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Gene targeting can be used to make modifications at a specific region in a plant’s genome and create high-precision tools for plant biotechnology and breeding. However, its low efficiency is a major barrier to its use in plants. The discovery of CRISPR (clustered regularly interspaced short palindromic repeats)–Cas-based site-specific nucleases capable of inducing double-strand breaks in desired loci resulted in the development of novel approaches for plant gene targeting. Several studies have recently demonstrated improvements in gene targeting efficiency through cell-type-specific expression of Cas nucleases, the use of self-amplified gene-targeting-vector DNA, or manipulation of RNA silencing and DNA repair pathways. In this review, we summarize recent advances in CRISPR/Cas-mediated gene targeting in plants and discuss potential efficiency improvements. Increasing the efficiency of gene targeting technology will help pave the way for increased crop yields and food safety in environmentally friendly agriculture.

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 speciesArabidopsis 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 theSlSCZandSlEXO1transcription 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 roots acquire nutrients and water while managing interactions with the soil microbiota. The root endodermis provides an extracellular diffusion barrier through a network of lignified cell walls called Casparian strips, supported by subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, suberin lamellae display plasticity, which is crucial for root adaptative responses. Although suberin is a major plant polymer, fundamental aspects of its biosynthesis and turnover have remained obscure. Plants shape their root system via lateral root formation, an auxin-induced process requiring local breaking and re-sealing of endodermal lignin and suberin barriers. Here, we show that differentiated endodermal cells have a specific, auxin-mediated transcriptional response dominated by cell wall remodelling genes. We identified two sets of auxin-regulated GDSL lipases. One is required for suberin synthesis, while the other can drive suberin degradation. These enzymes have key roles in suberization, driving root suberin plasticity.

SUMMARYPlant 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 organismArabidopsis 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.

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 side of the same cell, and this asymmetry is required for endodermal remodeling and lateral root initiation. We identify that MICROTUBULE ASSOCIATED PROTEIN 70-5 (MAP70-5) 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.

Publisher Copyright: Copyright © 2021 The Authors, some rights reserved; In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

Stress granules (SGs) are ribonucleoprotein functional condensates that form under stress conditions in all eukaryotic cells. Although their stress-survival function is far from clear, SGs have been implicated in the regulation of many vital cellular pathways. Consequently, SG dysfunction is thought to be a mechanistic point of origin for many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Additionally, SGs are thought to play a role in pathogenic pathways as diverse as viral infection and chemotherapy resistance. There is a growing consensus on the hypothesis that understanding the mechanistic regulation of SG physical properties is essential to understanding their function. Although the internal dynamics and condensation mechanisms of SGs have been broadly investigated, there have been fewer investigations into the timing of SG formation and clearance in live cells. Because the lifetime of SG persistence can be a key factor in their function and tendency toward pathological dysregulation, SG clearance mechanisms deserve particular attention. Here we show that resveratrol and its analogues piceatannol, pterostilbene, and 3,4,5,4′-tetramethoxystilbene induce G3BP-dependent SG formation with atypically rapid clearance kinetics. Resveratrol binds to G3BP, thereby reducing its protein–protein association valency. We suggest that altering G3BP valency is a pathway for the formation of uniquely transient SGs.

BackgroundMultiplex 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 orderArabidopsis thalianamutants. Low entry, reliable assembly of CRISPR/Cas9 vectors and efficient mutagenesis is necessary to enable a maximum of researchers to break through the genetic redundancy within plant multi-gene families and allow for a plethora of gene function studies that have been previously unachievable. It will also allow routine de novo generation of mutations in ever more complex genetic backgrounds that make introgression of pre-existing alleles highly cumbersome.ResultsTo facilitate rapid and efficient use of CRISPR/Cas9 forArabidopsisresearch, we developed a CRISPR/Cas9-based toolbox for generating mutations 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 a FastRed, FastGreen or FastCyan fluorescent seed selection cassette. 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. Importantly, in the second generation after transformation,Cas9free plants are identified simply by screening the 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) fromA. thalianaor the constitutive promoter fromPetroselinum crispum(PcUBi4-2). Available enzymes are “classical”Cas9codon-optimized forA. thalianaand a recently reported, intron-containing version ofCas9codon-optimized forZea mays,zCas9i. We observed the highest efficiency in producing knockout phenotypes by using intron-containingzCas9idriven under egg-cell specificpEC1.2promoter. Finally, we introduced convenient restriction sites flanking promoter,Cas9and fluorescent selection cassette in some of the T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system.ConclusionA rapid, simple and flexible CISPR/Cas9cloning system was established that allows assembly of multi-guide RNA constructs in a robust and reproducible fashion, by avoiding generation of very big constructs. The system enables a flexible, fast and efficient screening of single or higher orderA. thalianamutants.

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 targetALTERED 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.

Significance Lignin is a defining polymer of vascular plants and of great physiological, ecological, and economical importance. Yet, its polymerization in the cell wall is still not understood. Lignin polymerizing enzymes, laccases and peroxidases, exist in vast numbers in plant genomes. By focusing on a specific lignin structure, the ring-like Casparian strips (CSs), we reduced candidate numbers and abolished essentially all laccases with detectable endodermal expression. Yet, not even slight defects in CS formation were detected. By contrast, a quintuple peroxidase mutant displayed a complete absence of CS. Our findings suggest that cells lignify differently depending on whether lignin is localized or ubiquitous and whether cells stay alive during and after lignification, as well as the composition of the cell wall., Lignin has enabled plants to colonize land, grow tall, transport water within their bodies, and protect themselves against various stresses. Consequently, this polyphenolic polymer, impregnating cellulosic plant cell walls, is the second most abundant polymer on Earth. Yet, despite its great physiological, ecological, and economical importance, our knowledge of lignin biosynthesis in vivo, especially the polymerization steps within the cell wall, remains vague—specifically, the respective roles of the two polymerizing enzymes classes, laccases and peroxidases. One reason for this lies in the very high numbers of laccases and peroxidases encoded by 17 and 73 homologous genes, respectively, in Arabidopsis. Here, we have focused on a specific lignin structure, the ring-like Casparian strips (CSs) within the root endodermis. By reducing candidate numbers using cellular resolution expression and localization data and by boosting stacking of mutants using CRISPR-Cas9, we mutated the majority of laccases in Arabidopsis in a nonuple mutant—essentially abolishing 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 lignin is localized or ubiquitous and whether cells stay alive during and after lignification, as well as the composition of the cell wall.

Plant roots acquire nutrients and water while managing interactions with the soil microbiota. The root endodermis provides an extracellular diffusion barrier through a network of lignified cell walls called Casparian strips, supported by subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, suberin lamellae display plasticity, which is crucial for root adaptative responses. Although suberin is a major plant polymer, fundamental aspects of its biosynthesis and turnover have remained obscure. Plants shape their root system via lateral root formation, an auxin-induced process requiring local breaking and re-sealing of endodermal lignin and suberin barriers. Here, we show that differentiated endodermal cells have a specific, auxin-mediated transcriptional response dominated by cell wall remodelling genes. We identified two sets of auxin-regulated GDSL lipases. One is required for suberin synthesis, while the other can drive suberin degradation. These enzymes have key roles in suberization, driving root suberin plasticity.

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 new tool with which target genes can efficiently and conditionally be knocked out by genome editing at any developmental stage. 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 having null alleles that are non-viable or show pleiotropic developmental defects.

Roots 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.

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.

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 immediate molecular action of ROS. A well‐understood ROS action in plants is to provide the co‐substrate for 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 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 indicate 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., Polarized activation of SGN1 kinase regulates Casparian strip formation in the root via direct phosphorylation of NADPH oxidases.

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.

In this Letter, owing to a copying error in Illustrator, the two centre panels in Extended Data Fig. 7a were identical. This error has been corrected online. The old, incorrect Extended Data Fig. 7 is shown in the Supplementary Information to this Amendment for transparency. Some typos ('occurence') in Figs. 1, 2 and 3 have also been corrected and the publication details for ref. 32 have been added.

The development and growth of plants, as well as their successful adaptation to a variety of environments, is highly dependent on the conduction of water, nutrients and other important molecules throughout the plant body. Xylem is a specialized vascular tissue that serves as a conduit of water and minerals and provides mechanical support for upright growth. Wood, also known as secondary xylem, constitutes the major part of mature woody stems and roots. In the past two decades, a number of key factors including hormones, signal transducers and (post)transcriptional regulators have been shown to control xylem formation. We outline the main mechanisms shown to be essential for xylem development in various plant species, with an emphasis on Arabidopsis thaliana, as well as several tree species where xylem has a long history of investigation. We also summarize the processes which have been shown to be instrumental during xylem maturation. This includes mechanisms of cell wall formation and cell death which collectively complete xylem cell fate.

Higher plant function is contingent upon the complex three‐dimensional (3D) architecture of plant tissues, yet severe light scattering renders deep, 3D tissue imaging very problematic. Although efforts to ‘clear’ tissues have been ongoing for over a century, many innovations have been made in recent years. Among them, a protocol called ClearSee efficiently clears tissues and diminishes chlorophyll autofluorescence while maintaining fluorescent proteins – thereby allowing analysis of gene expression and protein localisation in cleared samples. To further increase the usefulness of this protocol, we have developed a ClearSee‐based toolbox in which a number of classical histological stains for lignin, suberin and other cell wall components can be used in conjunction with fluorescent reporter lines. We found that a number of classical dyes are highly soluble in ClearSee solution, allowing the old staining protocols to be enormously simplified; these additionally have been unsuitable for co‐visualisation with fluorescent markers due to harsh fixation and clearing. Consecutive staining with several dyes allows 3D co‐visualisation of distinct cell wall modifications with fluorescent proteins – used as transcriptional reporters or protein localisation tools – deep within tissues. Moreover, the protocol is easily applied on hand sections of different organs. In combination with confocal microscopy, this improves image quality while decreasing the time and cost of embedding/sectioning. It thus provides a low‐cost, efficient method for studying thick plant tissues which are usually cumbersome to visualise. Our ClearSee‐adapted protocols significantly improve and speed up anatomical and developmental investigations in numerous plant species, and we hope they will contribute to new discoveries in many areas of plant research.

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.

International audience; In a striking case of evolutionary convergence, polarized cell layers with ring-like diffusion barriers have evolved in both plant and animal lineages independently. In plants, ring-like Casparian strips become localized by the CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs). The mechanism of this striking localization, however, has remained enigmatic. Here we present a genetic screen aimed at isolating determinants of CASP localization. One of the mutants, lord of the rings 2 (lotr2)/exo70a1, displays dramatic de-localization of CASPs into randomly localized microdomains. EXO70A1 is a subunit of the exocyst complex, a central component of secretion in eukaryotes. Irradiation of EXO70 subunit genes in plants has suggested specialization of this conserved complex. Intriguingly, lotr2/exo70a1 does neither affect secretion of the CASPs, nor that of other membrane proteins in the endodermis, thus separating exocyst activity in localization from a general defect in secretion. Our results establish EXO70A1 as a central player in Casparian strip formation, generating a transient positional information that will be translated into a precisely localized cell wall modification.

About 200 researchers from around the world attended the Third International Conference on Plant Vascular Biology (PVB 2013) held in July 2013 at the Rantapuisto Conference Center, in Helsinki, Finland (http://www.pvb2013.org). The plant vascular system, which connects every organ in the mature plant, continues to attract the interest of researchers representing a wide range of disciplines, including development, physiology, systems biology, and computational biology. At the meeting, participants discussed the latest research advances in vascular development, long- and short-distance vascular transport and long-distance signalling in plant defence, in addition to providing a context for how these studies intersect with each other. The meeting provided an opportunity for researchers working across a broad range of fields to share ideas and to discuss future directions in the expanding field of vascular biology. In this report, the latest advances in understanding the mechanism of vascular trafficking presented at the meeting have been summarized.

A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies.

The stems and roots of most dicot plants increase in diameter by radial growth, due to the activity of secondary meristems. Two types of meristems function in secondary plant body formation: the vascular cambium, which gives rise to secondary xylem and phloem, and the cork cambium, which produces a bark layer that replaces the epidermis and protects the plant stem from mechanical damage and pathogens. Cambial development, the initiation and activity of the vascular cambium, leads to an accumulation of wood, the secondary xylem tissue. The thick, cellulose-rich cell walls of wood provide a source of cellulose and have the potential to be used as a raw material for sustainable and renewable energy production. In this review, we will discuss what is known about the mechanisms regulating the cambium and secondary tissue development.

SummaryThe plant hormone cytokinin controls root growth by balancing the division and differentiation of stem cells. But what controls accumulation of cytokinin? A new study has identified a regulatory loop between a transcription factor, PHABULOSA, and cytokinin biosynthesis that creates robust domains of cytokinin activity.

Phloem, a plant tissue responsible for long-distance molecular transport, harbours specific junctions, sieve areas, between the conducting cells. To date, little is known about the molecular framework related to the biogenesis of these sieve areas. Here we identify mutations at the CHER1/AtCTL1 locus of Arabidopsis thaliana. The mutations cause several phenotypic abnormalities, including reduced pore density and altered pore structure in the sieve areas associated with impaired phloem function. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We show that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and during cytokinesis is associated with the phragmoplast. Consistent with its function in the elaboration of the sieve areas, CHER1 has a sustained, polar localization in the forming sieve plates. Our results indicate that the regulation of choline levels is crucial for phloem development and conductivity in plants. © 2014 Macmillan Publishers Limited. All rights reserved.

The development and growth of higher plants is highly dependent on the conduction of water and minerals throughout the plant by xylem vessels. In Arabidopsis roots the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. During vascular development, high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. Protoxylem specification is determined by both mobile, ground tissue-emanating miRNA165/6 species, which downregulate, and auxin concentrated by polar transport, which promotes HD-ZIP III expression. However, the factors promoting high HD-ZIP III expression for metaxylem identity have remained elusive. We show here that auxin biosynthesis promotes HD-ZIP III expression and metaxylem specification. Several auxin biosynthesis genes are expressed in the outer layers surrounding the vascular tissue in Arabidopsis root and downregulation of HD-ZIP III expression accompanied by specific defects in metaxylem development is seen in auxin biosynthesis mutants, such as trp2-12, wei8 tar2 or a quintuple yucca mutant, and in plants treated with L-kynurenine, a pharmacological inhibitor of auxin biosynthesis. Some of the patterning defects can be suppressed by synthetically elevated HD-ZIP III expression. Taken together, our results indicate that polar auxin transport, which was earlier shown to be required for protoxylem formation, is not sufficient to establish a proper xylem axis but that root-based auxin biosynthesis is additionally required.

The soil surface is a major natural system that accumulates pollutants and allows researchers to disclose the history and the present state of contamination of an area with toxic pollutants. This conclusion is based on the comparison of genotoxicity of aqueous extracts and DMSO extracts of topsoil from military territories in various locations left behind after the retreat of the Soviet Army from Lithuania, and several sites in the city of Vilnius, characterized by different history and current traffic intensity. The specific character of the soil-surface contamination was shown in a series of Tradescantia micronucleus (Trad-MN) and stamen-hair mutation (Trad-SHM) bioassays and tests. The most effective ones were the Trad-MN and, unexpectedly, the branched-hair tests. A preliminary result of the study is the somaclonal variation of individual Tradescantia plants revealed by the RAPD method of DNA analysis. A comparison of aqueous extracts and DMSO extracts of the soil showed the permanent character of the mobile forms of genotoxic pollutants in the soil surface, despite the fact that several military territories were already closed 20 years ago.