PREreview of Directional Cell-to-cell Transport in Plant Roots

Title of the manuscript:

Directional Cell-to-cell Transport in Plant Roots

Authors: Léa Jacquie, Celeste Fiorenza, Kevin Robe, Jian-Pu Han1, Fabienne Cléard, Christelle Fuchs, Priya Ramakrishna, Sylvain Loubéry, Linnka Lefebvre-Legendre, Marie Barberon.

The manuscript by Jacquie et al., describes the concept of unidirectional transport in differentiated roots.  The authors show evidence for the unidirectional transport of molecules below 27 kDa, by utilizing the symplastic dye CFDA and fluorescent protein sGFP2 driven by various cell type-specific promoters. The authors can in this way show that different fluorescent proteins driven by the same cell-type specific promoter results in similar transport of the molecule across cell layers. Next, the mechanism underlying this directionality is further investigated by analyzing the plasmodesmata aperture and functional barriers in the roots. One major resource of the paper is the contribution of an EMS-mutagenesis based genetic screen that aimed to identify a mutant with bidirectional and exacerbated transport between root layers, accompanied by plasmodesmata with larger apertures. The study further shows that this directionality can be overridden by changes in plasmodesmata aperture and also establishes a connection between pectin and plasmodesmata, which is a new addition in the field.  Overall, this paper is well-structured, well written and provides insights into the bidirectional and unidirectional transport in undifferentiated and differentiated roots. I picked it to present in my PhD Journal club at Max Planck Institute for Plant Breeding Research, Cologne. My peers and I agree that paper deserves to be in a high-impact journal.  However, there are few points which came up during the discussion that could potentially strengthen the manuscript further.   

Major Comments:

1.      The authors have utilized symplastic dyes and fluorescent proteins as proof of concept for the unidirectional transport in differentiated roots. However, to ensure that this happens, the authors may want to investigate flow of dyes in the opposite direction. this could be done using a caged version of the symplastic dye (CMNB-caged carboxyfluorescein) (Gao et al., 2020). The caged dye can be loaded and photo-activated in the pericycle in order to study movement to endodermis.

2.      The effect of nutritional inputs (particularly micro and macro elements varying in size) in changing the bulk flow and osmosis with further consequences on the dye movement can also be tested to strengthen the cell-to-cell unidirectional transport model, e.g., whether the movement of the dye changes if the medium of the plant growth is supplemented with excess of macronutrient (calcium) and micronutrient (molybdenum) in WT and ssm-1 mutant.

3.      It is mentioned in the discussion that based on the results, “the directionality could not be corelated with the morphological changes at the plasmodesmata levels”. However, the width of the plasmodesmata aperture is measured and shown in the results, but still the manuscript lacks a detailed assessment of the morphological features of plasmodesmata, such as a high-resolution 3D- construction of the plasmodesmata between the layers. So, it is suggested that the beforementioned sentence in the discussion part can be rephrased, e.g., the directionality could not be corelated with the pore size/ width of the plasmodesmata.

4.      The study identified the ssm1/rol1 mutant with a clear phenotype of bidirectional transport within the differentiated roots. Since this gene encodes UDP-L-Rhamnose synthase, the rhamnose content of this mutant should be quantified. Also, as previously reported (Ringli et al., 2008), rol1 mutants show an altered flavanol glycosylation profile. ssm1 mutants should also be checked for altered flavanol profile and whether that contributes to the bidirectional transport seen in ssm1.

5.      In P14, l10 it is mentioned that in the ground meristem a degree of disorganization is seen in the division planes of cortical-endodermal initial cells in ssm1/rol1 mutants with no cell-patterning defects. However, it is difficult to spot that in Fig 5C. The authors could provide a better representative picture for this with quantification for the disorganization in the meristem. Further, since formation of ground tissues and vasculature structure is highly dependent on movement of transcription factors and miRNAs through PD, it should also be tested if SHR/HDBZIPIII TFs and/or miRNA155/156 movement is also unaffected in the ssm1 mutant.

Minor Comments:

1.      The header “ssm1, a mutant with specific developmental and nutritional” seems incomplete and could be modified to, e.g., “ssm1, a mutant with a specific developmental and nutritional phenotype” for further clarity.

2.      The sentence in the line 8, under the header “ssm1, a mutant with specific developmental and nutritional” states that in the allelic mutants ssm1-1 and rol1- 2, root hairs formed in epidermal cells in contact with two cortical cells, resulting in an alternance of trichoblastic and atrichoblastic cell files at the root periphery comparable to WT plants. However, fig. 5 B shows that even in the WT root, one root hair is in contact with two cortical cells. This leads to confusion and the authors should find a better representative picture or a reference where this is normal. Perhaps, whether the expression of CAPRICE is differentially altered in WT and ssm1 mutants should also be checked; that could explain the altered trichoblast cell files.

3.      Supplementary Fig. S5E, shows a batch effect between experiment 1 and experiment 2. This experiment could be repeated to make the data more conclusive.

4.      Figure 5 F, shows the preferential accumulation of Molybdenum in the roots of ssm1 mutants. This should be mentioned in the manuscript.

5.      The manuscript should be checked thoroughly for misspellings in text and figures, e.g., on p17, l7, the word “bellow” should be replaced with ‘below’. In fig. 5 E& F, “Fresch Weight” should be ‘Fresh Weight’ and “root lenght” should be ‘root length’.  

 Swati Mahiwal and the Plant Microbe Interactions PhD journal club at MPIPZ, Cologne

Competing interests

The authors declare that they have no competing interests.