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A root phloem pole cell atlas reveals common transcriptional states in protophloem-adjacent cells

Abstract

Single-cell sequencing has recently allowed the generation of exhaustive root cell atlases. However, some cell types are elusive and remain underrepresented. Here we use a second-generation single-cell approach, where we zoom in on the root transcriptome sorting with specific markers to profile the phloem poles at an unprecedented resolution. Our data highlight the similarities among the developmental trajectories and gene regulatory networks common to protophloem sieve element (PSE)-adjacent lineages in relation to PSE enucleation, a key event in phloem biology. As a signature for early PSE-adjacent lineages, we have identified a set of DNA-binding with one finger (DOF) transcription factors, the PINEAPPLEs (PAPL), that act downstream of PHLOEM EARLY DOF (PEAR) genes and are important to guarantee a proper root nutrition in the transition to autotrophy. Our data provide a holistic view of the phloem poles that act as a functional unit in root development.

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Fig. 1: A root phloem pole cell atlas containing PSE, MSE, CC and PPP cells.
Fig. 2: MSE cells identification and identity of cluster 11.
Fig. 3: Developmental trajectories and mapping of the PSE enucleation point.
Fig. 4: Phloem cell types in the integrated UMAP.
Fig. 5: Similarities in gene expression between leaf phloem parenchyma and root pericycle.
Fig. 6: Identification of a gene expression pattern common to non-PSE cells frequent after PSE enucleation.
Fig. 7: PAPL genes are PEAR targets that influence root nutritional status.
Fig. 8: Difference in WT and 3papl metabolite levels in leaves and roots.

Data availability

Sequencing data from 10x Chromium single-cell RNA-seq are available from NCBI’s Gene Expression Omnibus through GEO accession number GSE181999. Sequencing data from bulk RNA-seq are available from NCBI’s GEO accession number GSE182672. All other data (phenotypic scoring, microscopy imaging and plasmid maps) are available from the Cambridge Apollo Repository (https://doi.org/10.17863/CAM.74836).

Code availability

Analysis code, with instructions on how to run it, is available from: https://github.com/tavareshugo/publication_Otero2022_PhloemPoleAtlas.

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Acknowledgements

We thank W. Frommer and J.-Y. Kim for providing pSWEET11:SWEET11-2A-GFP seeds; C. Hardtke for providing pMAKR5:MAKR5-GFP seeds and plasmids; C. Cossetti, R. Schulte and all the staff from Flow Cytometry Core Facility at CIMR for their technical support with cell sorting; K. Kania from Genomics Unit at CRUK for preparing single-cell RNA-seq libraries; B. Guillotin and K. Birnbaum for helpful insights on single-cell analysis; R. Wightman and G. Evans for technical support with microscopy experiments; G. Hindle, J. Salmon and S. Ward for media preparation; K. Blajecka for technical assistance; K. Petkovic and R. Alcaina for technical support; and S. Schornack for helpful comments. S.O. was supported by a Herchel Smith postdoctoral fellowship from the University of Cambridge (2017–2020). L.K. received funding from the SNSF (P2LAP3_178062) and a Marie Curie IEF (No. 795250). This work was supported by the Finnish CoE in Molecular Biology of Primary Producers (Academy of Finland CoE programme 2014–2019) decision no. 271832, the Gatsby Foundation (GAT3395/PR3), the University of Helsinki (award 799992091) and the ERC Advanced Investigator Grant SYMDEV (No. 323052). T.L. was supported by the German Research foundation (DFG) under Germany’s Excellence Strategy (CIBSS-EXC-2189-Project ID 390939984) and by grant La606/18-1. A.R.F and V.D.V. acknowledge support from the Max-Planck Society.

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Authors and Affiliations

Authors

Contributions

S.O. performed the experiments; I.G. identified PAPL1 and At3g16330 expression patterns which appeared as PEAR targets in microarray data; P.R. provided pear1pear2 double mutant, pPEAR(del)::3xYFP and advised on experimental design; Y.L. and H.T. analysed gene regulatory networks; P.R., M.B., L.K., B.B. and J.H. participated in sample collection for sorting and metabolomic profiling; M.B. imaged pSUC2:GFP; J.H. provided the pSAPL::YFPer line; V.D.V. and A.R.F. carried out metabolic profiling and data analysis; F.P. and T.L. provided the papl2 and papl1-2 alleles; H.T. designed and performed the single-cell data and statistical analysis; S.O., H.T. and Y.H. conceptualized and designed the study; S.O. wrote the manuscript with input from Y.H., H.T., P.R. and L.K. All authors read, edited and discussed the manuscript.

Corresponding authors

Correspondence to Hugo Tavares or Yka Helariutta.

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The authors declare no competing interests.

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Nature Plants thanks Jia-Wei Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Markers used for sorting phloem pole cells and new CC genes identified.

a) MAKR5 translational fusion highlights all the cells surrounding PSE73 from the quiescent centre (QC) (often stronger from cell number 3) until the differentiation zone, where it becomes weaker. Some weak expression is also found in PSE. In mature parts of the root this marker spreads to the whole pericycle. Published marker was fused to 3xYFP to increase signal. S171 is expressed in PPP from the unloading zone. pAPL::3xYFP is expressed first in PSE and after enucleation switches to all the cells around PSE, stronger in CC. This line is not fully reflecting ALTERED PHLOEM DEVELOPMENT (APL) endogenous expression, since it has some expression in the outer layers but it is a very strong phloem pole marker. sAPL is expressed in CC and MSE (weaker) from 90–120 µm from the QC. pPEAR1(del)::3xYFP is a modified version of the PEAR1 promoter that is expressed in early PSE, MSE and a procambial cell resulting from the same division plus columella cells. See Roszak et al.33 for the detailed expression pattern b) Newly identified genes expressed in CC (PLC5, At5g58690, and At2g38640). c) Expression of mature CC (NAKR1, SUC2), mature PSE (NAC086, NAC045) and mature PPP (S17, MES7) genes at the terminal clusters. UMAPs show the particular cluster-weighted normalised expression of each gene in the phloem pole cell atlas and microscopy pictures are representative images of the transcriptional reporter lines where the gene promoter is fused to VENUSer. Scale bar in the longitudinal sections is 25 µm while it is 10 µm in the cross sections. White arrowheads point to PSE cells as a reference point. The numbers in each panel indicate samples with similar results, of the total independent biological samples observed.

Extended Data Fig. 2 Validation of the temporal information in the UMAP by using complementary tools.

a) Color-coded UMAP according to the longitudinal sections in Brady et al. 2007, showing a developmental progression in each cell lineage. b) Bar plot indicating the percentage of cells contributed by each cluster to each of the Slingshot trajectories shown in the main text. Bars are coloured by trajectory. c) RNA velocity analysis using scVelo, with velocity vectors projected on our UMAP d) Confocal pictures of pAPL::VENUSer showing continuous expression in PSE in the early root meristem with a patchy expression in the neighbouring cells that gets stable in CC and MSE shootward. As observed in the zoom picture, the signal in PPP gets weaker after PSE enucleation. The pictures are accompanied by a UMAP showing APL cluster-weighted normalised expression in the phloem pole cell atlas. Scale bar in the longitudinal sections is 25 µm while it is 10 µm in the cross sections and zoom. White arrowheads point to PSE cells as a reference point. 25 independent seedlings from three different lines expressing this construct were observed e) Probability of a cell to be assigned to different trajectories, ranging from 0 to 1. In the image we are showing a few clusters as an example. f) The expression of APL and PSE enucleation markers NAC086 and NEN4 was plotted along the PPP, CC and PSE trajectories, with the cells coloured by cluster number in the UMAP. NAC086, an APL target, appears later than APL in the PSE trajectory, and NEN4, a NAC086 target, appears later than NAC086, indicating our approach matches the temporal aspects observed in PSE biology in the roots. The dots representing the cells are coloured according to cluster colours in panel c and Fig. 1b. The black line is a smoothed trend estimated from a non-parametric generalised additive model. g) Initiation of S17 and SAPL gene expression. Distance from QC (µm) was measured to the first cell expressing each gene in 7 days post sowing (dps) seedlings, n = 10 for S17 and n = 11 for SAPL.

Extended Data Fig. 3 Expression of CC, SE, PPP and ring genes in the integrated dataset faceted by source.

a) Orange arrowheads point to MSE cluster 24. Red arrowhead points to cluster 27, cells not present in other dataset that represent cluster 5 of the UMAP and the cells surrounding PSE around enucleation time. b) Black arrowheads point to PSE cluster 28. Orange arrowheads point to the blue cells in MSE cluster 24. The phloem pole cell atlas provides a majority of PSE cells.

Extended Data Fig. 4 Gene co-expression network analysis identified 16 gene modules.

Gene expression of each module is summarised by the eigengene profile, which is the first principal component score from a PCA done on the expression matrix and are plotted on the UMAP and different trajectories to visualise module expression across various cell types and along developmental trajectories in the phloem pole. Module 1 consists of 1367 genes (with variance explained score 21.4%) and shows an increasing expression in both PPP and CC trajectories, while lower than average expression in PSE. Modules 2, 3 and 7 contain 995, 878 and 225 genes respectively, which are highly expressed in the early cells. Module 4 with 778 genes shows broad expression in CC, PPP and PSE, but was lowly expressed on all three trajectories. Module 5, containing 368 genes, shows expression in cell clusters 12 and 13 while the 291 genes in Module 6 are mostly expressed specifically for PSE cells. The 164 genes in Module 8 were mostly expressed in the outer layers, with some lowly expressed in the mature PPP and CC cells. The eigengene profile of Module 9 (134 genes) shows expression in all cell types except CC, but particularly higher in early cells while Module 10 contains 18 genes highly expressed in CC. Modules 11–16 contain no more than 10 genes and the exact sizes are 6, 5, 3, 2, 2, and 2 genes respectively.

Extended Data Fig. 5 Module 1 also groups genes with an extended or partial ring expression pattern.

a) New genes with an expression pattern validating the module eigengene analysis. All the genes presented in this panel are expressed forming a ring pattern (At3g1633802 or extended ring (At4g27435, note the expression in protoxylem, and PER30, note the expression in procambium) at the time of PSE enucleation. They are all grouped in module 1, except At4g27435, which belongs to module 4. UMAPs show the particular cluster-weighted normalised expression of each gene in the phloem pole cell atlas and microscopy pictures are representative images of the transcriptional reporter lines where the gene promoter is fused to VENUSer. Scale bar in the longitudinal sections is 25 µm while it is 10 µm in the cross sections. White arrowheads point to PSE cells as a reference point. Each gene has also been plotted in PPP (green), CC (orange) and PSE (purple) trajectories, showing average expression values in the Y-axis and pseudotime in the X-axis. The numbers in each panel indicate samples with similar results, of the total independent biological samples observed.

Extended Data Fig. 6 Analysis on sub-network of Module 1 genes identified 15 sub-modules via Louvain algorithm.

The eigengene profile of sub-module 1, containing 326 genes, shows expression in both PPP and CC with relatively low expression in PSE, similar to the pattern revealed by Module 1 eigengene. 8 out of 9 genes with ring-specific expression pattern found in Module 1 fall in this sub-module, while At3g16330 falls in sub-module 3, which shifts slightly towards mature pericycle cells. Sub-module 2 contains 318 genes specifically for pericycle cells. Genes in sub-module 4 and 6 are highly expressed in PPP cells and some in out layers. For sub-module 5 and 7, the eigengene profiles show relatively broader expression in both PPP and CC, as well as the out layers. The other 8 sub-modules contain no more than 9 genes.

Extended Data Fig. 7 Detailed analysis of PAPL gene expression.

a) pPAPL1::3xYFP showing a strong expression in all the cells surrounding PSE and a weaker expression in the neighbouring procambial layer. The latter is only observed with the 3xYFP reporter. b) Nuclear localization of pPAPL1::PAPL1-YFP in epidermis and PSE-adjacent cells which recapitulates the 3xYFP fusion pattern (c), indicating that PAPL1 is not mobile. Occasionally some nuclei appear highlighted in the neighbouring procambial layer, where this gene is expressed weakly as shown in a. d) Phloem meristematic expression of pPAPL1::PAPL1-3xYFP disappears in pear sext. while the epidermis signal stays. Occasionally the reporter was also observed in a central xylem cell. e) pPAPL2::PAPL2-YFP recapitulates PAPL1 translational expression pattern. f) pPAPL2::VENUSer expression mirrors the PAPL1 ring transcriptional expression, although it has a broader domain close to QC and it is expressed in columella and epidermis. g) Like PAPL1, PAPL2 ring expression domain gets delayed in pear1pear2 mutant. h) pCDF2:VENUSer has a broader expression than PAPL genes i) Fluorescent signal (mean grey value) in the ring and PSE cells in seedlings of pPAPL1::3xYFP j) Transcriptional reporter lines where the promoter was fused to VENUSer were transformed into pear sext mutant background and pRPS5A::PEAR2-GR, a line overexpressing ectopically PEAR2 in the whole meristem. PEAR2 was sufficient to induce the expression of the different genes in these layers. Primed letters show the cross section of each respective letter. Scale bar in the longitudinal sections is 25 µm while it is 10 µm in the cross sections. White arrowheads point to PSE cells as a reference point. µm in the cross sections indicate the distance from QC. The number in each confocal picture indicates samples with similar results of the total independent biological samples observed.

Extended Data Fig. 8 PAPL genes do not induce periclinal cell divisions.

2 different lines for pWOL::XVE»PAPL1 in pear1pear2 mutant background were induced in beta-estradiol for either 5 days (ad) or 20 h (eh). The same construct was transformed in the Col0 background, with 2 lines carried forward. Seedlings were induced for either 7 days (il) or 20 h (m–p). In the mock treatment, DMSO was added to the media instead of beta-estradiol. Primed letters show the cross sections of each respective letter. Scale bar in the longitudinal sections is 25 µm while it is 10 µm in the cross sections. The number in each panel indicates samples with similar results of the total independent biological samples analysed.

Extended Data Fig. 9 PAPL genes seem to be important for a correct root nutrition.

a) Root length in cm for 5dps 3papl and wt seedlings in different conditions (1% sucrose, 0.5% sucrose, 0% sucrose and 24-hour light regime). For 0.5% sucrose, 42 wt and 36 3papl seedlings were used. For 1% sucrose, 39 wt and 29 3papl seedlings, for 24 hours, 41 wt and 25 3papl and for 0% sucrose, 34 wild type and 30 3papl seedlings b) Root length in cm for 6 dps seedlings of wt, 3papl mutant, 3 complementation lines, double mutants and cdf2 single mutant grown in media depleted of sucrose. 36 seedlings were measured for wt, 37 seedlings for cdf4cog1–7, 33 for cdf4cog1–6, 32 for cdf2, 28 for 3papl, 38 for 3papl complementation PAPL2 line 7.3, 37 for 3papl complementation PAPL2 line 1.1 and 34 for 3papl complementation PAPL1 line 3.2 c) Replicate of the transfer experiment between sucrose and sucrose-depleted plates of 3papl seedlings (stock 8). Time (in days) spent in sucrose and without sucrose is represented by a grey and purple bar respectively. Transfer was done days 1–5 and all roots were measured at 8 dps. The bars are divided in 8 portions representing the days in each condition. 203 seedlings were grown as a control without sucrose, 156 were grown as a control with sucrose and a total of 99 seedlings were transferred on day 1, 75 on day 2, 70 on day 3, 93 in day 4 and 96 in day 5 d) Confocal pictures of 7 dps wt and 3papl seedlings grown in sucrose containing media or media without sucrose. Scale bar is 25 µm e) Overall root length (cm) of the different phloem mutant phenotypes grown with (red) and without sucrose (blue) at 6 days post germination. The points denote the median and error bars the 95% confidence interval estimated by bootstrap (500 samples; see methods). The number of seedlings measured was: 195 3papl; 269 3papl-2; 314 pear sextuple; 57 apl; 272 pear1pear2; 311 wt. These were spread across 3 experimental batches with N = 11–62 (median = 46) seedlings per combination of batch and treatment. f) Scans of 8 dps seedlings (wt, stock 1, and 3papl, stock 8) grown in 1% sucrose media or without sucrose. g) Transfer experiment between sucrose and sucrose-depleted plates of wild type seedlings. Time (in days) spent in sucrose and without sucrose is represented by a grey and purple bar respectively. Transfer was done days 1–5 and all roots were measured at 8 dps. The bars are divided in 8 portions representing the days in each condition. 135 seedlings were measured for wt control (stock1) and an average of 29 wild type total seedlings per transfer experiment. h) Same data as in Fig. 4g, but showing the variation across experiment and seed stock batches (only wt and 3papl are shown for illustration, but similar variation was observed for the complementation lines). Mean and 95% confidence interval per experiment were estimated by bootstrap (500 samples). A total of 1986 seedlings were measured split across 5 experimental batches and, for some genotypes, derived from different seed stocks. The median number of seedlings per experimental batch and seed stock combination was 36 with a range from 24–46. The number in each confocal picture indicates samples with similar results of the total independent biological samples analysed.

Extended Data Fig. 10 Phloem marker genes are expressed in 3papl mutant.

a) Confocal pictures of 3 dps roots expressing reporters for pPAPL1::GFP/GUS, pPAPL2::VENUSer and pCDF2::VENUSer in Col0 background with and without sucrose. b) Confocal pictures of pMAKR5::MAKR5-3xYFP in Col0, 3papl and pear1pear2 mutants. c) Confocal pictures of pSBT4.12::YFPer in Col0 and 3papl mutant d) Confocal pictures of pAPL::3xYFP in 3papl crossed to Col0 (F1 +/−) or crossed to 3papl (F1 −/− e) Confocal pictures of pSBT4.12::YFPer in 3papl mutant in the presence and absence of sucrose f) Confocal pictures of pMAKR5::MAKR5-3xYFP in 3papl mutant in the presence and absence of sucrose. g) Confocal pictures of pAPL:3xYFP in 3papl mutant in the presence and absence of sucrose h) Confocal pictures of 3dps roots expressing pSUC2::GFP in wt and 3papl in the presence and absence of sucrose. The confocal signal was observed in the number of roots indicated in each picture.

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Otero, S., Gildea, I., Roszak, P. et al. A root phloem pole cell atlas reveals common transcriptional states in protophloem-adjacent cells. Nat. Plants 8, 954–970 (2022). https://doi.org/10.1038/s41477-022-01178-y

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