Conservation and Divergence of YODA MAPKKK Function in Regulation of Grass Epidermal Patterning

Abstract

10All multicellular organisms must properly pattern cell types to generate functional tissues and organs. 11The organized and predictable cell lineages of the Brachypodium leaf enabled us to characterize the 12 role of the MAPK kinase kinase gene BdYODA1 in regulating asymmetric cell divisions. We find that 13 YODA genes promote normal stomatal spacing patterns in both Arabidopsis and Brachypodium, 14 despite species-specific differences in those patterns. Using lineage tracing and cell fate markers, we 15 show that, unexpectedly, patterning defects in bdyoda1 mutants do not arise from faulty physical 16 asymmetry in cell divisions but rather from improper enforcement of alternative cellular fates after 17 division. These cross-species comparisons allow us to refine our interpretations of MAPK activities 18 during plant asymmetric cell divisions.19 Keywords 20 Asymmetric Cell Division, MAPK pathway, stomata, Brachypodium, comparative development 21 Summary Statement 22 Analysis of Brachypodium leaf epidermis development reveals that the MAPKKK, BdYODA1, 23 regulates asymmetric divisions by enforcing resultant cell fates rather than driving initial physical 24 asymmetries.25 56 ( Fig. 1A-B). The common deployment of asymmetric divisions in multiple epidermal lineages in 57 grasses requires that stomatal fate be later superimposed in particular cell files to specify the smaller 58 daughter cells as stomatal precursors (Raissig et al., 2016). 59Asymmetric divisions during development are guided by fate, polarity, and signaling inputs. In 60 3 Arabidopsis stomatal production, bHLH transcription factors, the polarity protein BASL, and a 61 signaling pathway comprising ligands, receptors, and a mitogen-activated protein kinase (MAPK) 62 cascade have been connected to these roles (Lau and Bergmann, 2012; Pillitteri et al., 2016). The 63 MAPK pathway is headed by the MAPK kinase kinase (MAPKKK) YODA (AtYDA) (Bergmann et 64 al., 2004). Genetic and biochemical data indicate that AtYDA responds to positional information 65 provided by peptide ligands of the EPIDERMAL PATTERING FACTOR (EPF) family via 66 transmembrane receptors TOO MANY MOUTHS (TMM) and members of the ERECTA (ER) family 67(ERf) (Nadeau and Sack, 2002; Kim et al., 2012; Hunt and Gray, 2009; Kondo et al., 2010; Sugano et 68 al., 2010; Hara et al., 2007; Shpak et al., 2005). AtYDA then relays this information through 69 downstream kinases MKK4/5 and MAPK3/6 (Lampard et al., 2009; Wang et al., 2007) to 70 phosphorylate and inhibit the bHLH transcription factor SPEECHLESS (AtSPCH), the primary 71 regulator of entry into the stomatal lineage pathway (Lampard et al., 2008). Loss of AtYDA activity 72 results in the production of excess stomata arranged in large clusters, whereas overactivity suppresses 73 development of stomata (Bergmann et al., 2004), phenotypes opposite of those ascribed to loss and 74 gain of AtSPCH function (Lampard et al., 2009; Macalister et al., 2007). 75 In grasses, the stomatal lineage requires homologues of the bHLH transcription facto...

Stomatal development in the grasses: lessons from models and crops (and crop models)

“…Selfrenewing divisions are not present in grass stomatal lineages but, in the absence of BdYDA1, greater numbers of cells are committed to stomatal fate, suggesting that both daughters of the asymmetric division commit to the lineage. Additionally, the persistence of physically asymmetric divisions in the bdyoda1 mutant suggests it acts more to enforce differential cell fates postdivision rather than to set up asymmetry predivision (Abrash et al, 2018). In light of these findings, it may be valuable to re-evaluate previous interpretations of when YODA activity is required in Arabidopsis, at least for the stomatal lineage.…”

“…Interestingly, the BdYDA1 mutants completed normal asymmetric stomatal entry divisions, but both the small and large daughter cells acquire stomatal fate. The larger daughter cells also appeared to occasionally undergo additional divisions that eventually acquire stomatal fate, and abnormal recruitment of SCs often resulted in a single SC spanning two stomatal complexes (Abrash et al, 2018). Excess divisions are seen in the stomatal lineages in both Arabidopsis and Brachypodium yoda mutants.…”

“…Grass stomatal patterning is also characterised by a one-cell spacing pattern, but the origin is different, as cells are in files and lack amplifying divisions, so lineage, rather than neighbour cell communication could play a more dominant role. Homologues of TMM and two ER family RLKs have been identified in Brachypodium (Abrash et al, 2018), and two rice SERK family members have been characterised for their function in plant defence response and BR signalling (Khurana et al, 2009;Zuo et al, 2014), but roles for any of these receptor-like proteins in stomatal development have yet to be reported in any grasses.…”

“…At least two YODA homologues have been identified in both rice and Brachypodium (Peterson et al, 2010;Abrash et al, 2018). A genetic screen in Brachypodium yielded a mutation in one of the duplicated YODA homologues, BdYDA1, that also displayed short stature and sterility (Abrash et al, 2018). Interestingly, the BdYDA1 mutants completed normal asymmetric stomatal entry divisions, but both the small and large daughter cells acquire stomatal fate.…”

“…An alternative Pooidae species, Brachypodium distachyon (Brachypodium) has gained momentum as a grass model based on its diminutive size and genome, rapid life cycle, and amenability to genetic transformation (Brkljacic et al, 2011;Scholthof et al, 2018). Recent advances in the generation of translational and transcriptional reporters, application of CRISPR/ Cas9 technology, and completion of forward genetic screens in Brachypodium have made it possible to use this plant model to provide a mechanistic framework for grass stomatal development (Raissig et al, 2016(Raissig et al, , 2017Abrash et al, 2018). This framework can be combined with judicious use of more labour-intensive crop manipulations to translate useful discoveries into tangible crop advancement.…”