , Chang HS , Zhu T , Chris V ( 2003 ) Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis . J Biol Chem 278 : 47644 – 47653 . Google Scholar Crossref Search ADS PubMed WorldCat Hippler FWR , Cipriano DO , Boaretto RM , Quaggio JA , Gaziola SA , Azevedo RA , Mattos D Jr ( 2016 ) Citrus rootstocks regulate the nutritional status and antioxidant system of trees under copper stress . Environ Exp Bot 130 : 42 – 52 . Google Scholar Crossref Search ADS WorldCat Hossain MA , Piyatida P , da JAT , Fujita M ( 2012 ) Molecular mechanism of heavy metal toxicity and tolerance in plants: central role of glutathione in detoxification of reactive oxygen species and methylglyoxal and in heavy metal chelation . J Bot 2012 : 1 – 37 . Google Scholar Crossref Search ADS WorldCat Huang G , Jin Y , Zheng J , Kang W , Hu H , Liu Y , Zou T ( 2016 ) Accumulation and distribution of copper in castor bean (Ricinus communis L.) callus cultures: in vitro . Plant Cell Tiss Org 128 : 177 – 186 . Google Scholar Crossref Search ADS WorldCat Jung HI , Gayomba SR , Rutzke MA , Craft E , Kochian LV , Vatamaniuk OK ( 2012 ) COPT6 is a plasma membrane transporter that functions in copper homeostasis in Arabidopsis and is a novel target of SQUAMOSA promoter-binding protein-like 7 . J Biol Chem 287 : 33252 – 33267 . Google Scholar Crossref Search ADS PubMed WorldCat Kabata PA , Pendias H ( 1992 ) Trace elements in soils and plants , 2nd edn . CRC Press Inc. , Boca Raton, FL, USA . Google Preview WorldCat COPAC Kang W , Bao J , Zheng J , Hu H , Du J ( 2015 ) Distribution and chemical forms of copper in the root cells of castor seedlings and their tolerance to copper phytotoxicity in hydroponic culture . Environ Sci Pollut Res Int 22 : 7726 – 7734 . Google Scholar Crossref Search ADS PubMed WorldCat Klaumann S , Nickolaus SD , Furst SH , Starck S , Schneider S , Ekkehard Neuhaus H , Trentmann O ( 2011 ) The tonoplast copper transporter COPT5 acts as an exporter and is required for interorgan allocation of copper in Arabidopsis thaliana . New Phytol 192 : 393 – 404 . Google Scholar Crossref Search ADS PubMed WorldCat Komarek M , Cadkova E , Chrastny V , Bordas F , Bollinger JC ( 2010 ) Contamination of vineyard soils with fungicides: a review of environmental and toxicological aspects . Environ Int 36 : 138 – 151 . Google Scholar Crossref Search ADS PubMed WorldCat Lange B , Delhaye G , Boisson S , Verbruggen N , Meerts P , Faucon MP ( 2018 ) Variation in copper and cobalt tolerance and accumulation among six populations of the facultative metallophyte Anisopappus chinensis (Asteraceae) . Environ Exp Bot 153 : 1 – 9 . Google Scholar Crossref Search ADS WorldCat Lai HY ( 2015 ) Subcellular distribution and chemical forms of cadmium in Impatiens walleriana in relation to its phytoextraction potential . Chemosphere 138 : 370 – 376 . Google Scholar Crossref Search ADS PubMed WorldCat Lei YB , Korpelainen H , Li CY ( 2007 ) Physiological and biochemical responses to high Mn concentrations in two contrasting Populus cathayana populations . Chemosphere 68 : 686 – 694 . Google Scholar Crossref Search ADS PubMed WorldCat Leng X , Wang P , Zhu X , Li X , Zheng T , Shangguan L , Fang J ( 2017 ) Ectopic expression of CSD1 and CSD2 targeting genes of miR398 in grapevine is associated with oxidative stress tolerance . Funct Integr Genomics 17 : 697 – 710 . Google Scholar Crossref Search ADS PubMed WorldCat Li W , Zhang M , Shu H ( 2005 ) Distribution and fractionation of copper in soils of apple orchards . Environ Sci Pollut Res Int 12 : 168 – 172 . Google Scholar Crossref Search ADS PubMed WorldCat Li X , Wu Y , Li B , He W , Yang Y , Yang Y ( 2018 ) Genome-wide identification and expression analysis of the cation diffusion facilitator gene family in turnip under diverse metal ion stresses . Front Genet 9 : 103 . Google Scholar Crossref Search ADS PubMed WorldCat Lin YF , Aarts MGM ( 2012 ) The molecular mechanism of zinc and cadmium stress response in plants . Cell Mol Life Sci 69 : 3187 – 3206 . Google Scholar Crossref Search ADS PubMed WorldCat Liu CS , Sun BY , Kan SH , Zhang YZ , Deng SH , Yang G ( 2011 ) Copper toxicity and accumulation in potted seedlings of three apple rootstock species: implications for safe fruit production on copper-polluted soils . J Plant Nutr 34 : 1268 – 1277 . Google Scholar Crossref Search ADS WorldCat Livak KJ , Schmittgen TD ( 2001 ) Analysis of relative gene expression data using real-time quantitative PCR and the 2 -∆∆CT method . Methods 25 : 402 – 408 . Google Scholar Crossref Search ADS PubMed WorldCat Luo J , Zhou J , Li H , Shi W , Polle A , Lu M , Sun X , Luo ZB ( 2015 ) Global poplar root and leaf transcriptomes reveal links between growth and stress responses under nitrogen starvation and excess . Tree Physiol 35 : 1283 – 1302 . Google Scholar Crossref Search ADS PubMed WorldCat Luo ZB , Calfapietra C , Scarascia-Mugnozza G , Liberloo M , Polle A ( 2008 ) Carbon-based secondary metabolites and internal nitrogen pools in Populus nigra under free air CO2 enrichment (FACE) and nitrogen fertilisation . Plant and Soil 304 : 45 – 57 . Google Scholar Crossref Search ADS WorldCat Luo ZB , He J , Polle A , Rennenberg H ( 2016 ) Heavy metal accumulation and signal transduction in herbaceous and woody plants: paving the way for enhancing phytoremediation efficiency . Biotechnol Adv 34 : 1131 – 1148 . Google Scholar Crossref Search ADS PubMed WorldCat Ma Y , He J , Ma C , Luo J , Li H , Liu T , Polle A , Peng C , Luo ZB ( 2014 ) Ectomycorrhizas with Paxillus involutus enhance cadmium uptake and tolerance in Populus × canescens . Plant Cell Environ 37 : 627 – 642 . Google Scholar Crossref Search ADS PubMed WorldCat Marastoni L , Sandri M , Pii Y , Valentinuzzi F , Brunetto G , Cesco S , Mimmo T ( 2019 ) Synergism and antagonisms between nutrients induced by copper toxicity in grapevine rootstocks: Monocropping vs. intercropping . Chemosphere 214 : 563 – 578 . Google Scholar Crossref Search ADS PubMed WorldCat Marzilli M , Di P , Palumbo G , Maiuro L , Paura B , Tognetti R , Cocozza C ( 2018 ) Cd and cu accumulation, translocation and tolerance in Populus alba clone (Villafranca) in autotrophic in vitro screening . Environ Sci Pollut Res Int 25 : 10058 – 10068 . Google Scholar Crossref Search ADS PubMed WorldCat Mellado M , Contreras RA , González A , Dennett G , Moenne A ( 2012 ) Copper-induced synthesis of ascorbate, glutathione and phytochelatins in the marine alga Ulva compressa (Chlorophyta) . Plant Physiol Bioch 51 : 102 – 108 . Google Scholar Crossref Search ADS WorldCat Merlos MA , Zitka O , Vojtech A , Azcon-Aguilar C , Ferrol N ( 2016 ) The arbuscular mycorrhizal fungus Rhizophagus irregularis differentially regulates the copper response of two maize cultivars differing in copper tolerance . Plant Sci 253 : 68 – 76 . Google Scholar Crossref Search ADS PubMed WorldCat Migocka M , Malas K ( 2018 ) Plant responses to copper: molecular and regulatory mechanisms of copper uptake, distribution and accumulation in plants. In: Plant micronutrient use efficiency . Academic Press; Elsevier, Inc. , London , pp. 71 – 86 . Mostofa MG , Hossain MA , Fujita M , Tran LS ( 2015 ) Physiological and biochemical mechanisms associated with trehalose-induced copper-stress tolerance in rice . Sci Rep 5 : 11433 ). Google Scholar Crossref Search ADS PubMed WorldCat Mwamba TM , Li L , Gill RA , Islam F , Nawaz A , Ali B , Farooq MA , Lwalaba JL , Zhou W ( 2016 ) Differential subcellular distribution and chemical forms of cadmium and copper in Brassica napus . Ecotox Environ Safe 134 : 239 – 249 . Google Scholar Crossref Search ADS WorldCat Niu L , Shen Z , Luo C , Deng YE , Wang C ( 2012 ) Accumulation mechanisms and subcellular distribution of cu in maize grown on soil treated with [S, S]-ethylenediamine disuccinic acid . Plant and Soil 351 : 237 – 247 . Google Scholar Crossref Search ADS WorldCat Printz B , Lutts S , Hausman JF , Sergeant K ( 2016 ) Copper trafficking in plants and its implication on cell wall dynamics . Front Plant Sci 7 : 601 . Google Scholar Crossref Search ADS PubMed WorldCat Puig S , Andres-Colas NMA , Penarrubia L ( 2010 ) Copper and iron homeostasis in Arabidopsis: responses to metal deficiencies, interactions and biotechnological applications . Plant Cell Environ 30 : 271 – 290 . Google Scholar Crossref Search ADS WorldCat Sancenón V , Puig S , Mateu-Andrés I , Dorcey E , Thiele DJ , Peñarrubia L ( 2004 ) The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development . J Biol Chem 279 : 15348 – 15355 . Google Scholar Crossref Search ADS PubMed WorldCat Sanz A , Pike S , Khan MA , Carrio-Segui A , Mendoza-Cozatl DG , Penarrubia L , Gassmann W ( 2018 ) Copper uptake mechanism of Arabidopsis thaliana high-affinity COPT transporters . Protoplasma 256 : 161 – 170 . Google Scholar Crossref Search ADS PubMed WorldCat Seth CS , Remans T , Keunen E , Jozefczak M , Gielen H , Opdenakker K , Weyens N , Vangronsveld J , Cuypers A ( 2012 ) Phytoextraction of toxic metals: a central role for glutathione . Plant Cell Environ 35 : 334 – 346 . Google Scholar Crossref Search ADS PubMed WorldCat Shahid M , Pourrut B , Dumat C , Nadeem M , Aslam M , Pinelli E ( 2014 ) Heavy-metal-induced reactive oxygen species: phytotoxicity and physicochemical changes in plants . Rev Environ Contam T 232 : 1 – 44 . WorldCat Sharma SS , Dietz KJ , Mimura T ( 2016 ) Vacuolar compartmentalization as indispensable component of heavy metal detoxification in plants . Plant Cell Environ 39 : 1112 – 1126 . Google Scholar Crossref Search ADS PubMed WorldCat Shi WG , Li H , Liu TX , Polle A , Peng CH , Luo ZB ( 2015 ) Exogenous abscisic acid alleviates zinc uptake and accumulation in Populus × canescens exposed to excess zinc . Plant Cell Environ 38 : 207 – 223 . Google Scholar Crossref Search ADS PubMed WorldCat Szalai G , Kellős T , Galiba G , Kocsy G ( 2009 ) Glutathione as an antioxidant and regulatory molecule in plants under abiotic stress conditions . J Plant Growth Regul 28 : 66 – 80 . Google Scholar Crossref Search ADS WorldCat Takahashi M , Terada Y , Nakai I , Nakanishi H , Yoshimura E , Mori S , Nishizawa NK ( 2003 ) Role of nicotianamine in the intracellular delivery of metals and plant reproductive development . Plant Cell 15 : 1263 – 1280 . Google Scholar Crossref Search ADS PubMed WorldCat Thounaojam TC , Panda P , Mazumdar P , Kumar D , Sharma GD , Sahoo L , Panda SK ( 2012 ) Excess copper induced oxidative stress and response of antioxidants in rice . Plant Physiol Biochem 53 : 33 – 39 . Google Scholar Crossref Search ADS PubMed WorldCat Wang P , Sun X , Li C , Wei Z , Liang D , Ma F ( 2013 ) Long-term exogenous application of melatonin delays drought-induced leaf senescence in apple . J Pineal Res 54 : 292 – 302 . Google Scholar Crossref Search ADS PubMed WorldCat Wang QY , Liu JS , Cheng S ( 2015a) Heavy metals in apple orchard soils and fruits and their health risks in Liaodong peninsula, Northeast China . Environ Monit Assess 187 : 4178 . Google Scholar Crossref Search ADS PubMed WorldCat Wang QY , Liu JS , Wang Y , Yu HW ( 2015b) Accumulations of copper in apple orchard soils: distribution and availability in soil aggregate fractions . J Soil Sediment 15 : 1075 – 1082 . Google Scholar Crossref Search ADS WorldCat Wang QY , Liu JS , Hu B ( 2016 ) Integration of copper subcellular distribution and chemical forms to understand copper toxicity in apple trees . Environ Exp Bot 123 : 125 – 131 . Google Scholar Crossref Search ADS WorldCat Waters BM , Chu HH , Didonato RJ , Roberts LA , Eisley RB , Lahner B , Salt DE , Walker EL ( 2006 ) Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds . Plant Physiol 141 : 1446 – 1458 . Google Scholar Crossref Search ADS PubMed WorldCat Watson C , Pulford ID , Riddell-Black D ( 2003 ) Screening of willow species for resistance to heavy metals: comparison of performance in hydroponics system and field trials . Int J Phytoremediation 5 : 351 – 365 . Google Scholar Crossref Search ADS PubMed WorldCat Wintz H , Fox T , Wu YY , Feng V , Chen W , Chang HS , Zhu T , Vulpe C ( 2003 ) Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis . J Biol Chem 278 : 47644 – 47653 . Google Scholar Crossref Search ADS PubMed WorldCat Won-Yong S , Jiyoung P , Mendoza-Cózatl DG , et al. ( 2010 ) Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters . Proc Natl Acad Sci USA 107 : 21187 – 21192 . Google Scholar Crossref Search ADS PubMed WorldCat Xu Q , Qiu H , Chu W , Fu Y , Cai S , Min H , Sha S ( 2013 ) Copper ultrastructural localization, subcellular distribution, and phytotoxicity in Hydrilla verticillata (L.f.) Royle . Environ Sci Pollut Res Int 20 : 8672 – 8679 . Google Scholar Crossref Search ADS PubMed WorldCat Yadav P , Kaur R , Kanwar MK , Bhardwaj R , Sirhindi G , Wijaya L , Alyemeni MN , Ahmad P ( 2017 ) Ameliorative role of castasterone on copper metal toxicity by improving redox homeostasis in Brassica juncea L . J Plant Growth Regul 4 : 1 – 16 . WorldCat Yadav P , Kaur R , Kanwar MK , Sharma A , Verma V , Sirhindi G , Bhardwaj R ( 2018 ) Castasterone confers copper stress tolerance by regulating antioxidant enzyme responses, antioxidants, and amino acid balance in B. juncea seedlings . Ecotox Environ Safe 147 : 725 – 734 . Google Scholar Crossref Search ADS WorldCat Yang W , Zhao F , Zhang X , Ding Z , Wang Y , Zhu Z , Yang X ( 2015 ) Variations of cadmium tolerance and accumulation among 39 Salix clones: implications for phytoextraction . Environ Earth Sci 73 : 3263 – 3274 . Google Scholar Crossref Search ADS WorldCat Yemm EW , Willis AJ ( 1954 ) The estimation of carbohydrates in plant extracts by anthrone . Biochem J 57 : 508 – 514 . Google Scholar Crossref Search ADS PubMed WorldCat Yruela I ( 2009 ) Copper in plants: acquisition, transport and interactions . Funct Plant Biol 36 : 409 – 430 . Google Scholar Crossref Search ADS WorldCat Zacchini M , Pietrini F , Mugnozza GS , Iori V , Pietrosanti L , Massacci A ( 2009 ) Metal tolerance, accumulation and translocation in poplar and willow clones treated with cadmium in hydroponics . Water Air Soil Poll 197 : 23 – 34 . Google Scholar Crossref Search ADS WorldCat Zhang H , Yang J , Wang W , Li D , Hu X , Wang H , Wei M , Liu Q , Wang Z , Li C ( 2015 ) Genome-wide identification and expression profiling of the copper transporter gene family in Populus trichocarpa . Plant Physiol Bioch 97 : 451 – 460 . Google Scholar Crossref Search ADS WorldCat Zhang S , Lu S , Xu X , Korpelainen H , Li C ( 2010 ) Changes in antioxidant enzyme activities and isozyme profiles in leaves of male and female Populus cathayana infected with Melampsora larici-populina . Tree Physiol 30 : 116 – 128 . Google Scholar Crossref Search ADS PubMed WorldCat Zhou J , Wan H , Qin S , He J , Lyu D , Li H ( 2016 ) Net cadmium flux and gene expression in relation to differences in cadmium accumulation and translocation in four apple rootstocks . Environ Exp Bot 130 : 95 – 105 . Google Scholar Crossref Search ADS WorldCat Zhou J , Wan H , He J , Lyu D , Li H ( 2017 ) Integration of cadmium accumulation, subcellular distribution, and physiological responses to understand cadmium tolerance in apple rootstocks . Front Plant Sci 8 : 966 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Copper accumulation, subcellular partitioning and physiological and molecular responses in relation to different copper tolerance in apple rootstocks
JF - Tree Physiology
DO - 10.1093/treephys/tpz042
DA - 2019-07-18
UR - https://www.deepdyve.com/lp/oxford-university-press/copper-accumulation-subcellular-partitioning-and-physiological-and-5Mht06JqQf
SP - 1215
VL - 39
IS - 7
DP - DeepDyve
ER -