Phosphosite charge rather than shootward localization determines OCTOPUS activity in root protophloem

Abstract

Polar cellular localization of proteins is often associated with their function and activity. In plants, relatively few polar-localized factors have been described. Among them, the plasma membrane-associated Arabidopsis proteins OCTOPUS (OPS) and BREVIS RADIX (BRX) display shootward and rootward polar localization, respectively, in developing root protophloem cells. Both ops and brx null mutants exhibit defects in protophloem differentiation. Here we show that OPS and BRX act genetically in parallel in this process, although OPS dosage increase mends defects caused by brx loss-of-function. OPS protein function is ancient and conserved in the most basal angiosperms; however, many highly conserved structural OPS features are not strictly required for OPS function. They include a BRASSINOSTEROID INSENSITIVE 2 (BIN2) interaction domain, which supposedly mediates gain-of-function effects obtained through ectopic OPS overexpression. However, engineering an increasingly positive charge in a critical phosphorylation site, S318, progressively amplifies OPS activity. Such hyperactive OPS versions can even complement the severe phenotype of brx ops double mutants, and the most active variants eventually trigger gain-of-function phenotypes. Finally, BRX-OPS as well as OPS-BRX fusion proteins localize to the rootward end of developing protophloem cells, but complement ops mutants as efficiently as shootward localized OPS. Thus, our results suggest that S318 phosphorylation status, rather than a predominantly shootward polar localization, is a primary determinant of OPS activity.T he subcellular localization of proteins is often directly linked to their function. Asymmetric, polar protein localization has garnered particular attention because it is frequently associated with crucial developmental decisions in multicellular organisms. For example, the polar localization of PAR proteins is required to guide asymmetric cell divisions in animals (1). In plants, relatively few polar-localized factors have been described. The most prominent examples are the PIN-FORMED (PIN) proteins, which are integral plasma membrane efflux carriers for the plant hormone auxin (2). The polar localization of PINs determines the direction of intercellular auxin transport, which is instructive in many adaptive as well as developmental processes (3). PINs are themselves regulated by polar-localized cytoplasmic protein kinases (4), which associate with the plasma membrane through the interaction of their basic hydrophobic patches with phosphoinositides (5). Other examples for polar-localized proteins comprise auxin influx carriers (6), nutrient transporters (7, 8), regulatory proteins involved in Casparian strip formation (9), and developmental switches that determine daughter-cell fate during stomata formation (10). In all of these cases, polar localization is assumed to play an important role in the protein's activity, and in some cases, this has been demonstrated experimentally.In the Arabidopsis root, two plasma membrane-associated proteins, BREVIS...

A Cellular Insulator against CLE45 Peptide Signaling

“…Ectopic overexpression of OPS triggers severe developmental phenotypes [30,31], which could be explained by OPS interference with brassinosteroid signaling in the case of hypocotyl elongation [30]. However, a pertinent OPS deletion variant can still complement the ops root protophloem phenotype [31,32]. The most remarkable feature of OPS is its strongly quantitative action [31].…”

“…However, a pertinent OPS deletion variant can still complement the ops root protophloem phenotype [31,32]. The most remarkable feature of OPS is its strongly quantitative action [31]. For example, OPS gain-of-function mutations as well as increased OPS dosage can rescue brx protophloem (and thus root growth) defects [8,28,31].…”

CLE25 peptide regulates phloem initiation in Arabidopsis through a CLERK‐CLV2 receptor complex

“…Mutation in BAM3 rescued the short-root phenotypes of brx and ops (Rodriguez-Villalon et al 2014), suggesting that both BRX and OPS act upstream and negatively on BAM3. The ops brx double mutant exhibits an enhanced short-root phenotype (Breda et al 2017), suggesting that OPS and BRX act in parallel.…”

VISUAL-CC system uncovers the role of GSK3 as an orchestrator of vascular cell type ratio in plants

“…Although GSK3s act as a central regulator of SE/CC development, genetic experiments suggested the involvement of other signaling than BR mediated by GSK3s in this regulation. GSK3s have been implicated in regulation of phloem development through the interaction with OCTOPUS (OPS), which genetically functions together with BREVIS RADIX (BRX) 31 . Further studies on such interacting proteins and reverse genetic approaches combined with VISUAL-CC transcriptome data will be helpful for elucidating a new signaling cascade controlling the SE/CC ratio.…”