An atlas of Brachypodium distachyon lateral root development.

The root system of plants is a vital part for successful development and adaptation to different soil types and environments. A major determinant of the shape of a plant root system is the formation of lateral roots, allowing for expansion of the root system. Arabidopsis thaliana, with its simple root anatomy, has been extensively studied to reveal the genetic program underlying root branching. However, to get a more general understanding of lateral root development, comparative studies in species with a more complex root anatomy are required. Here, by combining optimized clearing methods and histology, we describe an atlas of lateral root development in Brachypodium distachyon, a wild, temperate grass species. We show that lateral roots initiate from enlarged phloem pole pericycle cells and that the overlying endodermis reactivates its cell cycle and eventually forms the root cap. In addition, auxin signaling reported by the DR5 reporter was not detected in the phloem pole pericycle cells or young primordia. In contrast, auxin signaling was activated in the overlying cortical cell layers, including the exodermis. Thus, Brachypodium is a valuable model to investigate how signaling pathways and cellular responses have been repurposed to facilitate lateral root organogenesis.

Lignin accumulation in cell wall plays a role in clubroot resistance.

Clubroot, caused by Plasmodiophora brassicae, is a significant disease affecting brassica crops worldwide and poses a threat to canola (Brassica napus) production in western Canada. Management of this disease heavily relies on the use of resistant cultivars, but resistance erosion is a serious concern due to the highly diverse pathogen populations. Understanding resistance mechanisms may aid in better deployment/rotation of clubroot resistance (CR) genes and improve resistance resilience. In this study, we conducted a comparative analysis using resistant canola varieties carrying either a single (Rcr1) or double CR genes (Rcr1+Crr1rutb ) to decipher the resistance modes associated with these genes. Cell wall (CW) biopolymeric compounds in different root layers were mapped and quantified using Fourier-transform mid-infrared microspectroscopy for changes in CW elements associated with clubroot resistance. Transmission electron and confocal microscopy were used to assess root infection details and relative transcript abundance was analyzed to determine the activation of the lignin-related pathway in relation to resistance. Neither resistant variety affected the primary infection of root hairs/epidermal cells compared to the susceptible "Westar", but both exhibited strong inhibition of cortical infection, effectively 'trapping' the pathogen in the exodermis. The most prominent change observed was increased lignin accumulation associated with resistance. In Westar, the pathogen was able to degrade CW lignin, facilitating access to the root cortex by secondary plasmodia of P. brassicae. In contrast, resistant varieties showed clear lignin accumulation around the penetration site on the exodermis, accompanied by elevated expression of genes involved in the phenylpropanoid pathway. These results suggest that induced lignin accumulation plays a role in clubroot resistance mediated by the CR genes Rcr1 and Crr1rutb in canola, providing cellular and structural evidence that supports the data from earlier transcriptomic studies.

The exodermis: A forgotten but promising apoplastic barrier.

The endodermis and exodermis are widely recognized as two important barriers in plant roots that play a role in regulating the movement of water and ions. While the endodermis is present in nearly all plant roots, the exodermis, characterized by Casparian strips and suberin lamellae is absent in certain plant species. The exodermis can be classified into three types: uniform, dimorphic, and inducible exodermis. Apart from its role in water and ion transport, the exodermis acts as a protective barrier against harmful substances present in the external environment. Furthermore, the exodermis is a complex barrier influenced by various environmental factors, and its resistance to water and ions varies depending on the type of exodermis and the maturity of the root. Therefore, investigations concerning the exodermis necessitate a plant-specific approach. However, our current understanding of the exodermal physiological functions and molecular mechanisms governing its development is limited due to the absence of an exodermis in the model plant Arabidopsis. Due to that, unfortunately, the exodermis has been largely overlooked until now. In this review, we aim to summarize the current fundamental knowledge regarding the exodermis in common research used crop species and propose suggestions for future research endeavors.

The barrier to radial oxygen loss protects roots against hydrogen sulphide intrusion and its toxic effect.

The root barrier to radial O2 loss (ROL) is a key root trait preventing O2 loss from roots to anoxic soils, thereby enabling root growth into anoxic, flooded soils. We hypothesized that the ROL barrier can also prevent intrusion of hydrogen sulphide (H2 S), a potent phytotoxin in flooded soils. Using H2 S- and O2 -sensitive microsensors, we measured the apparent permeance to H2 S of rice roots, tested whether restricted H2 S intrusion reduced its adverse effects on root respiration, and whether H2 S could induce the formation of a ROL barrier. The ROL barrier reduced apparent permeance to H2 S by almost 99%, greatly restricting H2 S intrusion. The ROL barrier acted as a shield towards H2 S; O2 consumption in roots with a ROL barrier remained unaffected at high H2 S concentration (500 µM), compared to a 67% decline in roots without a barrier. Importantly, low H2 S concentrations induced the formation of a ROL barrier. In conclusion, the ROL barrier plays a key role in protecting against H2 S intrusion, and H2 S can act as an environmental signalling molecule for the induction of the barrier. This study demonstrates the multiple functions of the suberized/lignified outer part of the rice root beyond that of restricting ROL.

Cell-Type-Dependent but CME-Independent Polar Localization of Silicon Transporters in Rice.

Silicon (Si) is an important nutrient required for sustainable and high production of rice and its uptake is mediated by a pair of influx (OsLsi1)-efflux (OsLsi2) transporters showing polar localization. However, the mechanisms underlying their polarity are unknown. Here, we revealed that the polarity of the Si transporters depends on cell types. The polar localization of both OsLsi1 and OsLsi2 was not altered by Si supply, but their protein abundance was reduced. Double immunostaining showed that localization of OsLsi1 and OsLsi2 was separated at the edge of the lateral polar domain by Casparian strips in the endodermis, whereas they were slightly overlapped at the transversal side of the exodermis. When OsLsi1 was ectopically expressed in the shoots, it showed polar localization at the xylem parenchyma cells of the basal node and leaf sheath, but not at the phloem companion cells. Ectopic expression of non-polar Si transporters, barley HvLsi2 and maize ZmLsi2 in rice, resulted in their polar localization at the proximal side. The polar localization of OsLsi1 and OsLsi2 was not altered by inhibition of clathrin-mediated endocytosis (CME) by dominant-negative induction of dynamin-related protein1A and knockout of mu subunit of adaptor protein 2 complex, although the knockout mutants of OsAP2M gene showed dwarf phenotype. These results indicate that CME is not required for the polar localization of Si transporters. Taken together, our results indicate that CME-independent machinery controls the polar localization of Si transporters in exodermis, endodermis of root cells and xylem parenchyma cells.

Rice OsCASP1 orchestrates Casparian strip formation and suberin deposition in small lateral roots to maintain nutrient homeostasis.

Arabidopsis Casparian strip membrane domain proteins (CASPs) form a transmembrane scaffold to recruit lignin biosynthetic enzymes for Casparian strip (CS) formation. Rice is a semi-aquatic plant with a more complex root structure than Arabidopsis to adapt its growing conditions, where the different deposition of lignin and suberin is crucial for adaptive responses. Here, we observed the structure of rice primary and small lateral roots (SLRs), particularly the deposition patterns of lignin and suberin in wild type and Oscasp1 mutants. We found that the appearance time and structure of CS in the roots of rice are different from those of Arabidopsis and observed suberin deposition in the sclerenchyma in wild type roots. Rice CASP1 is highly similar to AtCASPs, but its expression is concentrated in SLR tips and can be induced by salt stress especially in the steles. The loss of OsCASP1 function alters the expression of the genes involved in suberin biosynthesis and the deposition of suberin in the endodermis and sclerenchyma and leads to delayed CS formation and uneven lignin deposition in SLRs. These different depositions may alter nutrient uptake, resulting in ion imbalance in plant, withered leaves, fewer tillers, and reduced tolerance to salt stress. Our findings suggest that OsCASP1 could play an important role in nutrient homeostasis and adaptation to the growth environment.

Abscisic acid is required for exodermal suberization to form a barrier to radial oxygen loss in the adventitious roots of rice (Oryza sativa).

To acclimate to waterlogged conditions, wetland plants form a barrier to radial oxygen loss (ROL) that can enhance oxygen transport to the root apex. We hypothesized that one or more hormones are involved in the induction of the barrier and searched for such hormones in rice. We previously identified 98 genes that were tissue-specifically upregulated during ROL barrier formation in rice. The RiceXPro database showed that most of these genes were highly enhanced by exogenous abscisic acid (ABA). We then examined the effect of ABA on ROL barrier formation by using an ABA biosynthesis inhibitor (fluridone, FLU), by applying exogenous ABA and by examining a mutant with a defective ABA biosynthesis gene (osaba1). FLU suppressed barrier formation in a stagnant solution that mimics waterlogged soil. Under aerobic conditions, rice does not naturally form a barrier, but 24 h of ABA treatment induced barrier formation. osaba1 did not form a barrier under stagnant conditions, but the application of ABA rescued the barrier. In parallel with ROL barrier formation, suberin lamellae formed in the exodermis. These findings strongly suggest that ABA is an inducer of suberin lamellae formation in the exodermis, resulting in an ROL barrier formation in rice.

Convergent evolution of gene regulatory networks underlying plant adaptations to dry environments.

Plants transitioned from an aquatic to a terrestrial lifestyle during their evolution. On land, fluctuations on water availability in the environment became one of the major problems they encountered. The appearance of morpho-physiological adaptations to cope with and tolerate water loss from the cells was undeniably useful to survive on dry land. Some of these adaptations, such as carbon concentrating mechanisms (CCMs), desiccation tolerance (DT) and root impermeabilization, appeared in multiple plant lineages. Despite being crucial for evolution on land, it has been unclear how these adaptations convergently evolved in the various plant lineages. Recent advances on whole genome and transcriptome sequencing are revealing that co-option of genes and gene regulatory networks (GRNs) is a common feature underlying the convergent evolution of these adaptations. In this review, we address how the study of CCMs and DT has provided insight into convergent evolution of GRNs underlying plant adaptation to dry environments, and how these insights could be applied to currently emerging understanding of evolution of root impermeabilization through different barrier cell types. We discuss examples of co-option, conservation and innovation of genes and GRNs at the cell, tissue and organ levels revealed by recent phylogenomic (comparative genomic) and comparative transcriptomic studies.

Innovation, conservation, and repurposing of gene function in root cell type development.

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

Root vacuolar sequestration and suberization are prominent responses of Pistacia spp. rootstocks during salinity stress.

Understanding the mechanisms of stress tolerance in diverse species is needed to enhance crop performance under conditions such as high salinity. Plant roots, in particular in grafted agricultural crops, can function as a boundary against external stresses in order to maintain plant fitness. However, limited information exists for salinity stress responses of woody species and their rootstocks. Pistachio (Pistacia spp.) is a tree nut crop with relatively high salinity tolerance as well as high genetic heterogeneity. In this study, we used a microscopy-based approach to investigate the cellular and structural responses to salinity stress in the roots of two pistachio rootstocks, Pistacia integerrima (PGI) and a hybrid, P. atlantica x P. integerrima (UCB1). We analyzed root sections via fluorescence microscopy across a developmental gradient, defined by xylem development, for sodium localization and for cellular barrier differentiation via suberin deposition. Our cumulative data suggest that the salinity response in pistachio rootstock species is associated with both vacuolar sodium ion (Na+) sequestration in the root cortex and increased suberin deposition at apoplastic barriers. Furthermore, both vacuolar sequestration and suberin deposition correlate with the root developmental gradient. We observed a higher rate of Na+ vacuolar sequestration and reduced salt-induced leaf damage in UCB1 when compared to P. integerrima. In addition, UCB1 displayed higher basal levels of suberization, in both the exodermis and endodermis, compared to P. integerrima. This difference was enhanced after salinity stress. These cellular characteristics are phenotypes that can be taken into account during screening for sodium-mediated salinity tolerance in woody plant species.

Role of roots in adaptation of soil-indifferent Proteaceae to calcareous soils in south-western Australia.

Very few of the >650 Proteaceae species in south-western Australia cope with the high calcium (Ca) levels in young, calcareous soils (soil indifferent); most are Ca sensitive and occur on nutrient-impoverished, acidic soils (calcifuge). We assessed possible control points for Ca transport across roots of two soil-indifferent (Hakea prostrata and Banksia prionotes) and two calcifuge (H. incrassata and B. menziesii) Proteaceae. Using quantitative X-ray microanalysis, we investigated cell-specific elemental Ca concentrations at two positions behind the apex in relation to development of apoplastic barriers in roots of plants grown in nutrient solution with low or high Ca supply. In H. prostrata, Ca accumulated in outer cortical cells at 20 mm behind the apex, but [Ca] was low in other cell types. In H. incrassata, [Ca] was low in all cells. Accumulation of Ca in roots of H. prostrata corresponded to development of apoplastic barriers in the endodermis. We found similar [Ca] profiles in roots and similar [Ca] in leaves of two contrasting Banksia species. Soil-indifferent Hakea and Banksia species show different strategies to inhabit calcareous soils: H. prostrata intercepts Ca in roots, reducing transport to shoots, whereas B. prionotes allocates Ca to specific leaf cells.

Salt tolerance and exclusion in the mangrove plant Avicennia marina in relation to root apoplastic barriers.

Salt tolerance and the possible functions of suberization on salt exclusion and secretion were examined in a dominant mangrove plant, Avicennia marina. The results showed that low salinities (10‰ and 20‰) almost has no negative effect on A. marina, however significant growth inhibitions were observed in the seedlings grown in higher salinities (30‰ and 40‰). With the increases of salinity, increased tissue Na+ content and enhanced salt secretion by glands were observed. Obvious suberization thickening were detected both in the exodermis and endodermis of the roots after salt pretreatment when compared to the roots without salt treatment. More importantly, the present data further confirmed that these root apoplastic barriers would directly decrease Na+ loading into xylem. Higher salt tolerance was observed in the seedlings pre-cultivated by salty tide when compared to fresh water cultivated A. marina. In summary, this study suggests a barrier property of suberization in dealing with salt exclusion in mangroves, a moderate salt pre-treatment may benefit plant withstanding high salinity.

The Full-Size ABCG Transporter of Medicago truncatula Is Involved in Strigolactone Secretion, Affecting Arbuscular Mycorrhiza.

Strigolactones (SLs) are plant-derived signaling molecules that stimulate the hyphal branching of arbuscular mycorrhizal fungi (AMF), and consequently promote symbiotic interaction between the fungus and the plant. Currently, our knowledge on the molecular mechanism of SL transport is restricted to the Solanaceae family. In the Solanaceae family, SL translocation toward the rhizosphere occurs through the exodermis via hypodermal passage cells and involves a member of the G subfamily, of the ATP-binding cassette (ABC) membrane transporters. Most Fabaceae species, including those that are agriculturally important, have a different root anatomy compared to most angiosperm plants (i.e., lacking an exodermis). Thus, we have investigated how SL transport occurs in the model legume Medicago truncatula. Here, we show that overexpression of a SL transporter from petunia (PaPDR1) enhances AMF colonization rates in M. truncatula. This result demonstrates the importance of ABCG proteins for the translocation of orobanchol-type molecules to facilitate arbuscular mycorrhiza, regardless of root anatomy and phylogenetic relationships. Moreover, our research has led to the identification of Medicago ABCG59, a close homologue of Petunia PDR1, that exhibits root specific expression and is up-regulated by phosphate starvation as well as in the presence of rac-GR24, a synthetic SL. Its promoter is active in cortical cells, root tips, and the meristematic zone of nodules. The mtabcg59 loss-of-function mutant displayed a reduced level of mycorrhization compared to the WT plants but had no impact on the number of nodules after Sinorhizobium meliloti inoculation. The reduced mycorrhization indicates that less SLs are secreted by the mutant plants, which is in line with the observation that mtabcg59 exudates exhibit a reduced stimulatory effect on the germination of the parasitic plant Phelipanche ramosa compared to the corresponding wild type.

Groups of multi-cellular passage cells in the root exodermis of Echinochloa crus-galli varieties lack not only suberin lamellae but also lignin deposits.

Passage cells are frequently found in the exodermis and the endodermis of the roots. Because passage cells lack an apoplastic diffusion barrier, they are thought to provide pathways for the transport of nutrients and the entrance of endomycorrhizal fungi. Exodermal passage cells possess Casparian strips but not suberin lamellae. So far, exodermal passage cells have not been associated with a particular internal structure. In some wetland plants, the outer part of the root (i.e., epidermis, exodermis, and sclerenchyma) of emerging lateral root primordia has an oxygen leaky zone called a window. The exodermis at the window site also lacks suberin lamellae, but it remains unclear whether the exodermis at the window site also lacks Casparian strips. Here, we report that several of the exodermal cells in the window of Echinochloa crus-galli grown under aerated or deoxygenated stagnant agar nutrient solution also lack lignin, which is a major constituent of Casparian strips. The sclerenchyma cells that form part of the window also lacked lignin deposits. Sites at which lateral root primordia developed were highly permeable to an apoplastic tracer (periodic acid). These observations indicate that windows consist of a novel type of passage cell at the exodermis that lacks lignin as well as suberin lamellae.

Exodermis and Endodermis Respond to Nutrient Deficiency in Nutrient-Specific and Localized Manner.

The exodermis is a common apoplastic barrier of the outer root cortex, with high environmentally-driven plasticity and a protective function. This study focused on the trade-off between the protective advantages provided by the exodermis and its disadvantageous reduction of cortical membrane surface area accessible by apoplastic route, thus limiting nutrient acquisition from the rhizosphere. We analysed the effect of nutrient deficiency (N, P, K, Mg, Ca, K, Fe) on exodermal and endodermal differentiation in maize. To differentiate systemic and localized effects, nutrient deficiencies were applied in three different approaches: to the root system as a whole, locally to discrete parts, or on one side of a single root. Our study showed that the establishment of the exodermis was enhanced in low-N and low-P plants, but delayed in low-K plants. The split-root cultivation proved that the effect is non-systemic, but locally coordinated for individual roots. Within a single root, localized deficiencies didn't result in an evenly differentiated exodermis, in contrast to other stress factors. The maturation of the endodermis responded in a similar way. In conclusion, N, P, and K deficiencies strongly modulated exodermal differentiation. The response was nutrient specific and integrated local signals of current nutrient availability from the rhizosphere.

Investigation of Salt Tolerance Mechanisms Across a Root Developmental Gradient in Almond Rootstocks.

The intensive use of groundwater in agriculture under the current climate conditions leads to acceleration of soil salinization. Given that almond is a salt-sensitive crop, selection of salt-tolerant rootstocks can help maintain productivity under salinity stress. Selection for tolerant rootstocks at an early growth stage can reduce the investment of time and resources. However, salinity-sensitive markers and salinity tolerance mechanisms of almond species to assist this selection process are largely unknown. We established a microscopy-based approach to investigate mechanisms of stress tolerance in and identified cellular, root anatomical, and molecular traits associated with rootstocks exhibiting salt tolerance. We characterized three almond rootstocks: Empyrean-1 (E1), Controller-5 (C5), and Krymsk-86 (K86). Based on cellular and molecular evidence, our results show that E1 has a higher capacity for salt exclusion by a combination of upregulating ion transporter expression and enhanced deposition of suberin and lignin in the root apoplastic barriers, exodermis, and endodermis, in response to salt stress. Expression analyses revealed differential regulation of cation transporters, stress signaling, and biopolymer synthesis genes in the different rootstocks. This foundational study reveals the mechanisms of salinity tolerance in almond rootstocks from cellular and structural perspectives across a root developmental gradient and provides insights for future screens targeting stress response.

Phenotypic Plasticity in the Structure of Fine Adventitious Metasequoia glyptostroboides Roots Allows Adaptation to Aquatic and Terrestrial Environments.

Metasequoia glyptostroboides (Cupressaceae) is a rare deciduous conifer which grows successfully in both aquatic and terrestrial environments. This tree has a narrow natural distribution in central China but is cultivated worldwide. Using histochemical staining and microscopy (both brightfield and epifluorescent), we investigated whether the phenotypic anatomical and histochemical plasticity in the fine adventitious roots of M. glyptostroboides has promoted the adaptation of this plant to aquatic and terrestrial environments. The fine root development and cortex sloughing of M. glyptostroboides occurs later in aquatic habitats than in terrestrial habitats. Anatomical and histochemical analyses have revealed that the apoplastic barriers in the primary growth of the fine roots consist of the endodermis and exodermis with Casparian bands, suberin lamellae, and secondarily lignified cell walls. There were also lignified phi (Φ) thickenings in the cortex. In both aquatic and terrestrial roots, secondary growth was observed in the vascular cambium, which produced secondary xylem and phloem, as well as in the phellogen, which produced cork. As compared to terrestrial adventitious roots, aquatic adventitious roots had multiple lignified Φ thickenings throughout the cortex, larger air spaces, dilated parenchyma, and dense suberin and lignin depositions in the exodermis. Our results thus indicate that phenotypic plasticity in the anatomical features of the fine adventitious roots, including apoplastic barriers, air spaces, and lignified Φ thickenings, might support the adaptation of M. glyptostroboides to both aquatic and terrestrial environments.

Prevention of Radial Oxygen Loss Is Associated With Exodermal Suberin Along Adventitious Roots of Annual Wild Species of Echinochloa.

Internal aeration is crucial for root growth under waterlogged conditions. Some wetland plants have a structural barrier that impedes oxygen leakage from the basal part of roots called a radial oxygen loss (ROL) barrier. The ROL barrier reduces loss of oxygen transported via the aerenchyma to the root tips, enabling root growth into anoxic soil. The roots of some plants develop an ROL barrier under waterlogged conditions, while they remain leaky to oxygen under well-drained or aerated conditions. The main components of the inducible ROL barrier are thought to be suberin and lignin deposited at the outer cellular space (apoplast) in the outer part of roots. On the other hand, a few wetland plants including a species of Echinochloa form a constitutive ROL barrier, i.e., it is formed even in the absence of waterlogging. However, little is known about the components of constitutive ROL barriers. An ROL barrier is considered to be a characteristic of wetland species because it has not been found in any non-wetland species so far. Here, we examined whether Echinochloa species from non-waterlogged fields also form an inducible or constitutive ROL barrier. We found that three species of Echinochloa from non-waterlogged fields constitutively developed an ROL barrier under aerated conditions. Over 85% of their root exodermis cells were covered with suberin lamellae and had well-developed Casparian strips. These substances inhibited the infiltration of an apoplastic tracer (periodic acid), suggesting that the ROL barrier can also prevent the entry of phytotoxic compounds from the soil. Unlike the other Echinochloa species, E. oryzicola, which mainly inhabits rice paddies, was found to lack a constitutive ROL barrier under aerated conditions. Although close to 90% of its sclerenchyma was well lignified, it leaked oxygen from the basal part of roots. A high percentage (55%) of the root exodermis cells were not fortified with suberin lamellae. These results suggest that suberin is an important component of constitutive ROL barriers.

Composite Transport Model and Water and Solute Transport across Plant Roots: An Update.

The present review examines recent experimental findings in root transport phenomena in terms of the composite transport model (CTM). It has been a well-accepted conceptual model to explain the complex water and solute flows across the root that has been related to the composite anatomical structure. There are three parallel pathways involved in the transport of water and solutes in roots - apoplast, symplast, and transcellular paths. The role of aquaporins (AQPs), which facilitate water flows through the transcellular path, and root apoplast is examined in terms of the CTM. The contribution of the plasma membrane bound AQPs for the overall water transport in the whole plant level was varying depending on the plant species, age of roots with varying developmental stages of apoplastic barriers, and driving forces (hydrostatic vs. osmotic). Many studies have demonstrated that the apoplastic barriers, such as Casparian bands in the primary anticlinal walls and suberin lamellae in the secondary cell walls, in the endo- and exodermis are not perfect barriers and unable to completely block the transport of water and some solute transport into the stele. Recent research on water and solute transport of roots with and without exodermis triggered the importance of the extension of conventional CTM adding resistances that arrange in series (epidermis, exodermis, mid-cortex, endodermis, and pericycle). The extension of the model may answer current questions about the applicability of CTM for composite water and solute transport of roots that contain complex anatomical structures with heterogeneous cell layers.

Exodermis and endodermis are the sites of xanthone biosynthesis in Hypericum perforatum roots.

Xanthones are specialized metabolites with antimicrobial properties, which accumulate in roots of Hypericum perforatum. This medicinal plant provides widely taken remedies for depressive episodes and skin disorders. Owing to the array of pharmacological activities, xanthone derivatives attract attention for drug design. Little is known about the sites of biosynthesis and accumulation of xanthones in roots. Xanthone biosynthesis is localized at the transcript, protein, and product levels using in situ mRNA hybridization, indirect immunofluorescence detection, and high lateral and mass resolution mass spectrometry imaging (AP-SMALDI-FT-Orbitrap MSI), respectively. The carbon skeleton of xanthones is formed by benzophenone synthase (BPS), for which a cDNA was cloned from root cultures of H. perforatum var. angustifolium. Both the BPS protein and the BPS transcripts are localized to the exodermis and the endodermis of roots. The xanthone compounds as the BPS products are detected in the same tissues. The exodermis and the endodermis, which are the outermost and innermost cell layers of the root cortex, respectively, are not only highly specialized barriers for controlling the passage of water and solutes but also preformed lines of defence against soilborne pathogens and predators.