Table 1.
List of yeast (Saccharomyces cerevisiae) strains used in this study.
Table 2.
List of primers used for RT-qPCR and GFP fusion constructs.
Figure 1.
Phylogenetic analysis of 16 MTP protein sequences from Hordeum vulgare (Hv), Oryza sativa (Os), Arabidopsis thaliana (At) and Stylosanthes hamata (Sh).
Alignment of full-length sequences was done as described for S1 Figure. The phylogenetic tree was drawn with the MEGA version 4.0 (http://www.megasoftware.net) [59]. Numbers represent bootstrap values for 1000 trees.
Figure 2.
Effect of MTP8.1 and MTP8.2 expression on Mn2+ tolerance and Mn accumulation in the yeast mutant pmr1Δ.
Serial dilutions (1.0, 0.1 and 0.01 OD600) of pmr1 cells transformed with MTP8.1, MTP8.2 or empty vector (pFL6.1) were spotted on SD selective media with increased Mn content (A). Plates were incubated for 3-5 days at 30°C. The results are from one representative experiment out of three independent yeast transformations. Liquid yeast growth assay with MTP8.1 (circle), MTP8.2 (square) and pFL6.1 (triangle) transformed pmr1Δ cells grown with increasing Mn concentrations over 48 hours, data are the means ± SE of three independent yeast samples (B). Mn accumulation in pmr1Δ cells determined by ICP-OES analysis, cells were cultivated for 17 h with either 2.4 µM (black bars) or 100 µM (grey bars) Mn2+ in medium (C). Data are the means ± SE of three independent yeast samples. Values with the same letter within the same treatment are not significantly different (P>0.05).
Figure 3.
C-terminal MTP8-GFP fusion proteins localizes to the Golgi apparatus.
As a control, GONST1-YFP were transiently co-expressed with MTP8.1-GFP and MTP8.2-GFP in onion epidermal cells (A and B), respectively. The right panels show co-localizations of GONST1-YFP with each MTP8-GFP protein. Scale bars were 75 and 100 µm for A and B, respectively.
Figure 4.
The effects of insufficient, control or two increasing Mn toxicity levels on Mn accumulation in the youngest fully developed leaves (A), shoot dry weight (B) and root dry weight (C) of barley plants.
Data are means ± SE (n = 3). Values with the same letter between treatments are not significantly different (P>0.05).
Figure 5.
Elemental analysis of the youngest leaves of barley plants grown with insufficient, control or two increasing Mn toxicity levels.
The concentrations of five macro- (A) and four micronutrients (B) were determined in the youngest leaves. Data are means ± SE (n = 3). Values with the same letter between treatments are not significantly different (P>0.05).
Table 3.
Leaf % dry weight of barley plants grown with varying Mn supplies. Data are means ± SE (n = 3).
Figure 6.
Quantitative expression analysis of MTP8 genes in barley plants grown with insufficient, control or two increasing Mn toxicity levels.
The expression level of MTP8.1 (A and C) and MTP8.2 (B and D) were determined in root (A and B) and shoot tissue (C and D). Data are normalized with respect to the expression of the reference gene ACTIN. Furthermore the relative changes in MTP8 expression levels are indexed to barley plants grown under control conditions. Data represent means (n = 3) ± SE. Values with the same letter between treatments for each MTP gene and plant organ are not significantly different (P>0.05).
Figure 7.
Quantitative expression analysis of IRT1 in root tissue of barley plants grown with insufficient, control or two increasing Mn toxicity levels.
Data are normalized with respect to the expression of the reference gene ACTIN. Furthermore the relative change in IRT1 expression is indexed to barley plants grown under control conditions. Data represent means ± SE (n = 3). Values with the same letter between treatments are not significantly different (P>0.05).