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Co-option of plant gene regulatory network in nutrient responses during terrestrialization

Nature Plants volume 10, pages 1955–1968 (2024)Cite this article

Abstract

Plant responses to nitrate, phosphate and sucrose form a complex molecular network crucial for terrestrial adaptation. However, the origins, functional diversity and evolvability of this network during plant terrestrialization remain scarcely understood. Here we compare the transcriptomic response to these nutrients in the bryophyte Marchantia polymorpha and the streptophyte alga Klebsormidium nitens. We show that the largely species-specific nutrient response pattern is driven by gene regulatory network (GRN) alterations. Intriguingly, while pathways governing the GRNs exhibit modest conservation, M. polymorpha GRNs exhibit more regulatory connections through the redeployment of ancient transcription factor CSD. In M. polymorpha, functional analyses reveal the involvement of pre-existing cytokinin machineries in downstream targets, orchestrating plastic morpho-physiological responses to nutrient status. Our findings implicate the genetic co-option events facilitating successful land plant establishment.

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Fig. 1: Dynamic response patterns to nutrients are species specific in M. polymorpha and K. nitens.
Fig. 2: Nutritional gene regulatory networks are rewired between M. polymorpha and K. nitens.
Fig. 3: Loss-of-function mutants of candidate master transcription factors showed less sensitivity to nutrient deficiency.
Fig. 4: The master transcription factors are involved in cytokinin signalling.
Fig. 5: Compromised transcriptional responses to nutrient deficiency in mutants are due to impaired cytokinin signalling.
Fig. 6: Redeployment of trans-acting factor MpCSD leads to nutritional GRN rewiring and subnetwork co-option during terrestrialization.

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Data availability

Raw and processed RNA-seq data can be found in the NCBI GEO with accession number GSE248900. The SMART database used for protein domain prediction is available at http://smart.embl-heidelberg.de/. Source data are provided with this paper.

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Acknowledgements

We thank our lab members for all helpful discussions on the manuscript. This work was supported by the Singapore-MIT Alliance for Research and Technology, National Research Foundation, Prime Minister’s Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) programme (D.U.).

Author information

Authors and Affiliations

  1. Temasek Life Sciences Laboratory, Singapore, Singapore

    Yating Dong, Shalini Krishnamoorthi, Grace Zi Hao Tan, Zheng Yong Poh & Daisuke Urano

  2. Department of Biological Sciences, National University of Singapore, Singapore, Singapore

    Daisuke Urano

Authors
  1. Yating Dong
  2. Shalini Krishnamoorthi
  3. Grace Zi Hao Tan
  4. Zheng Yong Poh
  5. Daisuke Urano

Contributions

D.U. conceived the project. Y.D. and D.U. designed the experiments. Y.D., S.K., G.Z.H.T. and Z.Y.P. conducted the experiments. Y.D., S.K. and G.Z.H.T analysed the results. Y.D. and D.U. wrote the manuscript. All authors edited and commented on the initial draft.

Corresponding author

Correspondence to Daisuke Urano.

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The authors declare no competing interests.

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Nature Plants thanks Sven Gould, Jan de Vries 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 Transcriptional response to nitrate, phosphate, and sucrose in M. polymorpha and K. nitens.

(a,b) Principal component analysis (PCA) of general transcriptomic profiles. PCA was calculated using the built-in methods provided by DESeq2 for variance stabilizing transformation of read counts. Plots show the first two components. (c,d) Heatmaps showing the number of overlapped DEGs across different nutrient starvation and resupply in M. polymorpha and K. nitens. Nutrient responsive genes were annotated according to three components: (1) genes significantly regulated by nutrient starvation or resupply treatment (S or R), (2) time-point after nutrient resupply (6 h, 24 h or 72 h), and (3) up- or down-regulated by nutrient treatment (for example, NS_up indicates genes upregulated in nitrate starvation; NR6h_dn indicates genes downregulated after 6 h of nitrate resupply). Actual counts of overlapped DEGs are shown in the boxes, ranging from 0 (white) to maximum number (red). The order of rows and columns are the same. Numbers in the diagonal indicate the total numbers of DEGs in each time point. For instance, 790 up-regulated genes overlapped between nitrate starvation and phosphate starvation in M. polymorpha. Inset bar charts indicate the DEG numbers from each pairwise comparison. (e) Overlap of genes regulated by nutrient starvation and subsequent resupply. The bar graphs show the proportion of DEGs identified during nutrient starvation that also appeared in the DEG sets after nutrient resupply, at one or more of 6-, 24- and 72-hour timepoints.

Extended Data Fig. 2 Nitrate and phosphate depletion treatments for M. polymorpha WT and mutants.

(a) Representative images of WT (Tak-1), Mpcsd, Mphd9, and Mperf20 plants subjected to full-strength Yamagami medium (control), nitrate starvation (0% NO3−), and phosphate starvation (0% Pi) treatments for two weeks. Bars = 5 mm. (b) Heatmaps presenting the significance of the overlap between the expression dynamics patterns of nutrient-responsive genes in WT (refer to Fig. 1c for more details) and the genes mis-regulated in Mpcsd, Mperf20, and Mphd9 under control conditions. Colored boxes represent the hypergeometric test results with P < 0.05. The clusters for nitrate (NO3−), phosphate (Pi), and sucrose (Suc) responses were selected as explained in Fig. 1c.

Extended Data Fig. 3 Enrichment of cis-regulatory motifs in genes mis-regulated in Mpcsd, Mperf20, and Mphd9.

Motif enrichment was assessed within 1-kb regions of the transcriptional start sites for both up-regulated (a) and down-regulated (b) genes by MpCSD, MpERF20, and MpHD9. The motif analysis utilized the findMotifs function in HOMER software. Motif sequences with P < 1E-04 were considered as enriched.

Extended Data Fig. 4 Morphological phenotypes of the M. polymorpha WT and Mpcsd mutants treated with exogenous BA.

(a) Side-view of two-week-old WT and Mpcsd mutants grown on 1⁄2 B5 plates supplemented with different BA concentrations (0, 10, 30, and 50 μM). Bars = 5 mm. (b) Quantification of thallus area of WT and Mpcsd grown under mock and BA treatment conditions for 21 days. Boxplots show the centerline of the median, box bounds of 25 to 75 percentile, whiskers of 1.5 x interquartile range with minimum-maximum distributions of the data (n = 15 independent plants). Different lowercase letters indicate a significant difference among different groups (two-way ANOVA; Tukey’s HSD post-hoc test; P < 0.05).

Source data

Extended Data Fig. 5 Exogenous application of BA promotes the sensitivity to nitrate and phosphate deficiency in WT and Mpcsd.

Top-view of WT (a) and Mpcsd-4 (b) thallus grown on full-strength or modified Yamagami plates supplemented with different BA concentrations (0, 10, and 30 μM) for 18 days. Bars = 5 mm. (c) Segmentation of thallus area for hyperspectral image analysis. (d) Quantification of chlorophyll index and auronidin index in WT. (e) Quantification of chlorophyll index and auronidin index in WT and Mpcsd-4. Note that experiments for (d) and (e) were conducted in different batches. Chlorophyll index was calculated using whole thallus. Auronidin_index represents data calculated using the central part of thallus, while Auronidin_index_whole represents data calculated using the whole thallus. Data in all bar plots are mean ± sd. In the right panel of (d), n = 10. Other bar charts, n = 5. Different lowercase letters indicate a significant difference among different groups (two-way ANOVA; Tukey’s HSD post-hoc test; P < 0.05). Asterisks represent significant difference by Student’s t-test (* P < 0.05; ** P < 0.01; ns, no significance).

Source data

Extended Data Fig. 6 The growth and gene expression response under cytokinin treatment in K. nitens.

(a) Representative images of K. nitens grown on full Yamagami media supplied with different concentrations of BA. (b) Pixel intensity, measured as a grayscale value ranging from 0 to 255, was used as an indicator of K. nitens growth. Data are shown in mean ± sd (n = 6 colonies). (c) Relative fold changes of KnRRA1, KnRRA2, and KnRRB affected by 30 μM BA for 1 hr or 6 hrs (n = 5 biological replicates). Data are shown in mean ± sd. The K. nitens CitS (citrate synthase) gene was used as the endogenous control, and gene expression was calculated by 2-ΔCT method. ns, no significance by Student’s t-test at P value of 0.05.

Source data

Extended Data Fig. 7 Response of K. nitens to nitrate, phosphate, and sucrose starvation.

(a) Representative images of K. nitens grown on full-strength Yamagami medium or Yamagami medium with 0%, 1%, or 10% deficiency in nitrate, phosphate, or sucrose. (b) Pixel intensity, measured as a grayscale value ranging from 0 to 255, was used as an indicator of K. nitens growth. Data are shown in mean ± sd (n = 5 - 6 colonies). Small dots represent individual raw data.

Source data

Extended Data Fig. 8 Transcriptional responses to nitrate, phosphate, and sucrose starvation in Mpcsd, Mperf20 and Mphd9.

(a) Bar plots indicating the number of upregulated and downregulated genes in response to nitrate, phosphate and sucrose starvation in WT and mutants. (b) Euler plots showing the overlap of DEGs between WT and mutants.

Extended Data Fig. 9 HD9-centered subnetwork in phosphate and sucrose conditions.

(a) Nodes significantly enriched with GO terms (FDR < 0.5) are color coded accordingly as shown in the legend at the bottom right. Bar charts on the top right represent the proportion of nodes that with or without reciprocal best blast hits between M. polymorpha and K. nitens. (b) Nodes with or without reciprocal best BLAST hits count.

Extended Data Fig. 10 RT-qPCR for 10 phosphate responsive genes after prolonged nutrient resupply time course to 7 days.

The expression fold changes for starvation and resupply from RNA-seq data were compared with the same untreated control samples; for qPCR, starvation and nutrient resupply were compared with their respective untreated control samples (that is, collected at the same time points). The K. nitens CitS (citrate synthase) gene was used as the endogenous control. 2-ΔCT method was used for qPCR data analysis.

Supplementary information

Supplementary Information

Overview of Supplementary Information, and Supplementary Figs. 1–16.

Supplementary Tables

Supplementary Tables 1–7.

Supplementary Data 1

Multiple sequence alignment file for CSD proteins.

Source data

Source Data

Statistical source data for Figs. 1, 3 and 4, and Extended Data Figs. 4–7.

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Dong, Y., Krishnamoorthi, S., Tan, G.Z.H. et al. Co-option of plant gene regulatory network in nutrient responses during terrestrialization. Nat. Plants 10, 1955–1968 (2024). https://doi.org/10.1038/s41477-024-01851-4

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