Biology subjects

Ursache, R.

Publications and source records attributed to Ursache, R..

9 recordsLinked to original sources

bioRxiv2022

Regulation and Function of a Polarly Localized Lignin Barrier in the Exodermis

Multicellular organisms control interactions with their environment through the development of specialized barriers in specific cell types. A conserved barrier in plant roots is the endodermal Casparian strip (CS). The CS is made of polymerized lignin and forms a ring-like structure that seals the apoplastic space between the endodermal cells. Most angiosperms also have another root cell type, the exodermis, that is reported to form a barrier. Our understanding of exodermal developmental and molecular regulation, as well as function, is limited as this cell type is absent from the model species Arabidopsis thaliana. Using tomato (Solanum lycopersicum) as a model system we demonstrate that in this species, the exodermis does not form a CS. Instead, it forms a polar lignin cap with an equivalent barrier function to the endodermal CS. We demonstrate that although endodermal regulators are conserved between Arabidopsis and tomato, exodermal differentiation occurs by a distinct regulatory pathway involving the SlSCZ and SlEXO1 transcription factors. Although the exodermis and endodermis both produce barriers that restrict mineral ion uptake, they have unique and overlapping roles in their selectivity. Whether conservation and similarities between the endodermis and exodermis exist in other species remains to be determined. Nonetheless, in tomato, these distinct lignin structures have a convergent function with different genetic regulations.

plant biology↗

bioRxiv2022

A suberized exodermis is required for tomato drought tolerance

Plant roots integrate environmental signals and developmental programs using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates the entry and exit of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation, but we find that it is absent in the tomato endodermis during normal development. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here, we uncover genes driving exodermal suberization and describe its effects on drought responses in tomato, unravelling the similarities and differences with the paradigmatic Arabidopsis endodermis. Cellular resolution imaging, gene expression, and mutant analyses reveal loss of this program from the endodermis, and its co-option in the exodermis. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response. Controlling the degree of exodermal suberization could be a new strategy for breeding climate-resilient plants.

plant biology↗

bioRxiv2021

Microtubule-based perception of mechanical conflicts controls plant organ morphogenesis

Precise coordination between cells and tissues is essential for differential growth in plants. During lateral root formation in Arabidopsis thaliana, the endodermis is actively remodeled to allow outgrowth of the new organ. Here, we show that microtubule arrays facing lateral root founder cells display a higher order compared to arrays on the opposite wall of the same cell, and this asymmetry is required for endodermal remodeling and lateral root initiation. We identify that MICROTUBULE ASSOCIATED PROTEIN 70-5 is necessary for the establishment of this spatially defined microtubule organization and endodermis remodeling, and thus contributes to lateral root morphogenesis. We propose that MAP70-5 and cortical microtubule arrays in the endodermis integrate the mechanical signals generated by lateral root outgrowth, facilitating the channeling of organogenesis.

plant biology↗

bioRxiv2021

Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants

Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis mutants. To facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating large deletions at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either FastRed, FastGreen or FastCyan fluorescent seed selection cassette. Both, FastRed and FastGreen binary vectors, can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors at the same time. Our approach provides fast and flexible cloning, avoids very big constructs and enables screening different order mutants in the same generation. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the dark, non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) or ubiquitous promoter (PcUBi4-2). Available enzymes are "classical" Cas9, a recently reported, intron-optimized version or Cpf1 (Cas12a). Finally, we have taken care to introduce convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in the final T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system.

plant biology↗

bioRxiv2021

Analysis of phloem trajectory links tissue maturation to cell specialization.

The mechanisms that allow cells in the plant meristem to coordinate tissue-wide maturation gradients with specialized cell networks are critical for indeterminate growth. Here, we reconstructed the protophloem developmental trajectory of 19 cells from cell birth to terminal differentiation at single cell resolution in the Arabidopsis root. We found that cellular specification is mediated near the stem cell niche by PHLOEM EARLY DOF (PEAR) transcription factors. However, the PEAR dependent differentiation program is repressed by a broad gradient of PLETHORA (PLT) transcription factors, which directly inhibit PEARs own direct target ALTERED PHLOEM DEVELOPMENT (APL). The dissipation of PLT gradient around 7 cells from the stem cell activates APL expression, and a subsequent transitional network that results in a "seesaw" pattern of mutual inhibition over developmental time. Together, we provide a mechanistic understanding of how morphogen-like maturation gradients interface with cell-type specific transcriptional regulators to stage cellular differentiation.

plant biology↗

bioRxiv2020

GDSL-domain containing proteins mediate suberin biosynthesis and degradation, enabling developmental plasticity of the endodermis during lateral root emergence

ABSTRACTRoots anchor plants and deliver water and nutrients from the soil. The root endodermis provides the crucial extracellular diffusion barrier by setting up a supracellular network of lignified cell walls, called Casparian strips, supported by a subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, formation of suberin lamellae was demonstrated to be dynamic, facilitating adaptation to different soil conditions. Plants shape their root system through the regulated formation of lateral roots emerging from within the endodermis, requiring local breaking and re-sealing of the endodermal diffusion barriers. Here, we show that differentiated endodermal cells have a distinct auxin-mediated transcriptional response that regulates cell wall remodelling. Based on this data set we identify a set of GDSL-lipases that are essential for suberin formation. Moreover, we find that another set of GDSL-lipases mediates suberin degradation, which enables the developmental plasticity of the endodermis required for normal lateral root emergence.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2020

High-order mutants reveal an essential requirement for peroxidases but not laccases in Casparian strip lignification

The invention of lignin has been at the heart of plants capacity to colonize land, allowing them to grow tall, transport water within their bodies and protect themselves against various stresses. Consequently, this polyphenolic polymer, that impregnates the cellulosic plant cell walls, now represents the second most abundant polymer on Earth, after cellulose itself. Yet, despite its great physiological, ecological and economical importance, our knowledge of lignin biosynthesis in vivo, especially the crucial last steps of polymerization within the cell wall, remains vague. Specifically, the respective roles and importance of the two main polymerizing enzymes classes, laccases and peroxidases have remained obscure. One reason for this lies in the very high numbers of laccases and peroxidases encoded by 17 and 73 homologous genes, respectively, in the Arabidopsis genome. Here, we have focused on a specific lignin structure, the ring-like Casparian strips (CS) within the endodermis of Arabidopsis roots. By reducing the number of possible candidate genes using cellular resolution expression and localization data and by boosting the levels of mutants that can be stacked using CRISPR/Cas9, we were able to knock-out more than half of all laccases in the Arabidopsis genome in a nonuple mutant - abolishing the vast majority of laccases with detectable endodermal-expression. Yet, we were unable to detect even slight defects in CS formation. By contrast, we were able to induce a complete absence of CS formation in a quintuple peroxidase mutant. Our findings are in stark contrast to the strong requirement of xylem vessels for laccase action and indicate that lignin in different cell types can be polymerized in very distinct ways. We speculate that cells lignify differently depending on whether they deposit lignin in a localized or ubiquitous fashion, whether they stay alive during and after lignification as well as the composition of the cell wall.

plant biology↗

bioRxiv2019

ROS production by localized SCHENGEN receptor module drives lignification at subcellular precision

Production of reactive-oxygen species (ROS) by NADPH oxidases (NOXs) impacts many processes in animals and plants and many plant receptor pathways involve rapid, NOX-dependent increases of ROS. Yet, their general reactivity has made it challenging to pinpoint the precise role and direct cellular targets of ROS. A well-understood ROS target in plants are lignin peroxidases in the cell wall. Lignin can be deposited with exquisite spatial control, but the underlying mechanisms have remained elusive. Here we establish a full kinase signaling relay that exerts direct, spatial control over ROS production and lignification within the cell wall. We show that polar localization of a single kinase component is crucial for pathway function. Our data indicates that an intersection of more broadly localized components allows for micrometer-scale precision of lignification and that this system is triggered through initiation of ROS production as a critical peroxidase co-substrate.

plant biology↗

bioRxiv2019

An inducible genome editing system for plants

Conditional manipulation of gene expression is a key approach to investigating the primary function of a gene in a biological process. While conditional and cell-type specific overexpression systems exist for plants, there are currently no systems available to disable a gene completely and conditionally. Here, we present a novel tool with which target genes can be efficiently conditionally knocked out at any developmental stage. The target gene is manipulated using the CRISPR-Cas9 genome editing technology, and conditionality is achieved with the well-established estrogen-inducible XVE system. Target genes can also be knocked-out in a cell-type specific manner. Our tool is easy to construct and will be particularly useful for studying genes which have null-alleles that are non-viable or show strong developmental defects.

plant biology↗