Dual role for ubiquitin in plant steroid hormone receptor endocytosis

Brassinosteroids are plant steroid hormones that control many aspects of plant growth and development, and are perceived at the cell surface by the plasma membrane-localized receptor kinase BRI1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Using both artificial ubiquitination of BRI1 and generation of an ubiquitination-defective BRI1 mutant form, we demonstrate that ubiquitination promotes BRI1 internalization from the cell surface and is essential for its recognition at the trans-Golgi network/early endosomes (TGN/EE) for vacuolar targeting. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is an important control mechanism for brassinosteroid responses in plants. Altogether, our results identify ubiquitination and K63-linked polyubiquitin chain formation as a dual targeting signal for BRI1 internalization and sorting along the endocytic pathway, and highlight its role in hormonally controlled plant development

Alignment between PIN1 Polarity and Microtubule Orientation in the Shoot Apical Meristem Reveals a Tight Coupling between Morphogenesis and Auxin Transport

Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis

Spatiotemporal control of root immune responses during microbial colonization.

The entire evolutionary trajectory of plants towards large and complex multi-cellular organisms has been accompanied by incessant interactions with omnipresent unicellular microbes. This led to the evolution of highly complex microbial communities, whose members display the entire spectrum of pathogenic to mutualistic behaviors. Plant roots are dynamic, fractally growing organs and even small Arabidopsis roots harbor millions of individual microbes of diverse taxa. It is evident that microbes at different positions on a root surface could experience fundamentally different environments, which, moreover, rapidly change over time. Differences in spatial scales between microbes and roots compares to humans and the cities they inhabit. Such considerations make it evident that mechanisms of root-microbe interactions can only be understood if analyzed at relevant spatial and temporal scales. This review attempts to provide an overview of the rapid recent progress that has been made in mapping and manipulating plant damage and immune responses at cellular resolution, as well as in visualizing bacterial communities and their transcriptional activities. We further discuss the impact that such approaches will have for a more predictive understanding of root-microbe interactions

Root zone-specific localization of AMTs determines ammonium transport pathways and nitrogen allocation to shoots.

In plants, nutrient provision of shoots depends on the uptake and transport of nutrients across the root tissue to the vascular system. Nutrient delivery to the vasculature is mediated via the apoplastic transport pathway (ATP), which uses the free space in the cell walls and is controlled by apoplastic barriers and nutrient transporters at the endodermis, or via the symplastic transport pathway (STP). However, the relative importance of these transport routes remains elusive. Here, we show that the STP, mediated by the epidermal ammonium transporter 1;3 (AMT1;3), dominates the radial movement of ammonium across the root tissue when external ammonium is low, whereas apoplastic transport controlled by AMT1;2 at the endodermis prevails at high external ammonium. Then, AMT1;2 favors nitrogen (N) allocation to the shoot, revealing a major importance of the ATP for nutrient partitioning to shoots. When an endodermal bypass was introduced by abolishing Casparian strip (CS) formation, apoplastic ammonium transport decreased. By contrast, symplastic transport was increased, indicating synergism between the STP and the endodermal bypass. We further establish that the formation of apoplastic barriers alters the cell type-specific localization of AMTs and determines STP and ATP contributions. These results show how radial transport pathways vary along the longitudinal gradient of the root axis and contribute to nutrient partitioning between roots and shoots

Casparian strips prevent apoplastic diffusion of boric acid into root steles for excess B tolerance.

Casparian strips are ring-like structures consisting of lignin, sealing the apoplastic space between endodermal cells. They are thought to have important functions in controlling radial transport of nutrients and toxic elements in roots. However, Arabidopsis mutants with a defective Casparian strip structure have been found to maintain nutrient homeostasis in ranges supportive of growth under standard laboratory conditions. In this study, we investigated the function of Casparian strips under excess boron (B) conditions using sgn3 and sgn4 mutants with defective Casparian strip development but which do not exhibit excessive deposition of suberin, another endodermal diffusion barrier. The growth of sgn3 and sgn4 mutants did not differ significantly from that of wild-type (WT) plants under different B conditions in plate cultures; however, they were highly sensitive to B excess in hydroponic culture, where transpiration drives the translocation of boric acid toward the shoot. In hydroponic culture with sufficient to excess boric acid, B accumulation in shoots of the sgn3 and sgn4 mutants was higher than that in the WT. A time-course tracer study using <sup>10</sup> B-enriched boric acid at a sufficient or slightly excessive concentration showed higher translocation of B into shoots of the sgn3 and sgn4 mutants. Furthermore, a genetically encoded biosensor for boric acid expressed under a stele-specific promoter (proCIF2:NIP5;1 5'UTR : Eluc-PEST) visualized faster boric acid flux into the mutant steles. Collectively, our results demonstrate the importance of Casparian strips in preventing apoplastic diffusion of boric acid into the stele under excess supply

Efficient, cell-type-specific production of flavonols by multiplexed CRISPR activation of a suite of metabolic enzymes.

Synthetic biology in plants promises to transform basic and applied research by rewiring entire developmental modules, signaling cascades or metabolic pathways. Yet, this requires expression of many genes simultaneously, very difficult with classic transgenic approaches, especially for the generation of stable traits. CRISPR activation systems work in plants and could greatly facilitate multiplexed gene activation. Current CRISPR activation systems are efficient for transient or ubiquitous expression. Yet, to fulfill their potential, CRISPR activation needs to perform robustly in specific organs and tissue types. Here, we present a CRISPR activation system that efficiently drives expression in a cell-type-specific manner in stable lines, which requires assessing expression on a cellular basis using fluorescent reporter lines. Our CRISPR systems consistently re-wire gene expression at the cellular level, inducing genes with cell-type specific expression to efficiently express in a new cell layer, such as root endodermis or epidermis. We demonstrate the power of our system to drive functionally relevant, multiplexed gene activation by achieving endodermis-specific production of wild-type levels of flavonoids, detectable by in-situ fluorescence, in a root-flavonoid deficient myb12 mutant

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

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. &lt;i&gt;clv2&lt;/i&gt; as well as &lt;i&gt;crn&lt;/i&gt; null mutants are resistant to root-active CLE peptides, possibly because CLV2-CRN promotes expression of their cognate receptors. Here, we have identified the &lt;i&gt;CLE-RESISTANT RECEPTOR KINASE&lt;/i&gt; ( &lt;i&gt;CLERK&lt;/i&gt; ) 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, &lt;i&gt;CLERK&lt;/i&gt; also acts genetically independently of &lt;i&gt;CLV2-CRN&lt;/i&gt; We, thus, have discovered a novel hub for redundant CLE sensing in the root

The endodermis--development and differentiation of the plant's inner skin.

Controlling external compound entrance is essential for plant survival. To set up an efficient and selective sorting of nutrients, free diffusion via the apoplast in vascular plants is blocked at the level of the endodermis. Although we have learned a lot about endodermal specification in the last years, information regarding its differentiation is still very limited. A differentiated endodermal cell can be defined by the presence of the "Casparian strip" (CS), a cell wall modification described first by Robert Caspary in 1865. While the anatomical description of CS in many vascular plants has been very detailed, we still lack molecular information about the establishment of the Casparian strips and their actual function in roots. The recent isolation of a novel protein family, the CASPs, that localizes precisely to a domain of the plasma membrane underneath the CS represents an excellent point of entry to explore CS function and formation. In addition, it has been shown that the endodermis contains transporters that are localized to either the central (stele-facing) or peripheral (soil-facing) plasma membranes. These features suggest that the endodermis functions as a polar plant epithelium

Directed growth and fusion of membrane-wall microdomains requires CASP-mediated inhibition and displacement of secretory foci.

Casparian strips (CS) are aligned bands of lignin-impregnated cell walls, building an extracellular diffusion barrier in roots. Their structure profoundly differs from tight junctions (TJ), analogous structures in animals. Nonetheless, CS membrane domain (CSD) proteins 1-5 (CASP1-5) are homologues of occludins, TJ components. CASP-marked membranes display cell wall (matrix) adhesion and membrane protein exclusion. A full CASP knock-out now reveals CASPs are not needed for localized lignification, since correctly positioned lignin microdomains still form in the mutant. Ultra-structurally, however, these microdomains are disorganized, showing excessive cell wall growth, lack of exclusion zone and matrix adhesion, and impaired exocyst dynamics. Proximity-labelling identifies a Rab-GTPase subfamily, known exocyst activators, as potential CASP-interactors and demonstrate their localization and function at the CSD. We propose that CASP microdomains displace initial secretory foci by excluding vesicle tethering factors, thereby ensuring rapid fusion of microdomains into a membrane-cell wall band that seals the extracellular space