Recruitment and remodeling of an ancient gene regulatory network during land plant evolution

Abstract

The evolution of multicellular organisms was made possible by the evolution of underlying gene regulatory networks. In animals, the core of gene regulatory networks consists of kernels, stable subnetworks of transcription factors that are highly conserved in distantly related species. However, in plants it is not clear when and how kernels evolved. We show here that RSL (ROOT HAIR DEFECTIVE SIX-LIKE) transcription factors form an ancient land plant kernel controlling caulonema differentiation in the moss Physcomitrella patens and root hair development in the flowering plant Arabidopsis thaliana. Phylogenetic analyses suggest that RSL proteins evolved in aquatic charophyte algae or in early land plants, and have been conserved throughout land plant radiation. Genetic and transcriptional analyses in loss of function A. thaliana and P. patens mutants suggest that the transcriptional interactions in the RSL kernel were remodeled and became more hierarchical during the evolution of vascular plants. We predict that other gene regulatory networks that control development in derived groups of plants may have originated in the earliest land plants or in their ancestors, the Charophycean algae.

Keywords: auxin; bHLH; gametophyte; protonema; sporophyte.

Fig. 1.

RSL class I and RSL class II proteins control root hair development in A. thaliana. (A) Root hair phenotype of single and double mutants of RSL genes in A. thaliana. (Scale bar, 200 μm.) (B) Maximum-likelihood cladogram showing that the A. thaliana RSL genes fall into two classes. The tree was rooted with AtbHLH040 (27). (C) Promoter-GFP-protein constructs showing that RSL class I and class II proteins accumulate in the nuclei of root hair cells before and during root hair growth, respectively. GFP-AtRSL5 can only be detected after application of exogenous auxin. (Scale bars, 50 μm.)

Fig. 2.

RSL proteins are conserved across land plants. (A) Alignment of conserved regions of the A. thaliana and P. patens RSL proteins. The position of the bHLH RSL domains is indicated by colored boxes; identical amino acids are represented in black. The sequence logos represent the multiple alignment of RSL class I and class II amino acid sequences from 13 plant species (Table S1); heights are proportional to sequence conservation in each position. (B) Maximum-likelihood tree of A. thaliana (red) and P. patens (green) RSL proteins. The tree was based on the bHLH and RSL domains of the alignment shown in A, together with the bHLH sequence of the outgroups AtbHLH040 and PpbHLH069 (27); approximate likelihood ratio test support values are indicated in the nodes. See also Fig. S2. (C) Number of RSL class I and RSL class II genes in different plant species (Table S1). (D) Six-day-old seedling roots of the A. thaliana wild-type Col-0, Atrsl2 Atrsl4 double-mutant, and Atrsl2 Atrsl4 expressing the P. patens genes PpRSL3, PpRSL4, PpRSL5, and PpRSL6 under the control of the constitutive CaMV 35S promoter. (Scale bar, 200 μm in the main figure and 50 μm in the close ups.)

Fig. 3.

RSL class II proteins control caulonema development in P. patens. (A) Protonemata of Pprsl3, Pprsl4, Pprsl5, Pprsl6, Pprsl3 Pprsl4, and Pprsl5 Pprsl6 loss-of-function mutants and constitutively expressed PpRSL3 and PpRSL4 genes in P. patens. Spores were germinated on minimal media for 3 wk. (Scale bars, 1 mm.) (B) Diameter of plants, shown as a stacked graph of the relative sizes of the inner chloronema-rich and the peripheral caulonema filament-rich regions (mean ± SD, n = 30). (C) Number of protruding caulonema filaments per plant (mean ± SD, n = 30 plants). *Significantly different from wild-type (P < 0.01 with Bonferroni multiple comparison correction); gray asterisks refer to the caulonema-rich region alone.

Fig. 4.

RSL genes and auxin form regulatory networks in P. patens and A. thaliana (A–F) qRT-PCRs showing the relative expression level of RSL genes in different A. thaliana (A and D) and P. patens (B, C, E, and F) mutant backgrounds and after NAA (1-naphthaleneacetic acid, a synthetic auxin) treatments. The expression levels are relative to wild-type (A–C) or untreated plants (D–F). The putative PpRSL7 transcript was not detected. Asterisk means absent or not determined. Bars represent the SD of three independent replicates. (G and H) Schematic representation of the regulatory interactions between the different RSL class I genes (red), RSL class II genes (blue), and auxin (green) in A. thaliana (G) and P. patens (H).

Fig. 5.

Evolutionary history of the RSL network. RSL genes evolved in charophyte algae or in the first land plants, 500–1,000 million y ago. An ancestral RSL kernel was present in early land plants and was conserved during land plant evolution. Later, during vascular plant evolution, the RSL network was recruited to control the development of cellular projections from root epidermal cells (root hairs).