Dynamic cytokinin signalling landscapes during lateral root formation in Arabidopsis

By forming lateral roots, plants expand their root systems to improve anchorage and absorb more water and nutrients from the soil. Each phase of this developmental process in Arabidopsis is tightly regulated by dynamic and continuous signalling of the phytohormones cytokinin and auxin. While the roles of auxin in lateral root organogenesis and spatial accommodation by overlying cell layers have been well studied, insights on the importance of cytokinin is still somewhat limited. Cytokinin is a negative regulator of lateral root formation with versatile modes of action being activated at different root developmental zones. Here, we review the latest progress made towards our understanding of these spatially separated mechanisms of cytokinin-mediated signalling that shape lateral root initiation, outgrowth and emergence and highlight some of the enticing open questions

Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells.

In vascular plants, the root endodermis surrounds the central vasculature as a protective sheath that is analogous to the polarized epithelium in animals, and contains ring-shaped Casparian strips that restrict diffusion. After an initial lag phase, individual endodermal cells suberize in an apparently random fashion to produce 'patchy' suberization that eventually generates a zone of continuous suberin deposition. Casparian strips and suberin lamellae affect paracellular and transcellular transport, respectively. Most angiosperms maintain some isolated cells in an unsuberized state as so-called 'passage cells', which have previously been suggested to enable uptake across an otherwise-impermeable endodermal barrier. Here we demonstrate that these passage cells are late emanations of a meristematic patterning process that reads out the underlying non-radial symmetry of the vasculature. This process is mediated by the non-cell-autonomous repression of cytokinin signalling in the root meristem, and leads to distinct phloem- and xylem-pole-associated endodermal cells. The latter cells can resist abscisic acid-dependent suberization to produce passage cells. Our data further demonstrate that, during meristematic patterning, xylem-pole-associated endodermal cells can dynamically alter passage-cell numbers in response to nutrient status, and that passage cells express transporters and locally affect the expression of transporters in adjacent cortical cells

Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples

BACKGROUND: Fluorescence Resonance Energy Transfer (FRET) between the green fluorescent protein (GFP) variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET. METHODOLOGY/PRINCIPAL FINDINGS: To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP. CONCLUSIONS/SIGNIFICANCE: The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells

Translating ribosome affinity purification (trap) to investigate Arabidopsis thaliana root development at a cell type-specific scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation. As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained. However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq. TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation

Lateral root initiation in Arabidopsis thaliana: a force awakens

Osmotically driven turgor pressure of plant cells can be higher than that of a car tire. It puts tremendous forces onto cell walls and drives cell growth and changes in cell shape. This has given rise to unique mechanisms to control organ formation compared to metazoans. The fascinating interplay between forces and local cellular reorganization is still poorly understood. Growth of lateral roots is a prominent example of a developmental process in which mechanical forces between neighboring cells are generated and must be dealt with. Lateral roots initiate from a single cell layer that resides deep within the primary root. On their way out, lateral roots grow through the overlying endodermal, cortical, and epidermal cell layers. It was recently demonstrated that endodermal cells actively accommodate lateral root formation. Interfering genetically with these accommodating responses in the endodermis completely blocks cell proliferation in the pericycle. The lateral root system provides a unique opportunity to elucidate the molecular and cellular mechanisms whereby mechanical forces and intercellular communication regulate spatial accommodation during plant development

Breakout-lateral root emergence in Arabidopsis thaliana

Lateral roots are determinants of plant root system architecture. Besides providing anchorage, they are a plant's means to explore the soil environment for water and nutrients. Lateral roots form post-embryonically and initiate deep within the root. On its way to the surface, the newly formed organ needs to grow through three overlying cell layers; the endodermis, cortex and epidermis. A picture is emerging that a tight integration of chemical and mechanical signalling between the lateral root and the surrounding tissue is essential for proper organogenesis. Here we review the latest progress made towards our understanding of the fascinating biology underlying lateral root emergence in Arabidopsis

Perturbing phosphoinositide homeostasis oppositely affects vascular differentiation in Arabidopsis thaliana roots

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

Perturbing phosphoinositide homeostasis oppositely affects vascular differentiation in Arabidopsis thaliana roots.

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

In Vivo Imaging of Diacylglycerol at the Cytoplasmic Leaflet of Plant Membranes

Diacylglycerol (DAG) is an important intermediate in lipid biosynthesis and plays key roles in cell signaling, either as a second messenger itself or as a precursor of phosphatidic acid. Methods to identify distinct DAG pools have proven difficult because biochemical fractionation affects the pools, and concentrations are limiting. Here, we validate the use of a genetically encoded DAG biosensor in living plant cells. The sensor is composed of a fusion between yellow fluorescent protein and the C1a domain of protein kinase C (YFP-C1aPKC) that specifically binds DAG, and was stably expressed in suspension-cultured tobacco BY-2 cells and whole Arabidopsis thaliana plants. Confocal imaging revealed that the majority of the YFP-C1aPKC fluorescence did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that the biosensor works and that the basal DAG concentration in the cytoplasmic leaflet of membranes (i.e. accessible to the biosensor) is in general too low, and confirms that the known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the trans-Golgi network; (ii) the cell plate during cytokinesis; (iii) the plasma membrane of root epidermal cells in the transition zone, and (iv) the apex of growing root hairs. The results provide new insights into the spatiotemporal dynamics of DAG in plants and offer a new tool to monitor this in vivo