CLAVATA Was a Genetic Novelty for the Morphological Innovation of 3D Growth in Land Plants

Abstract

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.

The hornworts: morphology, evolution and development

“…MpCLE1 and its receptor gene MpTDR are expressed in distinct patterns across the gametophyte apical notch and act as a negative regulator of gametophytic apical cell meristematic activity . In the moss P. patens, there are nine (Goad et al, 2017;Whitewoods et al, 2018;Whitewoods, 2020) potential CLE genes ( Fig. 5) and two CLV1 genes.…”

“…All CLE genes belong to the CLV3like subclass while genes of the TDIF-like subclass are absent. Three CLE genes and their receptors PpCLV1a, PpCLV1b and PpRPK2 are co-expressed in the gametophyte shoot regulating orientation of stem cell division planes during the transition from two-to threedimensional growth in the gametophyte (Whitewoods et al, 2018). The role of CLV1 in regulating cell division plane orientation in P. patens has been shown to be shared with Arabidopsis thaliana but not with M. polymorpha.…”

“…Numbers in yellow boxes indicate the number of homologues in the corresponding species genome. Purple boxes with ‘x’ indicate the absence of the homologue (Miwa et al ., 2009; Nystedt et al ., 2013; Bowman et al ., 2017; Li et al ., 2018, 2020; Whitewoods et al ., 2018; Zhang et al ., 2020).…”

“…In the moss P . patens , there are nine (Goad et al ., 2017; Whitewoods et al ., 2018; Whitewoods, 2020) potential CLE genes (Fig. 5) and two CLV1 genes.…”

FASEB: The mechanisms in plant development

“…For example, in angiosperms, the regulation of meristematic growth by CLAVATA genes occurs via repression of WUSCHEL activity. However, in the moss Physcomitrella , CLAVATA genes still regulate apical growth but WUSCHEL is not present in the genome (Whitewoods et al , ). To resolve this, Zoe Nemec Venza (University of Bristol, UK) detailed her work on how plant hormone pathways may function directly downstream of CLAVATA to determine moss cell identity.…”