Mapping and engineering of auxin-induced plasma membrane dissociation in BRX family proteins

Abstract

Angiosperms have evolved the phloem for the long-distance transport of metabolites. The complex process of phloem development involves genes that only occur in vascular plant lineages. For example, in Arabidopsis thaliana, the BREVIS RADIX (BRX) gene is required for continuous root protophloem differentiation, together with PROTEIN KINASE ASSOCIATED WITH BRX (PAX). BRX and its BRX-LIKE (BRXL) homologs are composed of four highly conserved domains including the signature tandem BRX domains that are separated by variable spacers. Nevertheless, BRX family proteins have functionally diverged. For instance, BRXL2 can only partially replace BRX in the root protophloem. This divergence is reflected in physiologically relevant differences in protein behavior, such as auxin-induced plasma membrane dissociation of BRX, which is not observed for BRXL2. Here we dissected the differential functions of BRX family proteins using a set of amino acid substitutions and domain swaps. Our data suggest that the plasma membrane-associated tandem BRX domains are both necessary and sufficient to convey the biological outputs of BRX function and therefore constitute an important regulatory entity. Moreover, PAX target phosphosites in the linker between the two BRX domains mediate the auxin-induced plasma membrane dissociation. Engineering these sites into BRXL2 renders this modified protein auxin-responsive and thereby increases its biological activity in the root protophloem context.

A CLE–BAM–CIK signalling module controls root protophloem differentiation in Arabidopsis

“…For example, BREVIS RADIX ( BRX ) functions at the plasma membrane as a critical regulator of protophloem development. The brx mutants exhibit a smaller proximal root meristem, eventually leading to a short‐root phenotype, which is caused by a discontinued protophloem (Mouchel et al ., 2004; Scacchi et al ., 2010; Marhava et al ., 2018; Koh et al ., 2021).…”

Heterologous expression of a lycophyte protein enhances angiosperm seedling vigor

“…The encoded BRX family proteins consist of four distinct, highly conserved domains that are not found outside the green lineage (Briggs et al, 2006; Koh et al, 2021). They include the signature tandem “BRX-domains”, which are connected by a linker of variable sequence and size (Koh et al, 2021). All BRX family proteins monitored to date are primarily plasma-membrane-associated and display polar cellular localization (Bringmann and Bergmann, 2017; Koh et al, 2021; Marhava et al, 2020; Marhava et al, 2018; Rowe et al, 2019; Scacchi et al, 2009).…”

“…They include the signature tandem “BRX-domains”, which are connected by a linker of variable sequence and size (Koh et al, 2021). All BRX family proteins monitored to date are primarily plasma-membrane-associated and display polar cellular localization (Bringmann and Bergmann, 2017; Koh et al, 2021; Marhava et al, 2020; Marhava et al, 2018; Rowe et al, 2019; Scacchi et al, 2009). The biological functions of BRX family genes are diverse and point to sub-functionalization of individual family members (Koh et al, 2021; Li et al, 2019; Marhava et al, 2020; Rowe et al, 2019; Zhang et al, 2021).…”

“…All BRX family proteins monitored to date are primarily plasma-membrane-associated and display polar cellular localization (Bringmann and Bergmann, 2017; Koh et al, 2021; Marhava et al, 2020; Marhava et al, 2018; Rowe et al, 2019; Scacchi et al, 2009). The biological functions of BRX family genes are diverse and point to sub-functionalization of individual family members (Koh et al, 2021; Li et al, 2019; Marhava et al, 2020; Rowe et al, 2019; Zhang et al, 2021). For instance, the best-characterized members, AtBRX and AtBRXL2 , are interchangeable in stomata development (Rowe et al, 2019) but not in root protophloem development (Koh et al, 2021; Marhava et al, 2020), and this unequal redundancy has recently been associated with differences in protein behavior (Koh et al, 2021; Marhava et al, 2020).…”

“…The biological functions of BRX family genes are diverse and point to sub-functionalization of individual family members (Koh et al, 2021; Li et al, 2019; Marhava et al, 2020; Rowe et al, 2019; Zhang et al, 2021). For instance, the best-characterized members, AtBRX and AtBRXL2 , are interchangeable in stomata development (Rowe et al, 2019) but not in root protophloem development (Koh et al, 2021; Marhava et al, 2020), and this unequal redundancy has recently been associated with differences in protein behavior (Koh et al, 2021; Marhava et al, 2020). In the root, AtBRX guides the progression of protophloem sieve element differentiation by modulating the local trans-cellular flux of the phytohormone, auxin (Marhava et al, 2018; Moret et al, 2020).…”