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.

Spatiotemporal cues orchestrate the development of organs and cellular differentiation in multicellular organisms. For instance, in the root apical meristem an auxin gradient patterns the transition from stem cell maintenance to transit amplification and eventual differentiation. Among the proximal tissues generated by this growth apex, the early, so-called protophloem, is the first tissue to differentiate. This observation has been linked to increased auxin activity in the developing protophloem sieve element cell files as compared to the neighboring tissues. Here we review recent progress in the characterization of the unique mechanism by which auxin canalizes its activity in the developing protophloem and fine-tunes its own transport to guide proper timing of protophloem sieve element differentiation.

Exogenous application of CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE) peptides suppresses protophloem differentiation and leads to the consumption of the proximal root meristem. However, the exact CLE peptides and the corresponding receptor complex regulating protophloem differentiation have not yet been clarified. Through expression pattern and phylogenetic analyses, CLE25/26/45 were identified as candidate peptides. Further genetic analyses, physiological assays and specific protophloem marker observations indicated that CLE25/26/45, BARELY ANY MERISTEM1/3 (BAM1/3) and CLV3 INSENSITIVE KINASEs (CIKs) are involved in regulating protophloem differentiation. The cle25 26 45 and cik2 3 4 5 6 mutation can greatly rescue the root defects of brevis radix (brx) and octopus (ops) mutants. The protophloem differentiation and proximal root meristem consumption of clv1 bam1 3 and cik2 3 4 5 6 were insensitive to CLE25/26/45 treatments. cle25 26 45, clv1 bam1 3 and cik2 3 4 5 6 displayed similar premature protophloem. In addition, CLE25/26/45 induced the interactions between BAMs and CIKs in vivo. Furthermore, CLE25/26/45 enhanced the phosphorylation levels of CIKs, which were greatly impaired in clv1 bam1 3 mutant. Our work clarifies that the CLE25/26/45-BAM1/3-CIK2/3/4/5/6 signalling module genetically acts downstream of BRX and OPS to suppress protophloem differentiation.

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.

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.

The protocol allows to define and characterize mitosis distribution patterns in the plant root meristem. The method does not require genetic markers, which makes it applicable to plants of different non-transgenic genotypes, including ecotypes, mutants, and non-model plant species. Computer analysis of the mitosis distribution in three dimensions with iRoCS Toolbox identifies statistically significant changes in proliferation activity within specific root tissues and cell lineages.

Plants continuously elaborate their bodies through post-embryonic, reiterative organ formation by apical meristems [1]. Meristems harbor stem cells, which produce daughter cells that divide repeatedly before they differentiate. How transitions between stemness, proliferation, and differentiation are precisely coordinated is not well understood, but it is known that phytohormones as well as peptide signals play important roles [2-7]. For example, in Arabidopsis thaliana root meristems, developing protophloem sieve elements (PPSEs) express the secreted CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 45 (CLE45) peptide and its cognate receptor, the leucine-rich repeat receptor kinase (LRR-RK) BARELY ANY MERISTEM 3 (BAM3). Exogenous CLE45 application or transgenically increased CLE45 dosage impairs protophloem formation, suggesting autocrine inhibition of PPSE differentiation by CLE45 signaling. Since CLE45 and BAM3 are expressed throughout PPSE development, it remains unclear how this inhibition is eventually overcome. The OCTOPUS (OPS) gene is required for proper PPSE differentiation and therefore the formation of continuous protophloem strands. OPS dosage increase can mend the phenotype of other mutants that display protophloem development defects in association with CLE45-BAM3 hyperactivity [8, 9]. Here, we provide evidence that OPS protein promotes differentiation of developing PPSEs by dampening CLE45 perception. This markedly quantitative antagonism is likely mediated through direct physical interference of OPS with CLE45 signaling component interactions. Moreover, hyperactive OPS confers resistance to other CLE peptides, and ectopic OPS overexpression triggers premature differentiation throughout the root. Our results thus reveal a novel mechanism in PPSE transition toward differentiation, wherein OPS acts as an \"insulator\" to antagonize CLE45 signaling.

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.

In plants, a complex mixture of solutes and macromolecules is transported by the phloem. Here, we examined how solutes and macromolecules are separated when they exit the phloem during the unloading process. We used a combination of approaches (non-invasive imaging, 3D-electron microscopy, and mathematical modelling) to show that phloem unloading of solutes in Arabidopsis roots occurs through plasmodesmata by a combination of mass flow and diffusion (convective phloem unloading). During unloading, solutes and proteins are diverted into the phloem-pole pericycle, a tissue connected to the protophloem by a unique class of \'funnel plasmodesmata\'. While solutes are unloaded without restriction, large proteins are released through funnel plasmodesmata in discrete pulses, a phenomenon we refer to as \'batch unloading\'. Unlike solutes, these proteins remain restricted to the phloem-pole pericycle. Our data demonstrate a major role for the phloem-pole pericycle in regulating phloem unloading in roots.

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.