Figure 7.

Functional analysis of cacao NRAMP genes in yeast. Functional complementation for uptake of Fe²⁺, Mn²⁺ and Zn²⁺ in the yeast strains DEY1453 (fet3fet4), YOL122C (smf1) and ZHY3 (zrt1zrt2). Different yeast strains expressing TcNRAMP1, 1m (mutated version of TcNRAMP1), 2, 3, 5, 5s (splice variant of TcNRAMP5), 6 or empty vector pDR195 (EV) were cultured on synthetic defined (SD)-Ura plates containing 2% glucose in presence or absence of respective metal chelator. Transformed fet3fet4 cells were spotted on the plate (pH 4.0) supplemented with 10 μM FeCl₃ or 10 μM Fe chelator (a) and also grown (d) in low iron liquid medium (LIM10). Transformed smf1 cells were grown on the plate (pH 5.2) supplemented with 100 μM MnSO₄ or with 12.5 mM Mn chelator EGTA (b). The yeast mutant strain ZHY3 (zrt1zrt2) expressing the genes were assessed on the plate (pH 5.8) supplemented with 100 μM ZnCl₂ or with 100 μM of metal chelator EDTA, 10 μM each of FeCl₃ and ZnCl₂ (c) and also grown (e) in low zinc liquid medium (LZM100). The wild type yeast strain DY1457 transformed with the empty vector pDR195 (WT-EV) was included as a positive control. Data are means ± SD of three biological replicates.