Exploring apoplast reorganization in the nodules of Lotus corniculatus L. growing on old Zn-Pb calamine wastes.

Nodulation and symbiotic nitrogen fixation are important factors that determine legume growth. A pot experiment was carried out to determine the effects of Zn-Pb contamination on nodule apoplast (cell walls and intercellular spaces) of bird's foot trefoil (Lotus corniculatus L.) that spontaneously colonized old calamine wastes. The plants were grown in pots filled with sterile calamine substrate (M, metal treated) or expanded clay (NM, untreated) and inoculated with calamine-derived Lotus-nodulating Bradyrhizobium liaoningense. Apoplast reorganization in the nodules was examined using specific dyes for cellulose, pectin and lignin detection, and immuno-histochemical techniques based on monoclonal antibodies against xyloglucan (Lm25), pectins (Jim5 and Jim7), and structural proteins (arabinogalactan protein - Lm14 and extensin - Jim12). Microscopic analysis of metal-treated nodules revealed changes in the apoplast structure and composition of nodule cortex tissues and infected cells. Wall thickening was accompanied by intensified deposition of cellulose, xyloglucan, esterified pectin, arabinogalactan protein and extensin. The metal presence redirected also lignin and suberin deposition in the walls of the nodule cortex tissues. Our results showed reorganization of the apoplast of cortex tissues and infected cells of Lotus nodules under Zn-Pb presence. These changes in the apoplast structure and composition may have created actual barriers for the toxic ions. For this reason, they can be regarded as an element of legume defense strategy against metal stress that enables effective functioning of L. corniculatus-rhizobia symbiosis on Zn-Pb polluted calamine tailings.