Characteristics of cadmium accumulation and tolerance in apple plants grown in different soils.

Cadmium (Cd) is a nonessential element and highly toxic to apple tree. However, Cd accumulation, translocation and tolerance in apple trees planted in different soils remain unknown. To investigate soil Cd bioavailability, plant Cd accumulation, physiological changes as well as gene expression patterns in apple trees grown in five different soils, 'Hanfu' apple seedlings were planted in orchard soils collected from Maliangou village (ML), Desheng village (DS), Xishan village (XS), Kaoshantun village (KS) and Qianertaizi village (QT), and subjected to 500 µM CdCl2 for 70 d. Results showed that soils of ML and XS had higher content of organic matter (OM), clay and silt, and cation exchange capacity (CEC) but lower sand content than the other soils, thereby reduced Cd bioavailability, which could be reflected by lower concentrations and proportions of acid-soluble Cd but higher concentrations and proportions of reducible and oxidizable Cd. The plants grown in soils of ML and XS had relatively lower Cd accumulation levels and bio-concentration factors than those grown in the other soils. Excess Cd reduced plant biomass, root architecture, and chlorophyll content in all plants but to relatively lesser degree in those grown in soils of ML and XS. The plants grown in soils of ML, XS and QT had comparatively lower reactive oxygen species (ROS) content, less membrane lipid peroxidation, and higher antioxidant content and enzyme activity than those grown in soils of DS and KS. Transcript levels of genes regulating Cd uptake, transport and detoxification such as HA11, VHA4, ZIP6, IRT1, NAS1, MT2, MHX, MTP1, ABCC1, HMA4 and PCR2 displayed significant differences in roots of plants grown in different soils. These results indicate that soil types affect Cd accumulation and tolerance in apple plants, and plants grown in soils with higher OM content, CEC, clay and silt content and lower sand content suffer less Cd toxicity.

Malus rootstocks affect copper accumulation and tolerance in trees by regulating copper mobility, physiological responses, and gene expression patterns.

We investigated the roles of rootstocks in Cu accumulation and tolerance in Malus plants by grafting 'Hanfu' (HF) scions onto M. baccata (Mb) and M. prunifolia (Mp) rootstocks, which have different Cu tolerances. The grafts were exposed to basal or excess Cu for 20 d. Excess Cu-treated HF/Mb had less biomass, and pronounced root architecture deformation and leaf ultrastructure damage than excess Cu-challenged HF/Mp. Root Cu concentrations and bio-concentration factor (BCF) were higher in HF/Mp than HF/Mb, whereas HF/Mb had higher stem and leaf Cu concentrations than HF/Mp. Excess Cu lowered root and aerial tissue BCF and translocation factor (Tf) in all plants; however, Tf was markedly higher in HF/Mb than in HF/Mp. The subcellular distribution of Cu in the roots and leaves indicated that excess Cu treatments increased Cu fixation in the root cell walls, which decreased Cu mobility. Compared to HF/Mb, HF/Mp sequestered more Cu in its root cell walls and less Cu in leaf plastids, nuclei, and mitochondria. Moreover, HF/Mp roots and leaves had higher concentrations of water-insoluble Cu compounds than HF/Mb, which reduced Cu mobility and toxicity. Fourier transform infrared spectroscopy analysis showed that the carboxyl, hydroxyl and acylamino groups of the cellulose, hemicellulose, pectin and proteins were the main Cu binding sites in the root cell walls. Excess Cu-induced superoxide anion and malondialdehyde were 28.6% and 5.1% lower, but soluble phenolics, ascorbate and glutathione were 10.5%, 41.9% and 17.7% higher in HF/Mp than HF/Mb leaves. Compared with HF/Mb, certain genes involved in Cu transport were downregulated, while other genes involved in detoxification were upregulated in HF/Mp roots and leaves. Our results show that Mp inhibited Cu translocation and mitigated Cu toxicity in Malus scions by regulating Cu mobility, antioxidant defense mechanisms, and transcription of key genes involved in Cu translocation and detoxification.

Rootstock-Scion Interaction Affects Cadmium Accumulation and Tolerance of Malus.

To understand the roles of Malus rootstock, scion, and their interaction in Cd accumulation and tolerance, four scion/rootstock combinations consisting of the apple cultivars "Hanfu" (HF) and "Fuji" (FJ) grafted onto M. baccata (Mb) or M. micromalus "qingzhoulinqin" (Mm) rootstocks differing in relative Cd tolerance were exposed either to 0 µM or 50 µM CdCl2 for 18 d. Cd accumulation and tolerance in grafted Malus plants varied within rootstock, scion, and rootstock-scion interaction. Cd-induced decreases in photosynthesis, photosynthetic pigment level, and biomass were lower for HF grafted onto Mb than those for HF grafted onto Mm. Reductions in growth and photosynthetic rate were always the lowest for HF/Mb. Cd concentration, bioconcentration factor (BCF), and translocation factor (Tf ) were always comparatively higher in HF and FJ grafted onto rootstock Mm than in HF and FJ grafted on Mb, respectively. When HF and FJ were grafted onto the same rootstock, the root Cd concentrations were always higher in HF than FJ, whereas the shoot Cd concentrations displayed the opposite trend. The shoot Cd concentrations and Tf were lower for HF/Mb than the other scion/rootstock combinations. Rootstock, scion, and rootstock-scion interaction also affected subcellular Cd distribution. Immobilization of Cd in the root cell walls may be a primary Cd mobility and toxicity reduction strategy in Malus. The rootstock and scion also had statistically significant influences on ROS level and antioxidant activity. Cd induced more severe oxidative stress in HF and FJ grafted onto Mm than it did in HF and FJ grafted onto Mb. Compared with FJ, HF had lower foliar O2 -, root H2O2, and root and leaf MDA levels, but higher ROS-scavenging capacity. The rootstock, scion, and rootstock-scion interaction affected the mRNA transcript levels of several genes involved in Cd uptake, transport, and detoxification including HA7, FRO2-like, NRAMP1, NRAMP3, HMA4, MT2, NAS1, and ABCC1. Hence, the responses of grafted Malus plants to Cd toxicity vary with rootstock, scion, and rootstock-scion interaction.

Exogenous melatonin alleviates cadmium uptake and toxicity in apple rootstocks.

To examine the potential roles of melatonin in cadmium (Cd) uptake, accumulation and detoxification in Malus plants, we exposed two different apple rootstocks varying greatly in Cd uptake and accumulation to either 0 or 30 µM Cd together with 0 or 100 µM melatonin. Cadmium stress stimulated endogenous melatonin production to a greater extent in the Cd-tolerant Malus baccata Borkh. than in the Cd-susceptible Malus micromalus 'qingzhoulinqin'. Melatonin application attenuated Cd-induced reductions in growth, photosynthesis and enzyme activity, as well as reactive oxygen species (ROS) and malondialdehyde accumulation. Melatonin treatment more effectively restored photosynthesis, photosynthetic pigments and biomass in Cd-challenged M. micromalus 'qingzhoulinqin' than in Cd-stressed M. baccata. Exogenous melatonin lowered root Cd2+ uptake, reduced leaf Cd accumulation, decreased Cd translocation factors and increased root, stem and leaf melatonin contents in both Cd-exposed rootstocks. Melatonin application increased both antioxidant concentrations and enzyme activities to scavenge Cd-induced ROS. Exogenous melatonin treatment altered the mRNA levels of several genes regulating Cd uptake, transport and detoxification including HA7, NRAMP1, NRAMP3, HMA4, PCR2, NAS1, MT2, ABCC1 and MHX. Taken together, these results suggest that exogenous melatonin reduced aerial parts Cd accumulation and mitigated Cd toxicity in Malus plants, probably due to the melatonin-mediated Cd allocation in tissues, and induction of antioxidant defense system and transcriptionally regulated key genes involved in detoxification.

Copper accumulation, subcellular partitioning and physiological and molecular responses in relation to different copper tolerance in apple rootstocks.

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.

Integration of Cadmium Accumulation, Subcellular Distribution, and Physiological Responses to Understand Cadmium Tolerance in Apple Rootstocks.

Cadmium (Cd) is a nonessential and highly toxic element causing agricultural problems. However, little information is available about the variation in Cd tolerance among apple rootstocks and its underlying physiological regulation mechanisms. This study investigated Cd accumulation, subcellular distribution, and chemical forms as well as physiological changes among four apple rootstocks exposed to either 0 or 300 µM CdCl2. The results showed that variations in Cd tolerance existed among these rootstocks. Cd exposure caused decline in photosynthesis, chlorophyll and biomass in four apple rootstocks, which was less pronounced in M. baccata, indicating its higher Cd tolerance. This finding was corroborated with higher Cd tolerance indexes (TIs) of the whole plant in M. baccata than those in the other three apple rootstocks. Among the four apple rootstocks, M. baccata displayed the lowest Cd concentrations in roots, wood, and leaves, the smallest total Cd amounts as well as the lowest BCF. In apple rootstocks, it was found that to immobilize Cd in cell wall and soluble fraction (most likely in vacuole) and to convert it into pectate- or protein- integrated forms and undissolved Cd phosphate forms may be the primary strategies to reduce Cd mobility and toxicity. The physiological changes including ROS, carbohydrates and antioxidants were in line with the variations of Cd tolerance among four apple rootstocks. In comparison with the other three apple rootstocks, M. baccata had lower concentrations of ROS in roots and bark, H2O2 in roots and leaves and MDA in roots, wood and bark, but higher concentrations of soluble sugars in bark and starch in roots and leaves, and enhanced antioxidants. These results indicate that M. baccata are more tolerant to Cd stress than the other three apple rootstocks under the current experiment conditions, which is probably related to Cd accumulation, subcellular partitioning and chemical forms of Cd and well-coordinated antioxidant defense mechanisms.