Physiological And Ecological Mechanisms On Suppressed Root-shoot Translocation Of Copper In Willow By Soil Flooding

Copper（Cu）induced phytotoxicity has become a serious environmental problem,and excessive amounts of Cu in plant can cause phytotoxicity.Moreover,frequent or periodic flooded areas such as water and land staggered zones are more vulnerable to heavy metal contamination than other areas.Up to now,it is still a hot and difficult topic to study the accumulation and transport of heavy metals by woody plants worldwide.Willow is one of the suitable species for phytoremediation on heavy metal contaminated soil in wetlands.Research on the mechanism of accumulation,transport and elemental homeostasis of heavy metals in trees is of great significance to the phytoremediation in heavy metal contaminated soils.However,,moreover,most conclusions obtained from hydroponic solution or upland culture can not adequately represent the interaction mechanism between wetland plants willow and heavy metals in wetland soils.Thus,we investigated the response of Salix integra‘Yizhibi’to soil flooding and Cu stress.The willows were cultivated in Cu-polluted soils under flooding（F,with water level of 10 cm above the soil surface）and non-flooded（NF,normal soil moisture）conditions.The Cu treatments were set as control（no Cu addition）,50,150 and 450 mg?kg^-1 of Cu spiked with Cu SO4?5H2O.After flooding for 60 d and 90 d,we examined the variations of rhizosphere soil physical and chemical properties,different Cu fractions,microbial community composition and diversity.The growth and physiological/biochemical responses of willows were also observed.We also used the synchronous radiation X-ray fluorescence（μ-XRF）technique to investigate the distribution of Cu and other nutrient elements in willow tissues,revealing the mechanism of soil flooding effect on Cu transport in willow roots.Additionally,Cu concentrations in xylem sap and phloem exudates were extracted and determined by a hydroponic experiment.A series of experiments including foliar-applied ⁶⁵Cu and split-root experiments have clarified the mechanism of the xylem basted long-distance transport,phloem redistribution of Cu in Salix integra.The main results are as follows:（1）Soil flooding significantly reduced the content of available Cu in soil,but increased the content of soil organic matters.The activity of cellulase,invertase and urease were markedly decreased by 17.3%-34.1%,28.5%-59.0%and 55.0%-76.7%respectively,with increasing Cu levels.Flooding significantly increased the activity of polyphenol oxidase and peroxidase by 56.3%and 41.4%at the highest Cu level compared to its respective non-flooded condition.High Cu concentration significantly decreased the richness and diversity of the bacterial community,and fungi were more sensitive than bacteria under flooding conditions.Redundancy analysis suggests that available Cu and soil organic matter are the key determinants affecting the composition of microbial communities.（2）Under the combined stress of flooding and high Cu stress,the biomass of Salix integra was inhibited to a certain extent,but it still grew normally with a survival rate of100%.The adventitious roots and lenticels appeared in the base stem of Salix integra to adapt to flooding stress.Willow can maintain normal photosynthesis,and act positively by activating antioxidant enzyme system and osmotic regulators,which help to offset the injury caused by flooding and Cu stress.（3）Cu was preferentially accumulated in the root of Salix integra,and the root Cu concentration was 73,86 and 23-fold greater than that in the leaves,stems and cuttings,respectively.Most Cu distributed in the root cap and meristem region of the root tip.Soil flooding significantly increased the accumulation of Fe,Mn and S in roots,and precipitated more iron and manganese oxides and sulfides on the root surface,which may interfere with the absorption and fixation of Cu and some nutrient elements in the root.Soil flooding severely reduced Cu transport via the xylem vessels as a consequence of decreasing the transpiration rate of leaves.The alleviated Cu toxicity to willow plants by soil flooding can be attributed to the inhibited Cu uptake and transport via xylem.（4）High level of Cu in xylem sap and high Cu intensity in micro X-ray fluorescence imaging（μ-XRF）in xylem tissues directly confirm the significance of xylem transport of Cu from roots to shoots.In foliar-applied ⁶⁵Cu experiment,the ⁶⁵Cu spiked mature leaf exported approximately 78%⁶⁵Cu to newly emerged shoots,and approximately 22%downward to the new roots,confirming the bidirectional transport of Cu via phloem.In split-root experiment with willow seedlings exposed to ⁶⁵Cu,the ⁶⁵Cu concentration of the directly exposed roots was markedly higher than that on the non-⁶⁵Cu treated side.The Cu remobilized from old/mature leaves to newly growing leaves in Salix integra,which might be attributed to the high Cu requirement of the growing leaves.The results of our study are important to understand the accumulation and transport of Cu by willow,a fast-growing and flooding tolerance woody plant,in wetland soil.These findings could also enrich our understanding of the regularity of metal stress resistance physiology of trees,soil environment chemistry and so on,and provide theoretical basis for the application of fast-growing trees in wetland phytoremediation.Our findings also providenewinsight into the responses of soil microbes to Cu-contamination and contribute to our understanding of metal toxicity in soil-woody plant systems under flooded conditions.