Cheng et al. (Cell, 2019) sequenced the first subaerial Zygnematophyceae genomes (Spirogloea muscicola and Mesotaenium endlicherianum) and concluded that two gene families central to land-plant stress biology — GRAS transcription factors and PYR/PYL/RCAR abscisic-acid receptors — originated or expanded in the common ancestor of Zygnematophyceae and embryophytes and were gained by horizontal gene transfer (HGT) from soil bacteria. They left open four linked questions: whether GRAS homologs predate the Zygnematophyceae+embryophyte ancestor (a reported Chaetosphaeridium globosum GRAS homolog appeared absent from its genome by PCR); which bacterial lineages donated the genes; what the acquired genes functionally contribute to desiccation resistance, stress signaling, and development in the algal progenitors of land plants; and whether alternatives to HGT, such as ancient endosymbiotic gene transfer followed by differential loss, can be excluded. Later work has supported the placement of both families at the Zygnematophyceae+embryophyte ancestor without establishing whether the families extend earlier, functionally supported a bacterial origin of PYL-like proteins (bacterial PYL homologs bind PP2C phosphatases), established transformation and CRISPR capability in a zygnematophyte alga, and shown that the algal ABA-signaling chassis operates ABA-independently. However, no genome-grade data exist for Chaetosphaeridium or other Coleochaetophyceae, bacterial donor lineages remain unpinpointed, alternative transfer scenarios remain untested, and no in situ loss- or gain-of-function analysis of GRAS or PYR/PYL/RCAR has been performed in any Zygnematophyceae. The problem remains open.

Background

Land plants (embryophytes) evolved from within a grade of freshwater and terrestrial algae called streptophyte algae, and molecular phylogenies place the conjugating green algae of the class Zygnematophyceae (e.g., Spirogloea, Mesotaenium, Zygnema, Closterium) as the closest living algal relatives of land plants. Their transition to terrestrial life — terrestrialization — demanded pre-existing or newly acquired mechanisms for tolerating desiccation and other terrestrial stresses. A central actor in the land-plant response to water deficit is the hormone abscisic acid (ABA). ABA is perceived by soluble receptors of the PYR/PYL/RCAR family (named after the mutants and screens in which they were discovered: PYRABACTIN RESISTANCE 1, PYR1-LIKE, and REGULATORY COMPONENTS OF ABA RECEPTOR): hormone binding enables these receptors to inhibit type 2C protein phosphatases (PP2Cs), which releases SNF1-RELATED PROTEIN KINASE 2 (SnRK2) proteins to phosphorylate transcription factors that turn on stress-response genes. A second family, the GRAS proteins, are plant-specific transcription factors named after three founding members (GIBBERELLIC ACID INSENSITIVE, REPRESSOR OF gal-3, SCARECROW) that regulate development, arbuscular mycorrhizal symbiosis, and abiotic stress responses; the DELLA growth-repressor branch of gibberellin signaling also belongs to this family. Both families were long considered land-plant innovations. That view changed when de Vries et al. (2018) detected a canonical PYR/PYL/RCAR homolog in a Zygnema transcriptome — the first found outside land plants — and proposed that embryophyte stress signaling evolved in the algal progenitors of land plants. Cheng et al. (2019) then reported the first zygnematophyte genomes, of the subaerial species Spirogloea muscicola and Mesotaenium endlicherianum, and concluded that GRAS and PYR/PYL/RCAR originated or expanded in the common ancestor of Zygnematophyceae and embryophytes and were gained by horizontal gene transfer (HGT — the non-sexual transfer of genetic material between unrelated organisms) from soil bacteria. Their phylogenies placed plant GRAS and PYL sequences inside the diversity of bacterial homologs from soil-associated lineages (including the Deltaproteobacterium Sorangium cellulosum and Bacteroidetes Pontibacter species, plus Firmicutes, Actinobacteria and cyanobacteria), and they validated by PCR that a GRAS sequence previously reported from a Chaetosphaeridium globosum transcriptome (a member of the Coleochaetophyceae, another class of streptophyte algae closely related to land plants) is absent from C. globosum genomic DNA — suggesting that the transcriptome entry reflects contamination and leaving open whether GRAS predates the Zygnematophyceae+embryophyte ancestor. The interpretation as interdomain HGT also faces long-recognized confounds: HGT among soil bacteria is rampant, GRAS-like and PYL-like sequences appear to have been transferred repeatedly within Bacteria, and vertical inheritance with loss, or ancient endosymbiotic gene transfer followed by differential loss, have not been formally excluded. Finally, the functional significance of the acquired genes could not be tested when the paper appeared, because no genetically tractable model system existed in this algal class.

Problem Statement

Determine the evolutionary origin and the functional significance, for the colonization of land, of the GRAS and PYR/PYL/RCAR gene families, whose origin at the common ancestor of Zygnematophyceae and embryophytes and whose acquisition by horizontal gene transfer are the hypotheses under test. The problem has two linked parts, which together bear on a causal account of how interdomain gene acquisition assembled the ancestral land-plant stress-signaling toolkit. (1) Origin: establish whether GRAS and PYR/PYL/RCAR homologs predate the Zygnematophyceae+embryophyte ancestor; identify the bacterial donor lineages of the putative transfers from among soil bacteria, recognizing rampant HGT among bacteria and repeated transfers of GRAS-like and PYL-like sequences within Bacteria; and discriminate bona fide interdomain HGT into the streptophyte nuclear genome from alternative scenarios recognized in the literature, such as vertical inheritance with differential loss or ancient endosymbiotic gene transfer followed by differential loss (de Vries and Rensing 2020; Lu et al. 2025). (2) Function: determine what the putatively HGT-acquired GRAS and PYR/PYL/RCAR genes contribute to desiccation resistance, stress signaling, and development in the algal progenitors of land plants, and thereby whether these genes were causally important for terrestrialization. No in situ loss- or gain-of-function test of GRAS or PYR/PYL/RCAR has been performed in any Zygnematophyceae. Together, the origin and functional strands bear on whether horizontally acquired bacterial genes were a decisive innovation in the plant conquest of land. The origin and functional strands are jointly required to resolve the problem, so a result that resolves only one strand, or only some of the sub-questions within a strand, is a partial advance rather than a resolution of the task.

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