Populus deltoides Kunitz trypsin inhibitor 3 confers metal tolerance and binds copper, revealing a new defensive role against heavy metal stress

Abstract

The expression of Kunitz trypsin inhibitor (KTI) genes has been associated with multiple functions in plants, including defense and development. In particular, Populus deltoides KTI3 (PdKTI3) is highly upregulated when poplars are exposed to different copper stress conditions. However, the specific participation of PdKTI3 in cell protection against heavy metal-induced stress is still unknown. To assess its potential for conferring metal tolerance, we characterized the structure and function of PdKTI3. PdKTI3 was cloned and sequenced. Complementation assays were conducted in various Saccharomyces cerevisiae metal-sensitive strains cultured in solid media enriched with copper, cadmium, zinc, or nickel. Additionally, a homologous model of PdKTI3 was constructed and characterized at the molecular level in terms of its ability to bind copper. The gene expression of PdKTI3 and the sub-cellular localization of PdKTI3 were evaluated by RT-PCR in poplars treated with copper excess and by transient expression in onion cells, respectively. PdKTI3 conferred tolerance to a copper-sensitive strain when cultured in media enriched with copper or cadmium. Moreover, a molecular model of PdKTI3 predicted two putative copper-binding sites, indicating that PdKTI3 could chelate copper. Additionally, expression analyses revealed a significant upregulation of PdKTI3 in roots and leaves exposed to copper. Transient expression of PdKTI3 resulted in a homogenous distribution of protein in the cell involving transport via the endoplasmic reticulum. In conclusion, our results support a novel role for the evaluated KTI, demonstrating that PdKTI3 is involved in the defensive mechanisms induced by copper stress and can confer heavy metal tolerance by chelating metals.