Contribution of NtZIP1-like, NtZIP11 and a WAK-pectin based mechanism to the formation of Zn-related lesions in tobacco leaves

Previous studies on tobacco (Nicotiana tabacum var. Xanthi) have shown that in the presence of 200 μM Zn, lesions develop on the leaf blades from Zn accumulating mesophyll cells. This was interpreted as a tolerance mechanism against high Zn, protecting non-accumulating photosynthesizing cells. Underlying this phenomenon is the distinct capacity of mesophyll cells for Zn accumulation. To learn more about the involved mechanisms, here we searched for two key elements. First, for Zn transporters contributing to Zn accumulation in groups of mesophyll cells. Tissue-specific expression of two ZIP transporters previously identified in the leaves of tobacco plants exposed to high Zn was examined. Transgenic tobacco lines expressing GUS reporter gene under 2156 bp and 1922 bp of the promoter region of NtZIP1-like and NtZIP11, respectively, were used for analysis. Promoter activity of both genes in the leaves of high Zn-exposed plants was detected in groups of mesophyll cells, which correlates with the pattern of Zn accumulation. Second, we searched for putative components signalling the status of the cell wall. The WAKs/WAKLs and pectin fractions were the focus in the study, as it was known that upon binding pectin, WAK/WAKLs directly signals from the cell wall to the cytoplasm. Pectin microarray identified rhamnogalacturonan I (RG I) as the only pectin fraction that increased in the apoplast at high Zn (relative to control conditions), and immunocytochemistry demonstrated its presence in groups of cells. This was accompanied by increased expression of NtWAK2P-2 (putative NtWAK2), NtWAK4P-4, and NtWAK4P-2. Based on these data, a hypothesis was forwarded that RG I generated in the cell wall upon exposure to high Zn might bind to the extracellular domain of NtWAK2P-2, NtWAK4P-4, and/or NtWAK4P-2, which subsequently could transmit a signal on Zn status into the cytoplasm. It was also considered that regulation of NtZIP1-like and NtZIP11 expression by Zn availability might depend on the interaction between the RG I and/or NtWAK2P-2-, NtWAK4P-4, NtWAK4P-2.