Auxin influences plant development through several distinct concentration-dependent effects . 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. 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. 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.

Summary Patterning in plants relies on oriented cell divisions and acquisition of specific cell identities. Plants regularly endure wounds caused by abiotic or biotic environmental stimuli and have developed extraordinary abilities to restore their tissues after injuries. Here, we provide insight into a mechanism of restorative patterning that repairs tissues after wounding. Laser-assisted elimination of different cells in Arabidopsis root combined with live-imaging tracking during vertical growth allowed analysis of the regeneration processes in vivo . Specifically, the cells adjacent to the inner side of the injury re-activated their stem cell transcriptional programs. They accelerated their progression through cell cycle, coordinately changed the cell division orientation, and ultimately acquired de novo the correct cell fates to replace missing cells. These observations highlight existence of unknown intercellular positional signaling and demonstrate the capability of specified cells to re-acquire stem cell programs as a crucial part of the plant-specific mechanism of wound healing.

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. Finetuned 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-4phosphate 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.

Wound healing in plant tissues, consisting of rigid cell wall-encapsulated cells, represents a considerable challenge and occurs through largely unknown mechanisms distinct from those in animals. Owing to their inability to migrate, plant cells rely on targeted cell division and expansion to regenerate wounds. Strict coordination of these wound-induced responses is essential to ensure efficient, spatially restricted wound healing. Single-cell tracking by live imaging allowed us to gain mechanistic insight into the wound perception and coordination of wound responses after laser-based wounding inArabidopsisroot. We revealed a crucial contribution of the collapse of damaged cells in wound perception and detected an auxin increase specific to cells immediately adjacent to the wound. This localized auxin increase balances wound-induced cell expansion and restorative division rates in a dose-dependent manner, leading to tumorous overproliferation when the canonical TIR1 auxin signaling is disrupted. Auxin and wound-induced turgor pressure changes together also spatially define the activation of key components of regeneration, such as the transcription regulator ERF115. Our observations suggest that the wound signaling involves the sensing of collapse of damaged cells and a local auxin signaling activation to coordinate the downstream transcriptional responses in the immediate wound vicinity.

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 carried out a forward genetic screening of PIN2:PIN1-HA;pin2 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

Highlights d BR perception in developing phloem rescues the dwarfism of receptor mutants d BR perception antagonizes CLE45 peptide signaling in the root protophloem d Local and systemic effects of BR perception in the phloem are genetically distinct d Developing phloem can orchestrate cellular behavior of adjacent tissues

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.

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.