PREreview of NTMC2T5 links lipid homeostasis to plastid differentiation

PrePRint : NTMC2T5 links lipid homeostasis to plastid differentiation.

This work shows the importance of no vesicular lipid transfer during chloroplast biogenesis. The paper is very well written and easy to follow. The authors analysed and identified proteins containing the lipid-transfer MSP domain. They identify NTMC2T5 protein and generate N benthamiana knock-out mutants, investigate its subcellular location and find a clear chloroplast-develop phenotype in young plants. By electron microscopy, the internal membrane organization of the plastid is disturbed. Finally, the authors analyse its ER-Chloroplast localization.

1) Identification of SMP-domain proteins in Arabidopsis thaliana and Nicotiana benthamiana and their evolutionary relationships across eukaryotes. Results are shown and explained in a very clear way. We suggest to increase the size of plot A

2) NTMC2T5 proteins localize to the chloroplast envelope

If TM region localizes the protein to Chloroplast. What is the region of the protein that localizes at the ER? C2? HR?

Is perinuclear chloroplast localization detected only during stress signalling? Or during chloroplast biogenesis in de etiolation also happens? Is it possible that migration of chloroplast to perinuclear ER could be due to lipid precursor enzymes localizing at those regions more than cortical ER?

3) Loss of NTMC2T5 impairs cotyledon greening and chloroplast development during early seedling development and de-etiolation.

In Figure 3It would be useful to clarify how chlorophyll quantification was performed and the analysis performed. What’s the meaning of each point?

Why is quantification performed at 7 days and pictures shown at 6 days-old?

In figure 3, E and F there are many plants having 0 chloroplast. Would it be better to plot a categoric figure, like in figure 2b? Furthermore, the Dunnet test assumes normal distribution of data, which doesn’t seem to be the case.

4) Loss of NTMC2T5 disrupts the internal organization of plastids.

Since Mutant plant adults showed no phenotype, it would be nice to see if chloroplast architecture is recovered by electron microscopy too.

If the phenotype of the mutant is only observed in young plants, is it possible that the gene is transcriptionally regulated? Would it be possible to analyse expression levels of NTMC2T5 during plant development?

The authors claim that full NTMC2T5 induces ER-Chloroplast contact sites by confocal analysis. This claim could be also quantified by electron microscopy already taken.

5) NTMC2T5 proteins are located at ER–chloroplast interfaces.

In figure 5 the BIFC experiments are a bit confusing. Why do authors use C2 and full length from AtT5.2 and then switch to TM from AtT5.1? Why did not include HR in the analysis?

Co-expression of AtT5.1TM VINE vs C2-HR VICE would be nice to see. Additionally, no related Chloroplast outer membrane resident could be used as a control.

If the lab has access, immunogold Electron Microscopy would be an excellent way to localise NTMC2T5 at the ER-Chloroplast contact sites.

Bioinformatic analysis showed that the MSP domain of NTMC2T5 sequence is conserved, is it possible to swap N bethamiana MSP with Marchantia on for instance and try to restitute WT phenotype?

Finally, If NTMC2T5 is necessary for plastid biogenesis, would it be also implicated in amyloplastos/statolith? Would it be interesting to check gravitropism and amyloplastid morphology.

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Uso de Inteligencia Artificial (IA)

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