The DEK1 calpain Linker functions in three-dimensional body patterning in Physcomitrella patens

Abstract

The DEFECTIVE KERNEL1 (DEK1) calpain is a conserved 240-kD key regulator of three-dimensional body patterning in land plants acting via mitotic cell plane positioning. The activity of the cytosolic C-terminal calpain protease is regulated by the membrane-anchored DEK1 MEM, which is connected to the calpain via the 600-amino acid residue Linker. Similar to the calpain and MEM domains, the Linker is highly conserved in the land plant lineage, the similarity dropping sharply compared with orthologous charophyte sequences. Using site-directed mutagenesis, we studied the effect on Physcomitrella patens development by deleting the Linker and two conserved Linker motifs. The results show that removal of the Linker has nearly the same effect as removal of the entire DEK1 gene. In contrast, deletion of the conserved Laminin_G3 (LG3) domain had a milder effect, perturbing leafy gametophore patterning and archegonia development. The LG3 domain from Marchantia polymorpha is fully functional in P. patens, whereas angiosperm sequences are not functional. Deletion of a C-terminal Linker subsegment containing a potential calpain autolytic site severely disturbs gametophore development. Finally, changing one of the three calpain activesite amino acid residues results in the same phenotype as deleting the entire DEK1 gene. Based on the conserved nature of animal and DEK1 calpains, we propose that the DEK1 MEM-Linker complex inactivates the calpain by forcing apart the two calpain subunits carrying the three amino acids of the active site.The three-dimensional (3D) architecture of land plant bodies is determined by the orientation of cell walls deposited between dividing nuclei in developing organs. The ability to determine cell wall orientation in various planes was a novel feature that evolved in the transition from filamentous charophyte algae to the first land plants ( Pires and Dolan, 2012). In contrast to land plants, tip expansion and fixed division planes of the cells drove a two-dimensional (2D) growth pattern of charophyte members represented by filamentous or discoid forms. The membrane-anchored DEFECTIVE KERNEL1 (DEK1) calpain is a candidate protein involved in position-dependent cell wall deposition in the plant lineage (Perroud et al., 2014). Evidence for a conserved function in land plants comes from the observation that DEK1 is essential for cell wall orientation in land plants ranging from angiosperms to mosses, an evolutionary time span of 450 million years (Olsen et al., 2015). Support for a central function of DEK1 also comes from a recent review in which analyses of meristematic shoot tip transcriptomes of widely divergent extant vascular plant lineage members suggest that different genes were recruited to regulate similar meristematic processes during evolution. Exceptions identified include PIN-FORMED, DEK1, and LONELY GUY1 (Frank et al., 2015) proteins that represent potential genetic homologs in meristem function (Harrison, 2015).The DEK1 calpain derives its name from the maize (Zea mays) mutant dek1, whic...

An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing

“…3) were constructed from vector pBHRF∆LinkerG1 29 . To make the vector containing the WT LG3 sequence, nt 8347–11544 was first PCR-amplified using primers SP_Inf_6 N and ASP_Inf_6 N from genomic P .…”

“…patens dek1Δlg3 mutant, generated by in-frame removal of the sequence corresponding to the DEK1-LG3 domain. This mutant forms reduced gametophores with narrow phyllids lacking several features of the WT phyllids, including the midrib 29 .

Figure 1Intron-exon structure of the Physcomitrella patens DEK1 gene. DEK1 ORF consists of 30 exons (grey rectangles) encoding the MEM domain (blue line), the LINKER segment (red line), the LG3 domain (blue rectangle) and the CALPAIN domain (green line).

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Cutting in the middleman: hidden substrates at the interface between proteases and plant development

“…transcription factors, microtubule‐associated proteins, phosphatases, kinases or proteins involved in vesicular trafficking and hormonal signalling. It is notable that P. patens with Δdek1 shows perturbed expression of transcription factors regulating asymmetric divisions required for bud development (Johansen et al ., ). The previous finding does not, however, preclude other proteins which are not transcription factors from serving as DEK1 substrates.…”

“…The moss Physcomitrella patens with deleted DEK1 (Ddek1) shows faulty gametophore growth with increased number of buds ( Fig. 1b), pointing at a DEK1 function essential for a 3D body patterning, albeit not essential for the 2D growth of protonemata (Olsen et al, 2015;Johansen et al, 2016). The fact that 2D growth proceeds normally in Ddek1 allows the study of DEK1 functions in the asymmetric division during the 2D-to-3D patterning transition.…”

“…Perhaps DEK1 senses cell wall properties or tension through carbon binding, thereby translating mechanical cues to cell positioning. Furthermore, carbohydrate binding might be coupled with structural rearrangements of the TM, which drive Ca 2+ -dependent autoprocessing at the Linker and DEK1 activation (Johansen et al, 2016). Structural studies on DEK1 will allow all these scenarios to be assessed.…”