Conditional effects of the epigenetic regulator JUMONJI 14 in Arabidopsis root growth.

Methylation of lysine 4 in histone 3 (H3K4) is a post-translational modification that promotes gene expression. H3K4 methylation can be reversed by specific demethylases with an enzymatic Jumonji C domain. In Arabidopsis thaliana, H3K4-specific JUMONJI (JMJ) proteins distinguish themselves by the association with an F/Y-rich (FYR) domain. Here, we report that jmj14 mutations partially suppress reduced root meristem size and growth vigor of brevis radix (brx) mutants. Similar to its close homologs, JMJ15, JMJ16 and JMJ18, the JMJ14 promoter confers expression in mature root vasculature. Yet, unlike jmj14, neither jmj16 nor jmj18 mutation markedly suppresses brx phenotypes. Domain-swapping experiments suggest that the specificity of JMJ14 function resides in the FYR domain. Despite JMJ14 promoter activity in the mature vasculature, jmj14 mutation affects root meristem size. However, JMJ14 protein is observed throughout the meristem, suggesting that the JMJ14 transcript region contributes substantially to the spatial aspect of JMJ14 expression. In summary, our data reveal a role for JMJ14 in root growth in sensitized genetic backgrounds that depends on its FYR domain and regulatory input from the JMJ14 cistron

Local and Systemic Effects of Brassinosteroid Perception in Developing Phloem.

The plant vasculature is an essential adaptation to terrestrial growth. Its phloem component permits efficient transfer of photosynthates between source and sink organs but also transports signals that systemically coordinate physiology and development. Here, we provide evidence that developing phloem orchestrates cellular behavior of adjacent tissues in the growth apices of plants, the meristems. Arabidopsis thaliana plants that lack the three receptor kinases BRASSINOSTEROID INSENSITIVE 1 (BRI1), BRI1-LIKE 1 (BRL1), and BRL3 ("bri <sup>3</sup> " mutants) can no longer sense brassinosteroid phytohormones and display severe dwarfism as well as patterning and differentiation defects, including disturbed phloem development. We found that, despite the ubiquitous expression of brassinosteroid receptors in growing plant tissues, exclusive expression of the BRI1 receptor in developing phloem is sufficient to systemically correct cellular growth and patterning defects that underlie the bri <sup>3</sup> phenotype. Although this effect is brassinosteroid-dependent, it cannot be reproduced with dominant versions of known downstream effectors of BRI1 signaling and therefore possibly involves a non-canonical signaling output. Interestingly, the rescue of bri <sup>3</sup> by phloem-specific BRI1 expression is associated with antagonism toward phloem-specific CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 45 (CLE45) peptide signaling in roots. Hyperactive CLE45 signaling causes phloem sieve element differentiation defects, and consistently, knockout of CLE45 perception in bri <sup>3</sup> background restores proper phloem development. However, bri <sup>3</sup> dwarfism is retained in such lines. Our results thus reveal local and systemic effects of brassinosteroid perception in the phloem: whereas it locally antagonizes CLE45 signaling to permit phloem differentiation, it systemically instructs plant organ formation via a phloem-derived, non-cell-autonomous signal

Local auxin competition explains fragmented differentiation patterns.

Trajectories of cellular ontogeny are tightly controlled and often involve feedback-regulated molecular antagonism. For example, sieve element differentiation along developing protophloem cell files of Arabidopsis roots requires two antagonistic regulators of auxin efflux. Paradoxically, loss-of-function in either regulator triggers similar, seemingly stochastic differentiation failures of individual sieve element precursors. Here we show that these patterning defects are distinct and non-random. They can be explained by auxin-dependent bistability that emerges from competition for auxin between neighboring cells. This bistability depends on the presence of an auxin influx facilitator, and can be triggered by either flux enhancement or repression. Our results uncover a hitherto overlooked aspect of auxin uptake, and highlight the contributions of local auxin influx, efflux and biosynthesis to protophloem formation. Moreover, the combined experimental-modeling approach suggests that without auxin efflux homeostasis, auxin influx interferes with coordinated differentiation

Ectopic assembly of an auxin efflux control machinery shifts developmental trajectories.

Polar auxin transport in the Arabidopsis (Arabidopsis thaliana) root tip maintains high auxin levels around the stem cell niche that gradually decrease in dividing cells but increase again once they transition towards differentiation. Protophloem differentiates earlier than other proximal tissues and employs a unique auxin 'canalization' machinery that is thought to balance auxin efflux with retention. It consists of a proposed activator of PIN-FORMED (PIN) auxin efflux carriers, the AGC kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX); its inhibitor, BREVIS RADIX (BRX); and PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5 K) enzymes, which promote polar PAX and BRX localization. Because of dynamic PAX-BRX-PIP5 K interplay, the net cellular output of this machinery remains unclear. Here we deciphered the dosage-sensitive regulatory interactions between PAX, BRX and PIP5 K by their ectopic expression in developing xylem vessels. The data suggest that the dominant collective output of the PAX-BRX-PIP5 K module is a localized reduction in PIN abundance. This requires PAX-stimulated clathrin-mediated PIN endocytosis by site-specific phosphorylation, which distinguishes PAX from other AGC kinases. Ectopic assembly of the PAX-BRX-PIP5 K module is sufficient to cause cellular auxin retention and affects root growth vigor by accelerating the trajectory of xylem vessel development. Our data thus provide direct evidence that local manipulation of auxin efflux alters the timing of cellular differentiation in the root

A molecular rheostat adjusts auxin flux to promote root protophloem differentiation.

Auxin influences plant development through several distinct concentration-dependent effects <sup>1</sup> . In the Arabidopsis root tip, polar auxin transport by PIN-FORMED (PIN) proteins creates a local auxin accumulation that is required for the maintenance of the stem-cell niche <sup>2-4</sup> . Proximally, stem-cell daughter cells divide repeatedly before they eventually differentiate. This developmental gradient is accompanied by a gradual decrease in auxin levels as cells divide, and subsequently by a gradual increase as the cells differentiate <sup>5,6</sup> . However, the timing of differentiation is not uniform across cell files. For instance, developing protophloem sieve elements (PPSEs) differentiate as neighbouring cells still divide. Here we show that PPSE differentiation involves local steepening of the post-meristematic auxin gradient. BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX) are interacting plasma-membrane-associated, polarly localized proteins that co-localize with PIN proteins at the rootward end of developing PPSEs. Both brx and pax mutants display impaired PPSE differentiation. Similar to other AGC-family kinases, PAX activates PIN-mediated auxin efflux, whereas BRX strongly dampens this stimulation. Efficient BRX plasma-membrane localization depends on PAX, but auxin negatively regulates BRX plasma-membrane association and promotes PAX activity. Thus, our data support a model in which BRX and PAX are elements of a molecular rheostat that modulates auxin flux through developing PPSEs, thereby timing PPSE differentiation

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

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

Arabidopsis Flippases Cooperate with ARF GTPase Exchange Factors to Regulate the Trafficking and Polarity of PIN Auxin Transporters.

Cell polarity is a fundamental feature of all multicellular organisms. PIN auxin transporters are important cell polarity markers that play crucial roles in a plethora of developmental processes in plants. Here, to identify components involved in cell polarity establishment and maintenance in plants, we performed a forward genetic screening of PIN2:PIN1-HA;pin2 Arabidopsis (Arabidopsis thaliana) plants, which ectopically express predominantly basally localized PIN1 in root epidermal cells, leading to agravitropic root growth. We identified the regulator of PIN polarity 12 (repp12) mutation, which restored gravitropic root growth and caused a switch in PIN1-HA polarity from the basal to apical side of root epidermal cells. Next Generation Sequencing and complementation experiments established the causative mutation of repp12 as a single amino acid exchange in Aminophospholipid ATPase3 (ALA3), a phospholipid flippase predicted to function in vesicle formation. repp12 and ala3 T-DNA mutants show defects in many auxin-regulated processes, asymmetric auxin distribution, and PIN trafficking. Analysis of quintuple and sextuple mutants confirmed the crucial roles of ALA proteins in regulating plant development as well as PIN trafficking and polarity. Genetic and physical interaction studies revealed that ALA3 functions together with the ADP ribosylation factor GTPase exchange factors GNOM and BIG3 in regulating PIN polarity, trafficking, and auxin-mediated development