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 toward 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 cAMP-, cGMP- and Calcium-dependent (AGC) kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX); its inhibitor, BREVIS RADIX (BRX); and PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K) enzymes, which promote polar PAX and BRX localization. Because of a dynamic PAX-BRX-PIP5K interplay, the net cellular output of this machinery remains unclear. In this study, we deciphered the dosage-sensitive regulatory interactions among PAX, BRX, and PIP5K by their ectopic expression in developing xylem vessels. The data suggest that the dominant collective output of the PAX-BRX-PIP5K module is a localized reduction in PIN abundance. This requires PAX-stimulated clathrin-mediated PIN endocytosis upon site-specific phosphorylation, which distinguishes PAX from other AGC kinases. An ectopic assembly of the PAX-BRX-PIP5K 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.

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.

Metaphloem development in the Arabidopsis root tip.

The phloem transport network is a major evolutionary innovation that enabled plants to dominate terrestrial ecosystems. In the growth apices, the meristems, apical stem cells continuously produce early 'protophloem'. This is easily observed in Arabidopsis root meristems, in which the differentiation of individual protophloem sieve element precursors into interconnected conducting sieve tubes is laid out in a spatio-temporal gradient. The mature protophloem eventually collapses as the neighboring metaphloem takes over its function further distal from the stem cell niche. Compared with protophloem, metaphloem ontogenesis is poorly characterized, primarily because its visualization is challenging. Here, we describe the improved TetSee protocol to investigate metaphloem development in Arabidopsis root tips in combination with a set of molecular markers. We found that mature metaphloem sieve elements are only observed in the late post-meristematic root, although their specification is initiated as soon as protophloem sieve elements enucleate. Moreover, unlike protophloem sieve elements, metaphloem sieve elements only differentiate once they have fully elongated. Finally, our results suggest that metaphloem differentiation is not directly controlled by protophloem-derived cues but rather follows a distinct, robust developmental trajectory.

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.

Phloem development.

The evolution of the plant vascular system is a key process in Earth history because it enabled plants to conquer land and transform the terrestrial surface. Among the vascular tissues, the phloem is particularly intriguing because of its complex functionality. In angiosperms, its principal components are the sieve elements, which transport phloem sap, and their neighboring companion cells. Together, they form a functional unit that sustains sap loading, transport, and unloading. The developmental trajectory of sieve elements is unique among plant cell types because it entails selective organelle degradation including enucleation. Meticulous analyses of primary, so-called protophloem in the Arabidopsis thaliana root meristem have revealed key steps in protophloem sieve element formation at single-cell resolution. A transcription factor cascade connects specification with differentiation and also orchestrates phloem pole patterning via noncell-autonomous action of sieve element-derived effectors. Reminiscent of vascular tissue patterning in secondary growth, these involve receptor kinase pathways, whose antagonists guide the progression of sieve element differentiation. Receptor kinase pathways may also safeguard phloem formation by maintaining the developmental plasticity of neighboring cell files. Our current understanding of protophloem development in the A. thaliana root has reached sufficient detail to instruct molecular-level investigation of phloem formation in other organs.

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.

BAM1/2 receptor kinase signaling drives CLE peptide-mediated formative cell divisions in Arabidopsis roots.

Cell division is often regulated by extracellular signaling networks to ensure correct patterning during development. In Arabidopsis, the SHORT-ROOT (SHR)/SCARECROW (SCR) transcription factor dimer activates CYCLIND6;1 (CYCD6;1) to drive formative divisions during root ground tissue development. Here, we show plasma-membrane-localized BARELY ANY MERISTEM1/2 (BAM1/2) family receptor kinases are required for SHR-dependent formative divisions and CYCD6;1 expression, but not SHR-dependent ground tissue specification. Root-enriched CLE ligands bind the BAM1 extracellular domain and are necessary and sufficient to activate SHR-mediated divisions and CYCD6;1 expression. Correspondingly, BAM-CLE signaling contributes to the restriction of formative divisions to the distal root region. Additionally, genetic analysis reveals that BAM-CLE and SHR converge to regulate additional cell divisions outside of the ground tissues. Our work identifies an extracellular signaling pathway regulating formative root divisions and provides a framework to explore this pathway in patterning and evolution.

The transcription and export complex THO/TREX contributes to transcription termination in plants.

Transcription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3'UTR extensions in several endogenous genes. These results demonstrate that THO/TREX complex contributes to the regulation of transcription termination.

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.

CLERK is a novel receptor kinase required for sensing of root-active CLE peptides in Arabidopsis.

CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides are secreted endogenous plant ligands that are sensed by receptor kinases (RKs) to convey environmental and developmental inputs. Typically, this involves an RK with narrow ligand specificity that signals together with a more promiscuous co-receptor. For most CLEs, biologically relevant (co-)receptors are unknown. The dimer of the receptor-like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) conditions perception of so-called root-active CLE peptides, the exogenous application of which suppresses root growth by preventing protophloem formation in the meristem. clv2 as well as crn null mutants are resistant to root-active CLE peptides, possibly because CLV2-CRN promotes expression of their cognate receptors. Here, we have identified the CLE-RESISTANT RECEPTOR KINASE (CLERK) gene, which is required for full sensing of root-active CLE peptides in early developing protophloem. CLERK protein can be replaced by its close homologs, SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (SARK) and NSP-INTERACTING KINASE 1 (NIK1). Yet neither CLERK nor NIK1 ectodomains interact biochemically with described CLE receptor ectodomains. Consistently, CLERK also acts genetically independently of CLV2-CRN We, thus, have discovered a novel hub for redundant CLE sensing in the root.

Brassinosteroid signaling directs formative cell divisions and protophloem differentiation in Arabidopsis root meristems.

Brassinosteroids (BRs) trigger an intracellular signaling cascade through its receptors BR INSENSITIVE 1 (BRI1), BRI1-LIKE 1 (BRL1) and BRL3. Recent studies suggest that BR-independent inputs related to vascular differentiation, for instance root protophloem development, modulate downstream BR signaling components. Here, we report that protophloem sieve element differentiation is indeed impaired in bri1 brl1 brl3 mutants, although this effect might not be mediated by canonical downstream BR signaling components. We also found that their small meristem size is entirely explained by reduced cell elongation, which is, however, accompanied by supernumerary formative cell divisions in the radial dimension. Thus, reduced cell expansion in conjunction with growth retardation, because of the need to accommodate supernumerary formative divisions, can account for the overall short root phenotype of BR signaling mutants. Tissue-specific re-addition of BRI1 activity partially rescued subsets of these defects through partly cell-autonomous, partly non-cell-autonomous effects. However, protophloem-specific BRI1 expression essentially rescued all major bri1 brl1 brl3 root meristem phenotypes. Our data suggest that BR perception in the protophloem is sufficient to systemically convey BR action in the root meristem context.

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

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.

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses.

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium.

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

Perception of root-active CLE peptides requires CORYNE function in the phloem vasculature.

Arabidopsis root development is orchestrated by signaling pathways that consist of different CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide ligands and their cognate CLAVATA (CLV) and BARELY ANY MERISTEM (BAM) receptors. How and where different CLE peptides trigger specific morphological or physiological changes in the root is poorly understood. Here, we report that the receptor-like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) are necessary to fully sense root-active CLE peptides. We uncover BAM3 as the CLE45 receptor in the root and biochemically map its peptide binding surface. In contrast to other plant peptide receptors, we found no evidence that SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) proteins act as co-receptor kinases in CLE45 perception. CRN stabilizes BAM3 expression and thus is required for BAM3-mediated CLE45 signaling. Moreover, protophloem-specific CRN expression complements resistance of the crn mutant to root-active CLE peptides, suggesting that protophloem is their principal site of action. Our work defines a genetic framework for dissecting CLE peptide signaling and CLV/BAM receptor activation in the root.

Primary root protophloem differentiation requires balanced phosphatidylinositol-4,5-biphosphate levels and systemically affects root branching.

Protophloem is a specialized vascular tissue in growing plant organs, such as root meristems. In Arabidopsis mutants with impaired primary root protophloem differentiation, brevis radix (brx) and octopus (ops), meristematic activity and consequently overall root growth are strongly reduced. Second site mutation in the protophloem-specific presumed phosphoinositide 5-phosphatase cotyledon vascular pattern 2 (CVP2), but not in its homolog CVP2-like 1 (CVL1), partially rescues brx defects. Consistent with this finding, CVP2 hyperactivity in a wild-type background recreates a brx phenotype. Paradoxically, however, while cvp2 or cvl1 single mutants display no apparent root defects, the root phenotype of cvp2 cvl1 double mutants is similar to brx or ops, although, as expected, cvp2 cvl1 seedlings contain more phosphatidylinositol-4,5-biphosphate. Thus, tightly balanced phosphatidylinositol-4,5-biphosphate levels appear essential for proper protophloem differentiation. Genetically, OPS acts downstream of phosphatidylinositol-4,5-biphosphate levels, as cvp2 mutation cannot rescue ops defects, whereas increased OPS dose rescues cvp2 cvl1 defects. Finally, all three mutants display higher density and accelerated emergence of lateral roots, which correlates with increased auxin response in the root differentiation zone. This phenotype is also created by application of peptides that suppress protophloem differentiation, clavata3/embryo surrounding region 26 (CLE26) and CLE45. Thus, local changes in the primary root protophloem systemically shape overall root system architecture.

Broad spectrum developmental role of Brachypodium AUX1.

Targeted cellular auxin distribution is required for morphogenesis and adaptive responses of plant organs. In Arabidopsis thaliana (Arabidopsis), this involves the prototypical auxin influx facilitator AUX1 and its LIKE-AUX1 (LAX) homologs, which act partially redundantly in various developmental processes. Interestingly, AUX1 and its homologs are not strictly essential for the Arabidopsis life cycle. Indeed, aux1 lax1 lax2 lax3 quadruple knock-outs are mostly viable and fertile, and strong phenotypes are only observed at low penetrance. Here we investigated the Brachypodium distachyon (Brachypodium) AUX1 homolog BdAUX1 by genetic, cell biological and physiological analyses. We report that BdAUX1 is essential for Brachypodium development. Bdaux1 loss-of-function mutants are dwarfs with aberrant flower development, and consequently infertile. Moreover, they display a counter-intuitive root phenotype. Although Bdaux1 roots are agravitropic as expected, in contrast to Arabidopsis aux1 mutants they are dramatically longer than wild type roots because of exaggerated cell elongation. Interestingly, this correlates with higher free auxin content in Bdaux1 roots. Consistently, their cell wall characteristics and transcriptome signature largely phenocopy other Brachypodium mutants with increased root auxin content. Our results imply fundamentally different wiring of auxin transport in Brachypodium roots and reveal an essential role of BdAUX1 in a broad spectrum of developmental processes, suggesting a central role for AUX1 in pooideae.

Arabidopsis MAKR5 is a positive effector of BAM3-dependent CLE45 signaling.

Receptor kinases convey diverse environmental and developmental inputs by sensing extracellular ligands. In plants, one group of receptor-like kinases (RLKs) is characterized by extracellular leucine-rich repeat (LRR) domains, which interact with various ligands that include the plant hormone brassinosteroid and peptides of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) type. For instance, the CLE45 peptide requires the LRR-RLK BARELY ANY MERISTEM 3 (BAM3) to prevent protophloem formation in Arabidopsis root meristems. Here, we show that other proposed CLE45 receptors, the two redundantly acting LRR-RLKs STERILITY-REGULATING KINASE MEMBER 1 (SKM1) and SKM2 (which perceive CLE45 in the context of pollen tube elongation), cannot substitute for BAM3 in the root. Moreover, we identify MEMBRANE-ASSOCIATED KINASE REGULATOR 5 (MAKR5) as a post-transcriptionally regulated amplifier of the CLE45 signal that acts downstream of BAM3. MAKR5 belongs to a small protein family whose prototypical member, BRI1 KINASE INHIBITOR 1, is an essentially negative regulator of brassinosteroid signaling. By contrast, MAKR5 is a positive effector of CLE45 signaling, revealing an unexpected diversity in the conceptual roles of MAKR genes in different signaling pathways.

BIG BROTHER Uncouples Cell Proliferation from Elongation in the Arabidopsis Primary Root.

Plant organ size is sensitive to environmental conditions, but is also limited by hardwired genetic constraints. In Arabidopsis, a few organ size regulators have been identified. Among them, the BIG BROTHER (BB) gene has a prominent role in the determination of flower organ and leaf size. BB loss-of-function mutations result in a prolonged proliferation phase during leaf(-like) organ formation, and consequently larger leaves, petals and sepals. Whether BB has a similar role in root growth is unknown. Here we describe a novel bb allele which carries a P235L point mutation in the BB RING finger domain. This allele behaves similarly to described bb loss-of-function alleles and displays increased root meristem size due to a higher number of dividing, meristematic cells. In contrast, mature cell length is unaffected. The increased meristematic activity does not, however, translate into overall enhanced root elongation, possibly because bb mutation also results in an increased number of cell files in the vascular cylinder. These extra formative divisions might offset any growth acceleration by extra meristematic divisions. Thus, although BB dampens root cell proliferation, the consequences on macroscopic root growth are minor. However, bb mutation accelerates overall root growth when introduced into sensitized backgrounds. For example, it partially rescues the short root phenotypes of the brevis radix and octopus mutants, but does not complement their phloem differentiation or transport defects. In summary, we provide evidence that BB acts conceptually similarly in leaf(-like) organs and the primary root, and uncouples cell proliferation from elongation in the root meristem.

BEN3/BIG2 ARF GEF is Involved in Brefeldin A-Sensitive Trafficking at the trans-Golgi Network/Early Endosome in Arabidopsis thaliana.

Membrane traffic at the trans-Golgi network (TGN) is crucial for correctly distributing various membrane proteins to their destination. Polarly localized auxin efflux proteins, including PIN-FORMED1 (PIN1), are dynamically transported between the endosomes and the plasma membrane (PM) in the plant cells. The intracellular trafficking of PIN1 protein is sensitive to the fungal toxin brefeldin A (BFA), which is known to inhibit guanine nucleotide exchange factors for ADP ribosylation factors (ARF GEFs) such as GNOM. However, the molecular details of the BFA-sensitive trafficking pathway have not been fully revealed. In a previous study, we identified an Arabidopsis mutant BFA-visualized endocytic trafficking defective 3 (ben3) which exhibited reduced sensitivity to BFA in terms of BFA-induced intracellular PIN1 agglomeration. Here, we show that BEN3 encodes a member of BIG family ARF GEFs, BIG2. BEN3/BIG2 tagged with fluorescent proteins co-localized with markers for the TGN/early endosome (EE). Inspection of conditionally induced de novo synthesized PIN1 confirmed that its secretion to the PM is BFA sensitive, and established BEN3/BIG2 as a crucial component of this BFA action at the level of the TGN/EE. Furthermore, ben3 mutation alleviated BFA-induced agglomeration of another TGN-localized ARF GEF, BEN1/MIN7. Taken together, our results suggest that BEN3/BIG2 is an ARF GEF component, which confers BFA sensitivity to the TGN/EE in Arabidopsis.