O-glycosylation of the transcription factor SPATULA promotes style development in Arabidopsis.

O-linked ß-N-acetylglucosamine (O-GlcNAc) and O-fucose are two sugar-based post-translational modifications whose mechanistic role in plant signalling and transcriptional regulation is still largely unknown. Here we investigated how two O-glycosyltransferase enzymes of Arabidopsis thaliana, SPINDLY (SPY) and SECRET AGENT (SEC), promote the activity of the basic helix-loop-helix transcription factor SPATULA (SPT) during morphogenesis of the plant female reproductive organ apex, the style. SPY and SEC modify amino-terminal residues of SPT in vivo and in vitro by attaching O-fucose and O-GlcNAc, respectively. This post-translational regulation does not impact SPT homo- and heterodimerization events, although it enhances the affinity of SPT for the kinase PINOID gene locus and its transcriptional repression. Our findings offer a mechanistic example of the effect of O-GlcNAc and O-fucose on the activity of a plant transcription factor and reveal previously unrecognized roles for SEC and SPY in orchestrating style elongation and shape.

Heterologous expression of a lycophyte protein enhances angiosperm seedling vigor.

Seedling vigor is a key agronomic trait that determines juvenile plant performance. Angiosperm seeds develop inside fruits and are connected to the mother plant through vascular tissues. Their formation requires plant-specific genes, such as BREVIS RADIX (BRX) in Arabidopsis thaliana roots. BRX family proteins are found throughout the euphyllophytes but also occur in non-vascular bryophytes and non-seed lycophytes. They consist of four conserved domains, including the tandem BRX domains. We found that bryophyte or lycophyte BRX homologs can only partially substitute for Arabidopsis BRX (AtBRX) because they miss key features in the linker between the BRX domains. Intriguingly, however, expression of a BRX homolog from the lycophyte Selaginella moellendorffii (SmBRX) in an A. thaliana wild-type background confers robustly enhanced root growth vigor that persists throughout the life cycle. This effect can be traced to a substantial increase in seed and embryo size, is associated with enhanced vascular tissue proliferation, and can be reproduced with a modified, SmBRX-like variant of AtBRX. Our results thus suggest that BRX variants can boost seedling vigor and shed light on the activity of ancient, non-angiosperm BRX family proteins.

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

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.

Plasma Membrane Domain Patterning and Self-Reinforcing Polarity in Arabidopsis.

Cell polarity is a key feature in the development of multicellular organisms. For instance, asymmetrically localized plasma-membrane-integral PIN-FORMED (PIN) proteins direct transcellular fluxes of the phytohormone auxin that govern plant development. Fine-tuned auxin flux is important for root protophloem sieve element differentiation and requires the interacting plasma-membrane-associated BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX) proteins. We observed "donut-like" polar PIN localization in developing sieve elements that depends on complementary, "muffin-like" polar localization of BRX and PAX. Plasma membrane association and polarity of PAX, and indirectly BRX, largely depends on phosphatidylinositol-4,5-bisphosphate. Consistently, mutants in phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) display protophloem differentiation defects similar to brx mutants. The same PIP5Ks are in complex with BRX and display "muffin-like" polar localization. Our data suggest that the BRX-PAX module recruits PIP5Ks to reinforce PAX polarity and thereby the polarity of all three proteins, which is required to maintain a local PIN minimum.