Suberin plasticity to developmental and exogenous cues is regulated by a set of MYB transcription factors

Suberin is a hydrophobic biopolymer that can be deposited at the periphery of cells, forming protective barriers against biotic and abiotic stress. In roots, suberin forms lamellae at the periphery of endodermal cells where it plays crucial roles in the control of water and mineral transport. Suberin formation is highly regulated by developmental and environmental cues. However, the mechanisms controlling its spatiotemporal regulation are poorly understood. Here, we show that endodermal suberin is regulated independently by developmental and exogenous signals to fine tune suberin deposition in roots. We found a set of four MYB transcription factors (MYB41, MYB53, MYB92 and MYB93), that are regulated by these two signals, and are sufficient to promote endodermal suberin. Mutation of these four transcription factors simultaneously through genome editing, lead to a dramatic reduction of suberin formation in response to both developmental and environmental signals. Most suberin mutants analyzed at physiological levels are also affected in another endodermal barrier made of lignin (Casparian strips), through a compensatory mechanism. Through the functional analysis of these four MYBs we generated plants allowing unbiased investigations of endodermal suberin function without accounting for confounding effects due to Casparian strip defects, and could unravel specific roles of suberin in nutrient homeostasis

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

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

Upon bolting the GTR1 and GTR2 transporters mediate transport of glucosinolates to the inflorescence rather than roots

We recently described the glucosinolate transporters GTR1 and GTR2 as actively contributing to the establishment of tissue-specific distribution of the defense compounds glucosinolates in vegetative Arabidopsis plants. Upon bolting and thereby development of the inflorescence and initiation of seed setting, the spatial distribution of glucosinolates does undergo major changes. Here we investigate the role of GTR1 and GTR2 in establishment of glucosinolate source-sink relationships in bolting plants. By in vivo feeding the exogenous p-hydroxybenzylglucosinolate to a rosette leaf or the roots of wildtype and a gtr1 gtr2 mutant, we show that this glucosinolate can specifically translocate from the rosette and the roots to the inflorescence in a GTR1- and GTR2-dependent manner. This marks that, upon bolting, the inflorescence rather than the roots constitute the strongest sink for leaf glucosinolates compared with plants in vegetative state

Grafting Arabidopsis

In Arabidopsis thaliana, hypocotyl micrografting has been used to investigate transport of flowering signals, mobile silencing signals and other peptides, proteins and secondary compounds. The effects of transported signals on target tissues require that a good vascular connection is re-established across the graft junction between the cut hypocotyls (stumps) of the root (rootstock) and shoot (scion) tissues. We outline here a method that requires only that the cut stumps be placed in close proximity, so that they touch, followed by 3-5 days of undisturbed recovery time during which the grafts are allowed to dry out somewhat. This method is quick, easy to monitor and has up to 90% success rate

Anther development in Arabidopsis thaliana involves symplastic isolation and apoplastic gating of the tapetum-middle layer interface.

International audienceDuring flowering plant reproduction, anthers produce pollen grains, the development of which is supported by the tapetum, a nourishing maternal tissue that also contributes non-cell autonomously to the pollen wall, the resistant external layer on the pollen surface. How the anther restricts movement of the tapetum derived pollen wall components, whilst allowing metabolites such as sugars and amino acids to reach the developing pollen, remains enigmatic. Here we experimentally show that in Arabidopsis thaliana, the tapetum and developing pollen are symplastically isolated from each other, and from other sporophytic tissues, from meiosis onwards. We show that the peritapetal strip (PTS), an apoplastic structure, separates the tapetum and the pollen grains from other anther cell layers and can prevent the apoplastic diffusion of fluorescent proteins, again from meiosis onwards. The formation and selective barrier functions of the PTS require two NADPH oxidases, RBOHE and RBOHC, which play a key role in pollen formation. Together our results suggest that, together with symplastic isolation, gating of the apoplast around the tapetum may help generate metabolically distinct anther compartments

Plant Sterol Metabolism. Δ⁷-Sterol-C₅-Desaturase (STE1/DWARF7), Δ^5,7-Sterol-Δ⁷-Reductase (DWARF5) and Δ²⁴-Sterol-Δ²⁴-Reductase (DIMINUTO/DWARF1) Show Multiple Subcellular Localizations in <em>Arabidopsis thaliana</em> (Heynh) L

<div><p>Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids. The plant sterols exist as three major forms, free sterols, steryl glycosides and steryl esters. The storage of steryl esters in lipid droplets has been shown to contribute to cellular sterol homeostasis. To further document cellular aspects of sterol biosynthesis in plants, we addressed the question of the subcellular localization of the enzymes implicated in the final steps of the post-squalene biosynthetic pathway. In order to create a clear localization map of steroidogenic enzymes in cells, the coding regions of Δ⁷-sterol-C₅-desaturase (STE1/DWARF7), Δ²⁴-sterol-Δ²⁴-reductase (DIMINUTO/DWARF1) and Δ^5,7-sterol-Δ⁷-reductase (DWARF5) were fused to the yellow fluorescent protein (YFP) and transformed into <em>Arabidopsis thaliana</em> mutant lines deficient in the corresponding enzymes. All fusion proteins were found to localize in the endoplasmic reticulum in functionally complemented plants. The results show that both Δ^5,7-sterol-Δ⁷-reductase and Δ²⁴-sterol-Δ²⁴-reductase are in addition localized to the plasma membrane, whereas Δ⁷-sterol-C₅-desaturase was clearly detected in lipid particles. These findings raise new challenging questions about the spatial and dynamic cellular organization of sterol biosynthesis in plants.</p> </div