Objectives We investigated the distribution pattern of excessive copper (Cu) at the tissue and subcellular levels of two citrus species and the responses of the root cell wall to Cu toxicity with the aims of providing theoretical evidence of citrus Cu-tolerance mechanisms and providing scientific instructions for an optimized Cu nutrient management of citrus species.

Methods Seedlings of ‘Xuegan’ Citrus sinensis (L.) Osbeck and ‘Shatian pummelo’ Citrus grandis (L.) Osbeck were sandy cultured in 0.5 µmol/L Cu (control) or 300 µmol/L Cu (excessive Cu treatment) nutrient solution for 18 weeks. At the end of treatments, the Cu concentration and the distribution pattern at the tissue level (the primary roots, lateral roots, stems and leaves), subcellular level (the cell wall, organells and soluble fractions) of lateral roots and the cell wall polysaccharides (the pectin, hemicellulose Ⅰ, hemicellulose Ⅱ and cellulose) of lateral roots were analyzed. The alterations of cell wall ultrastructure of lateral roots (the thickness and chemical group constituents) were investigated by transmission electron microscopy (TEM) and Fourier Transform Infrared Spectroscopy (FTIR).

Results 1) At the tissue level, the lateral roots of citrus seedlings were the primary sites of Cu distribution of citrus seedlings under control and excessive Cu treatment. Compared to control, the ratio of Cu distributed in the lateral roots increased from 57.32% to 90.87% and from 51.31% to 91.99%. in C. sinensis and C. grandis, respectively. By contrast, the Cu distribution ratio decreased significantly in the stems and leaves of two citrus species by Cu treatment compared to control. 2) At the subcellular level of control seedlings, Cu was mainly distributed in the organells fractions of the lateral roots. However, the excessive Cu treatment decreased the Cu ratio of the control organells fractions from 58.61% to 14.34% and from 39.26% to 11.49% in C. sinensis and C. grandis, respectively. Compared to the control, the excessive Cu treatment increased the Cu concentration in the cell wall and soluble fractions of two citrus species. Strikingly, the Cu concentration was higher in the cell wall of C. grandis than C. sinensis under Cu toxicity. 3) The Cu concentration in the polysaccharides of lateral root cell wall was increased remarkably in both citrus species under excessive Cu compared to the control. Noticeably, the Cu ratio distributed in the pectin of lateral root cell wall increased from 44.69% (control) to 98.45% (excessive Cu treatment) in C. sinensis and from 37.15% (control) to 98.48% (excessive Cu treatment) in C. grandis. By contrast, excessive Cu treatment significantly decreased the Cu ratio distributed in the hemicellulose Ⅰ, hemicellulose Ⅱ and cellulose compared to the control. 4) The cell wall fractioning and TEM analysis revealed that the excessive Cu treatment increased the extraction rate of the lateral root cell wall and thickened the cell wall of two citrus species. Compared to C. sinensis, C. grandis had a remarkably higher cell wall extraction rate and cell wall thickness under Cu toxicity. 5) The results of FTIR analysis indicated that the chemical groups of hydroxy, carboxyl and amidogen of cell wall from C. grandis lateral roots contributed to Cu adsorption and immobilization.

Conclusions At subcellular level, excessive Cu is mainly distributed in the soluble fraction and cell wall of lateral roots in seedlings of two citrus species under 300 µmol/L Cu. The pectin of cell wall restricts the Cu translocation from roots to shoots. Compared to C. sinensis, a relatively higher Cu tolerance of C. grandis is attributed to an upregulated synthesis, increased thickness and higher Cu²⁺ adsorption of the cell wall.