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.

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.

OBJECTIVE: Primary vascular tissues of angiosperm and gymnosperm roots have significant anatomical differences. In gymnosperms, lack of protophloem sieve elements indicates a lengthy parenchymatous pathway for nutrient transport to the root apical meristem (RAM). Because F-actin is an essential component of transport in parenchyma cells, the distribution of F-actin was determined and compared among roots of several angiosperm and gymnosperm species.
METHODS: Roots were chemically fixed and sectioned by hand to enable rapid production of many sections for labeling F-actin with phalloidin.
RESULTS: In angiosperm and gymnosperm root tips, relative intensity of F-actin labeling was highest in primary vascular tissues. Parenchyma cells in and around protophloem tended to have more F-actin while cells in cortical and protoxylem tissues tended to have less. In gymnosperms, phloem parenchyma was intensely labeled for several millimeters distal to the root apical meristem (RAM), and the F-actin is mostly composed of bundles that lie parallel to the root longitudinal axis. This orientation differed from the multidirectional arrangement of F-actin filaments in cortical cells. In angiosperms, intense F-actin labeling of pericycle and phloem parenchyma cells occurred around the first mature sieve elements.
CONCLUSIONS: F-actin is concentrated in the vascular cylinder, commonly in primary phloem parenchyma. In gymnosperms, the absence of sieve elements suggests that cytoplasmic streaming has a role in some aspect of phloem transport or unloading. In angiosperms, the region of intense F-actin labeling in the phloem parenchyma is limited to the extreme terminal portion of primary phloem where unloading of the earliest mature sieve elements occurs.

Sedentary plant parasitic nematodes such as root-knot nematodes and cyst nematodes induce giant cells or syncytia, respectively, in their host plant\'s roots. These highly specialized structures serve as feeding sites from which exclusively the nematodes withdraw nutrients. While giant cells are symplastically isolated and obtain assimilates by transporter-mediated processes syncytia are massively connected to the phloem by plasmodesmata. To support the feeding sites and the nematode during their development, phloem is induced around syncytia and giant cells. In the case of syncytia the unloading phloem consists of sieve elements and companion cells and in the case of root knots it consists exclusively of sieve elements. We applied immunohistochemistry to identify the cells within the developing phloem that responded to auxin and cytokinin. Both feeding sites themselves did not respond to either hormone. We were able to show that in root knots an auxin response precedes the differentiation of these auxin responsive cells into phloem elements. This process appears to be independent of B-type Arabidopsis response regulators. Using additional markers for tissue identity we provide evidence that around giant cells protophloem is formed and proliferates dramatically. In contrast, the phloem around syncytia responded to both hormones. The presence of companion cells as well as hormone-responsive sieve elements suggests that metaphloem development occurs. The implication of auxin and cytokinin in the further development of the metaphloem is discussed.