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Review of Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells DOI: 10.1101/102426 (BioRxiv), 10.1111/pce.13035 (Plant Cell & Environment)

Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells

[Isidro Abreu, Angela Saez, Rosario Castro-Rodriguez, Viviana Escudero, Benjamin Rodriguez-Haas,  Marta Senovilla, Camille Laure, Daniel Grolimund, Manuel Tejada-Jimenez, Juan Imperial, Manuel Gonzalez-Guerrero , January 24, 2017 (preprint),  September 21, 2017 (in print), BioRxiv & Wiley-Blackwell]DOI: 10.1101/102426 (BioRxiv), 10.1111/pce.13035 (Plant Cell & Environment)Overview Abreu et al. have made contributions to the understanding of the transport of zinc in rhizobia infected nodule cells by  Medicago truncatula Zinc-Iron Permease 6 (MtZIP6, Medtr4g083570). Their results showed that MtZIP6 is expressed in the differentiation zone of the nodules and particularly in the plasma membrane of the infected cells. MtZIP6 knockdown plants had a significant reduction of nitrogen fixation activity. Moreover, accumulation of zinc was detected in the apoplastic regions of the differentiation zone showing the inability of these plants to transport zinc inside the infected cells. First two figures were critical in terms of providing evidence to support authors' initial claims. In Figure 2A authors show that the GUS reporter activity is seen only in the apical region of the nodule and a closer look at the longitudinal section of the nodule clearly indicates the presence of GUS activity in the older part of differentiation zone (Zone II) and the younger parts of the fixation zone (Zone III).  The expression profile of MtZIP6 is also shown to be mostly in Zone II and Zone III with the help of RNA-seq performed on the laser-capture microdissected M. truncatula nodule cells isolated from all the different zones. Figure 3 provides evidence for subcellular localization of MtZIP6. A composite multi-fluorophore image shows MtZIP6 to be localized in the plasma membrane.  Figure 4 and figure 5 compare the symbiosis between control and mtzip6 RNAi line under mineral supply and in absence of mineral supply respectively. In figure 4, the authors point out that there is no effect on the growth between the control and the mtzip6 RNAi line under a non-symbiotic condition with NH4NO3 supplementation. These figures clearly help to prove the point that MtZIP6 has some important function in the development of healthy symbiosis as supported by results about the number of red(functional) and white(nonfunctional) nodules, dry weight and Acetylene reduction assay. 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 indeed 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 figure 6D have been altered in order to avoid oversaturation in the image.   The authors present a proposed model to summarize their findings and their hypothesis for zinc uptake by nodule cells in figure 7. The model fits their results, which showed that MtZIP6 is most probably localized in the cell membrane of the differentiation cells and that knockdown of MtZIP6 accumulate zinc in the apoplastic regions. So, suggesting that MtZIP6 is responsible for transporting zinc inside cells infected with rhizobia seems like a plausible conclusion. In this model, the authors attempt also to fill in other missing pieces of the zinc uptake pathway in nodules.  Positive feedback: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. Major concerns:The title of this paper accurately reflects the results in most cases, alas, some results raised questions after reading the paper. In our first reading, the necessity of figure 1B was not clearly understood. It would be helpful to emphasize how unrelated MtZIP6 and GmZIP1 are from each other. They are highlighted in red in the figure, however, they are not commented in the figure legend. These are homologs with similar functions, yet they do not group together in a tree based on sequence similarity. Based on the evidence in figure 3, authors propose that MtZIP6-HA is localized in the stele. We think this was a very broad area to define localization of MtZIP6. We appreciated that this proposed localization was changed in the print version of the paper. We had concerns that Figures 4 and 5 could have been combined into a single figure. They address the same finding, and side by side comparison of Figure 4B and 5A, as well as 4C and 5B, may have emphasized differences and consolidated figure legends. There were questions about the statistical significance of findings presented in Figure 4. Regarding figure 6B, we are confused about what message the authors want to send. The figure supports the zinc-dependent concentration of MtZIP6, but it was relatively difficult to understand the rationale to test different concentrations of zinc. Since figure 6A showed that zinc concentration in WT nodules is lower compared to nodules in RNAi line, in figure 6C I was expecting to observe the same zinc concentration pattern shown in figure 6A. We wonder if the exposure times were changed during image acquisition to prevent oversaturation of the image.In figure 7,  uncharacterized transporters are denoted by question marks. We think it is helpful for the reader to be able to look at a summary model but the question marks in the suggested transporters might be misleading. After all, it is just a suggested model which summarizes what we know so far from studies in other species; it is obvious that further studies are needed to validate it. Some of the not yet characterized transporters were eliminated in the final publication, helping to streamline main findings.