The phloem performs essential systemic functions in tracheophytes, yet little is known about its molecular genetic specification. Here we show that application of the peptide ligand CLAVATA3/EMBRYO SURROUNDING REGION 45 (CLE45) specifically inhibits specification of protophloem in Arabidopsis roots by locking the sieve element precursor cell in its preceding developmental state. CLE45 treatment, as well as viable transgenic expression of a weak CLE45 G6T variant, interferes not only with commitment to sieve element fate but also with the formative sieve element precursor cell division that creates protophloem and metaphloem cell files. However, the absence of this division appears to be a secondary effect of discontinuous sieve element files and subsequent systemically reduced auxin signaling in the root meristem. In the absence of the formative sieve element precursor cell division, metaphloem identity is seemingly adopted by the normally procambial cell file instead, pointing to possibly independent positional cues for metaphloem formation. The protophloem formation and differentiation defects in brevis radix (brx) and octopus (ops) mutants are similar to those observed in transgenic seedlings with increased CLE45 activity and can be rescued by loss of function of a putative CLE45 receptor, BARELY ANY MERI-STEM 3 (BAM3). Conversely, a dominant gain-of-function ops allele or mild OPS dosage increase suppresses brx defects and confers CLE45 resistance. Thus, our data suggest that delicate quantitative interplay between the opposing activities of BAM3-mediated CLE45 signals and OPS-dependent signals determines cellular commitment to protophloem sieve element fate, with OPS acting as a positive, quantitative master regulator of phloem fate. stem cell | division plane switching

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.

A central question in developmental biology is how multicellular organisms coordinate cell division and differentiation to determine organ size. In Arabidopsis roots, this balance is controlled by cytokinin-induced expression of SHORT HYPOCOTYL 2 (SHY2) in the so-called transition zone of the meristem, where SHY2 negatively regulates auxin response factors (ARFs) by protein-protein interaction. The resulting down-regulation of PIN-FORMED (PIN) auxin efflux carriers is considered the key event in promoting differentiation of meristematic cells. Here we show that this regulation involves additional, intermediary factors and is spatio-temporally constrained. We found that the described cytokinin-auxin crosstalk antagonizes BREVIS RADIX (BRX) activity in the developing protophloem. BRX is an auxin-responsive target of the prototypical ARF MONOPTEROS (MP), a key promoter of vascular development, and transiently enhances PIN3 expression to promote meristem growth in young roots. At later stages, cytokinin induction of SHY2 in the vascular transition zone restricts BRX expression to down-regulate PIN3 and thus limit meristem growth. Interestingly, proper SHY2 expression requires BRX, which could reflect feedback on the auxin responsiveness of SHY2 because BRX protein can directly interact with MP, likely acting as a cofactor. Thus, cross-regulatory antagonism between BRX and SHY2 could determine ARF activity in the protophloem. Our data suggest a model in which the regulatory interactions favor BRX expression in the early proximal meristem and SHY2 prevails because of supplementary cytokinin induction in the later distal meristem. The complex equilibrium of this regulatory module might represent a universal switch in the transition toward differentiation in various developmental contexts.

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.

Soil acidification is a major agricultural problem that negatively affects crop yield. Root systems counteract detrimental passive proton influx from acidic soil through increased proton pumping into the apoplast, which is presumably also required for cell elongation and stimulated by auxin. Here, we found an unexpected impact of extracellular pH on auxin activity and cell proliferation rate in the root meristem of two Arabidopsis mutants with impaired auxin perception, axr3 and brx. Surprisingly, neutral to slightly alkaline media rescued their severely reduced root (meristem) growth by stimulating auxin signaling, independent of auxin uptake. The finding that proton pumps are hyperactive in brx roots could explain this phenomenon and is consistent with more robust growth and increased fitness of brx mutants on overly acidic media or soil. Interestingly, the original brx allele was isolated from a natural stock center accession collected from acidic soil. Our discovery of a novel brx allele in accessions recently collected from another acidic sampling site demonstrates the existence of independently maintained brx loss-of-function alleles in nature and supports the notion that they are advantageous in acidic soil pH conditions, a finding that might be exploited for crop breeding.

Highlights d An early PSE misspecification promotes identity hybridism between PSE and CC d Meristematic CC and MSE retain plastic identity to safeguard phloem functionality d RPK2 excludes PSE identity from PSE-surrounding cells within the root meristem d CLE45 maintains PSE and PSE-surrounding cells in a plastic identity stage

The plant vascular network consists of specialized phloem and xylem elements that undergo two distinct morphogenetic developmental programs to become transport-functional units. Whereas vacuolar rupture is a determinant step in protoxylem differentiation, protophloem elements never form a big central vacuole. Here, we show that a genetic disturbance of phosphatidylinositol 4,5-bis-phosphate [PtdIns(4,5)P2] homeostasis rewires cell trafficking towards the vacuole in Arabidopsis thaliana roots. Consequently, an enhanced phosphoinositide-mediated vacuolar biogenesis correlates with premature programmed cell death (PCD) and secondary cell wall elaboration in xylem cells. By contrast, vacuolar fusion events in protophloem cells trigger the abnormal formation of big vacuoles, preventing cell clearance and tissue functionality. Removal of the inositol 5′ phosphatase COTYLEDON VASCULAR PATTERN 2 from the plasma membrane (PM) by brefeldin A (BFA) treatment increases PtdIns(4,5)P2 content at the PM and disrupts protophloem continuity. Conversely, BFA application abolishes vacuolar fusion events in xylem tissue without preventing PCD, suggesting the existence of additional PtdIns(4,5)P2-dependent cell death mechanisms. Overall, our data indicate that tight PM phosphoinositide homeostasis is required to modulate intracellular trafficking contributing to oppositely regulate vascular differentiation.

The package SQUADD is freely available via the Bioconductor repository at http://www.bioconductor.org/help/bioc-views/release/bioc/html/SQUADD.html.