Figure 6c and d aimed to show the differences in zinc accumulation in a WT nodule compared to the MtZIP6 RNAi knockdown. It was stated that higher accumulation occurred in the differentiation zone of the mutant nodule. However, the images gave a differing impression. It is clear that the zinc does not disperse into the rest of the nodule and remains in the differentiation zone, but a higher zinc accumulation compared to WT nodule is not clear. Perhaps it should be noted in the figure legend that fig. 6d had been altered in order to avoid oversaturation in the image.
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Figure 1A and figure 2 were the key figures where authors had the chance to support their initial claims on gene expression of MtZIP6 in nodules and its localization to differentiation zone. Localization experiments displayed in figure 3 were necessary to support their claims on the localization of MtZIP6 to the plasma membrane. We appreciated the three-dimensional reconstruction to point out the localization more clearly.
Furthermore, we really liked the transient expression assay in Agrobacterium infiltrated tobacco leaves to show plasma membrane localization of MtZIP6. The result was clean and succinct, MtZIP6 overlayed perfectly with plasma membrane localized CFP. We wish it was included in Figure 3 along with other localization experiments.
Figure 4 and 5 provided much-needed insight into the function of MtZIP6. But we think that these two figures can be combined together and this will help to provide a better view of the overall picture about the vital need of MtZIP6. This would also have provided space to include the tobacco results in the paper instead of supplemental material.
Abreu et al. showed nicely in figure 6, the possible role of MtZIP6 as zinc transporter in rhizobia-infected nodules. We think it is very interesting that knockdown of MtZIP6 does not affect zinc concentration in the plant. This result suggests a nodule-specific function of the MtZIP6 transporter.
The novelty of MtZIP6 is a notable finding and gives exciting justification to characterize the protein. Overall the paper and its findings were easily understood, even of nonexperts.