Subcellular visualization of the distribution of atmospheric dinitrogen fixed by Gluconacetobacter diazotrophicus bacteria in maize

Reviewer #1 (Public Review):

The study uses nanoscale secondary ion mass spectrometry to show that maize plants inoculated with a bacteria, Gd, incorporated fixed nitrogen into the chloroplast. The authors then state that since "chloroplasts are the chief engines that drive plant growth," that it is this incorporation that explains the maize's enhanced growth with the bacteria.

But the authors don't present the total special distribution of nitrogen in plants. That is, if the majority of nitrogen is in the chloroplast (which, because of Rubisco, it likely is) then the majority of fixed nitrogen should go into the chloroplast.

Also, what are the actual controls? In the methods, the authors detail that the plants inoculated with Gd are grown without nitrogen. But how did the authors document the "enhanced growth rates of the plants containing this nitrogen fixing bacteria." Were there other plants grown without nitrogen and the Gd? If so, of course, they didn't grow as well. Nitrogen is essential for plant growth. If Gd isn't there to provide it in n-free media, then the plants won't grow. Do we need to go into the mechanism for this, really? And it's not just because nitrogen is needed in the chloroplast, even if that might be where the majority ends up.

Furthermore, it is not novel to say that nitrogen from a nitrogen fixing bacteria makes its way into the chloroplast. For any plant ever successfully grown on N free media with a nitrogen fixing bacteria, this must be the case. We don't need a fancy tool to know this.

The experimental setup does not suit the argument the authors are trying to make (and I'm not sure if the argument the authors are trying to make has any legitimacy). The authors contend that their study provides the basis of a "detailed agronomic analysis of the extent of fixed nitrogen fertilizer needs and growth responses in autonomous nitrogen-fixing maize plants." But what is a "fixed nitrogen fertilizer need"? The phrase makes no sense. A plant has nitrogen needs. This nitrogen can be provided via nitrogen fixing bacteria or fertilizer. But are there fixed nitrogen fertilizer needs? It sounds like the authors are suggesting that a plant can distinguish between nitrogen fixed by bacteria nearby and that provided by fertilizer. If that is the contention, then a new set of experiments is needed - with other controls grown on different levels of fertilizer.

What is interesting, and potentially novel, in this study is figure 1D (and lines 90-99). In that image, is the bacteria actually in the plant cell? Or is it colonizing the region between the cells? Either way, it looks to have made its way into the plant leaf, correct? I believe that would be a novel and fascinating finding. If the authors were to go into more detail into how Gd is entering into the symbiotic relationship with maize (e.g. fixing atmospheric nitrogen in the leaf tissue rather than in root nodules like legumes) I believe that would be very significant. But be sure to add to the field in relation to reference 9, and any new references since then.

Also, it would be helpful to have an idea of how fast these plants, grown in n free media but inoculated with the bacteria, grow compared to plants grown on various levels of fertilizer.

Reviewer #2 (Public Review):

Summary:
In agriculture, nitrogen fertilizers are used to allow for optimum growth and yield of crops. The use of these fertilizers has a large negative impact on the environment and climate. In this report McMahon et al. have inoculated maize seeds with a nitrogen fixing bacterium: Gluconacetobacter diazotrophicus. It has been demonstrated before that nitrogen fixed by this bacterium can be incorporated in a plant. In this study the spatial distribution of the incorporated nitrogen was revealed using NanoSIMS. The nitrogen was strongly enriched in the chloroplasts and especially the stromal region where the Calvin-Benson cycle enzymes are located.

Strengths:
The topic is very interesting as nitrogen supply is of great importance for agriculture. The study is well designed, and the data convincingly show enrichment of 15N (fixed by the bacterium) in the chloroplasts.

Weaknesses:
Some of the data that is discussed is not presented in the (supplement) of the paper. First, in the abstract it is mentioned "help explain the observation of enhanced growth rates in plants containing this nitrogen fixing bacterium". It is unclear if this refers to literature or to this study. Either, it should be mentioned in the introduction, or the data should be shown in the paper. Second, it is mentioned that the chloroplast had a significantly higher nitrogen isotope ratio value compared to the nuclei and the xylem cell walls. Please provide the numbers of the ratios (preferably also an image of the xylem cell wall) and the type of statistical analysis that has been performed.

The paper could benefit from a more in-depth analysis of why the nitrogen isotope ratio is higher in the chloroplast. It seems to be correlated with the local nitrogen abundance, did the authors plot the two against each other? What would it mean if it is correlated? What minimal nitrogen concentration/signal should there be to make a reliable estimate of the ratio? Does the higher ratio mean that the turnover rate of the Calvin-Benson cycle enzymes is higher than for other proteins?

For the small structures that could be the nitrogen fixing bacteria the 15N enrichment is up to 270x the natural ratio. Does this mean that 100% (270*0.0036=1) of their nitrogen is fixed from the provided atmosphere?

Could one also provide the absolute ratio in the chloroplasts? It would be nice if the authors discuss, based on their data, the potential of using nitrogen fixing bacteria to provide nitrogen to crops.