SummaryHow genes shape diverse plant and animal body forms is a key question in biology. Unlike animal cells, plant cells are confined by rigid cell walls, and cell division plane orientation and growth rather than cell movement determine overall body form. The emergence of plants on land coincided with a new capacity to rotate stem cell divisions through multiple planes, and this enabled three-dimensional (3D) forms to arise from ancestral forms constrained to 2D growth. The genes involved in this evolutionary innovation are largely unknown. The evolution of 3D growth is recapitulated during the development of modern mosses when leafy shoots arise from a filamentous (2D) precursor tissue. Here, we show that a conserved, CLAVATA peptide and receptor-like kinase pathway originated with land plants and orients stem cell division planes during the transition from 2D to 3D growth in a moss, Physcomitrella. We find that this newly identified role for CLAVATA in regulating cell division plane orientation is shared between Physcomitrella and Arabidopsis. We report that roles for CLAVATA in regulating cell proliferation and cell fate are also shared and that CLAVATA-like peptides act via conserved receptor components in Physcomitrella. Our results suggest that CLAVATA was a genetic novelty enabling the morphological innovation of 3D growth in land plants.

In this article, we characterized roles for CLAVATA in the development of a moss, Physcomitrella patens, focusing on the 2D to 3D growth transition. Ongoing work to further characterize mutant phenotypes identified some phenotype discrepancies among the Ppclv1a and Ppclv1b mutant lines published in the original paper. For this reason, we implemented further checks of the published manuscript and fully sequenced both PpCLV1a and PpCLV1b loci in all mutants originally reported in the Methods S1 figure ''CRISPR/Cas9 strategy for generating Ppclv1 mutants.'' Although the conclusions of the paper remain valid, our investigations revealed errors that we wish to correct. We found that Ppclv1a line 18 (Figures 4D and 4K; Methods S1, CRISPR figure, panel E) contained an 805 bp deletion at the PpCLV1a locus, but while Ppclv1a line 18 plants had phenotypes resembling WT plants, there was also a 9 bp deletion at PpCLV1b. We found no mutations at PpCLV1a or PpCLV1b in Ppclv1a line 29 or Ppclv1a line 32 (Methods S1, CRISPR figure, panels F and G), and these lines were indistinguishable from WT plants. The genotype of Ppclv1b line 2 (Figures 4E and 4L; Methods S1, CRISPR figure, panel H) was reported as a 2 bp deletion, but genome walking now confirms that there is a 4 bp deletion and >6 kb insertion at PpCLV1b, and the insertion comprises sequence integrated from the pACT::Cas9 expression vector used to engineer the lines [1]. The genotypes of Ppclv1b line 9 and Ppclv1b line 33 (Methods S1, CRISPR figure, panels I and J) were not previously reported, and while Ppclv1b line 9 has a 47 bp deletion at the PpCLV1b locus, Ppclv1b line 33 has no mutation at PpCLV1a or PpCLV1b, and an indistinguishable phenotype from WT plants. The reported genotypes of Ppclv1a1b lines 6, 8, and 12 were accurate (Methods S1, CRISPR figure, panels K-M). Consequently, we have re-engineered and fully sequenced PpCLV1a and PpCLV1b in three independent Ppclv1a and Ppclv1b mutant lines to verify the mutant phenotypes reported in Figure 4. The Ppclv1a and Ppclv1b mutant phenotypes previously reported hold true, and the genotypes and phenotypes of lines now in use are shown in Figure 1 below.

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Significance Plants grow from their tips. The gametophore (shoot-like organ) tip of the moss Physcomitrium patens is a single cell that performs the same functions as those of multicellular flowering plants, producing the cells that make leaves and regenerating new stem cells to maintain the shoot tip. Several pathways, including CLAVATA and cytokinin hormonal signaling, regulate stem cell abundance in flowering plants and in mosses, although the mechanisms whereby these pathways regulate stem cell abundance and their conservation between these plant lineages is poorly understood. Using moss, we investigated how Pp CLAVATA and cytokinin signaling interact. Overall, we found evidence that Pp CLAVATA and cytokinin signaling interact similarly in moss and flowering plants, despite their distinct anatomies, life cycles, and evolutionary distance.

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Plant shoots grow from stem cells within Shoot Apical Meristems (SAMs), which produce lateral organs while maintaining the stem cell pool. In the model flowering plant Arabidopsis, the CLAVATA (CLV) pathway functions antagonistically with cytokinin signaling to control the size of the multicellular SAM via negative regulation of the stem cell organizer WUSCHEL (WUS). Although comprising just a single cell, the SAM of the model moss Physcomitrium patens (formerly Physcomitrella) performs equivalent functions during stem cell maintenance and organogenesis, despite the absence of WUS-mediated stem cell organization. Our previous work showed that the stem cell-delimiting function of the CLV pathway receptors CLAVATA1 (CLV1) and RECEPTOR-LIKE PROTEIN KINASE2 (RPK2) is conserved in the moss P. patens. Here, we use P. patens to assess whether CLV-cytokinin crosstalk is also an evolutionarily conserved feature of stem cell regulation. Genetic analyses reveal that CLV1 and RPK2 regulate SAM proliferation via separate pathways in moss. Surprisingly, cytokinin receptor mutants also form ectopic stem cells in the absence of cytokinin signaling. Through modeling, we identified regulatory network archtectures that recapitulated the stem cell phenotypes of clv1 and rpk2 mutants, cytokinin application, cytokinin receptor mutations, and higher-order combinations of these perturbations. These models predict that CLV1 and RPK2 act through separate pathways wherein CLV1 represses cytokinin-mediated stem cell initiation and RPK2 inhibits this process via a separate, cytokinin-independent pathway. Our analysis suggests that crosstalk between CLV1 and cytokinin signaling is an evolutionarily conserved feature of SAM homeostasis that preceded the role of WUS in stem cell organization.

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Models of leaf development have long predicted the existence of an organ-wide polarity field. Now, a robust analysis in a developing Arabidopsis leaf reveals the presence of a general and persistent cell polarity coordinated over the entire leaf.