Do DEEPER ROOTING 1 Homologs Regulate the Lateral Root Slope Angle in Cucumber (Cucumis sativus)?

The architecture of the root system is fundamental to plant productivity. The rate of root growth, the density of lateral roots, and the spatial structure of lateral and adventitious roots determine the developmental plasticity of the root system in response to changes in environmental conditions. One of the genes involved in the regulation of the slope angle of lateral roots is DEEPER ROOTING 1 (DRO1). Its orthologs and paralogs have been identified in rice, Arabidopsis, and several other species. However, nothing is known about the formation of the slope angle of lateral roots in species with the initiation of lateral root primordia within the parental root meristem. To address this knowledge gap, we identified orthologs and paralogs of the DRO1 gene in cucumber (Cucumis sativus) using a phylogenetic analysis of IGT protein family members. Differences in the transcriptional response of CsDRO1, CsDRO1-LIKE1 (CsDRO1L1), and CsDRO1-LIKE2 (CsDRO1L2) to exogenous auxin were analyzed. The results showed that only CsDRO1L1 is auxin-responsive. An analysis of promoter-reporter fusions demonstrated that the CsDRO1, CsDRO1L1, and CsDRO1L2 genes were expressed in the meristem in cell files of the central cylinder, endodermis, and cortex; the three genes displayed different expression patterns in cucumber roots with only partial overlap. A knockout of individual CsDRO1, CsDRO1L1, and CsDRO1L2 genes was performed via CRISPR/Cas9 gene editing. Our study suggests that the knockout of individual genes does not affect the slope angle formation during lateral root primordia development in the cucumber parental root.

Lateral Root Initiation in Cucumber (Cucumis sativus): What Does the Expression Pattern of Rapid Alkalinization Factor 34 (RALF34) Tell Us?

In Arabidopsis, the small signaling peptide (peptide hormone) RALF34 is involved in the gene regulatory network of lateral root initiation. In this study, we aimed to understand the nature of the signals induced by RALF34 in the non-model plant cucumber (Cucumis sativus), where lateral root primordia are induced in the apical meristem of the parental root. The RALF family members of cucumber were identified using phylogenetic analysis. The sequence of events involved in the initiation and development of lateral root primordia in cucumber was examined in detail. To elucidate the role of the small signaling peptide CsRALF34 and its receptor CsTHESEUS1 in the initial stages of lateral root formation in the parental root meristem in cucumber, we studied the expression patterns of both genes, as well as the localization and transport of the CsRALF34 peptide. CsRALF34 is expressed in all plant organs. CsRALF34 seems to differ from AtRALF34 in that its expression is not regulated by auxin. The expression of AtRALF34, as well as CsRALF34, is regulated in part by ethylene. CsTHESEUS1 is expressed constitutively in cucumber root tissues. Our data suggest that CsRALF34 acts in a non-cell-autonomous manner and is not involved in lateral root initiation in cucumber.

Integrative Proteomics and Metabolomics Analysis Reveals the Role of Small Signaling Peptide Rapid Alkalinization Factor 34 (RALF34) in Cucumber Roots.

The main role of RALF small signaling peptides was reported to be the alkalization control of the apoplast for improvement of nutrient absorption; however, the exact function of individual RALF peptides such as RALF34 remains unknown. The Arabidopsis RALF34 (AtRALF34) peptide was proposed to be part of the gene regulatory network of lateral root initiation. Cucumber is an excellent model for studying a special form of lateral root initiation taking place in the meristem of the parental root. We attempted to elucidate the role of the regulatory pathway in which RALF34 is a participant using cucumber transgenic hairy roots overexpressing CsRALF34 for comprehensive, integrated metabolomics and proteomics studies, focusing on the analysis of stress response markers. CsRALF34 overexpression resulted in the inhibition of root growth and regulation of cell proliferation, specifically in blocking the G2/M transition in cucumber roots. Based on these results, we propose that CsRALF34 is not part of the gene regulatory networks involved in the early steps of lateral root initiation. Instead, we suggest that CsRALF34 modulates ROS homeostasis and triggers the controlled production of hydroxyl radicals in root cells, possibly associated with intracellular signal transduction. Altogether, our results support the role of RALF peptides as ROS regulators.

Effects of Phytotoxic Nonenolides, Stagonolide A and Herbarumin I, on Physiological and Biochemical Processes in Leaves and Roots of Sensitive Plants.

Phytotoxic macrolides attract attention as prototypes of new herbicides. However, their mechanisms of action (MOA) on plants have not yet been elucidated. This study addresses the effects of two ten-membered lactones, stagonolide A (STA) and herbarumin I (HBI) produced by the fungus Stagonospora cirsii, on Cirsium arvense, Arabidopsis thaliana and Allium cepa. Bioassay of STA and HBI on punctured leaf discs of C. arvense and A. thaliana was conducted at a concentration of 2 mg/mL to evaluate phenotypic responses, the content of pigments, electrolyte leakage from leaf discs, the level of reactive oxygen species, Hill reaction rate, and the relative rise in chlorophyll a fluorescence. The toxin treatments resulted in necrotic and bleached leaf lesions in the dark and in the light, respectively. In the light, HBI treatment caused the drop of carotenoids content in leaves on both plants. The electrolyte leakage caused by HBI was light-dependent, in contrast with that caused by STA. Both compounds induced light-independent peroxide generation in leaf cells but did not affect photosynthesis 6 h after treatment. STA (10 µg/mL) caused strong disorders in root cells of A. thaliana leading to the complete dissipation of the mitochondrial membrane potential one hour post treatment, as well as DNA fragmentation and disappearance of acidic vesicles in the division zone after 8 h; the effects of HBI (50 µg/mL) were much milder. Furthermore, STA was found to inhibit mitosis but did not affect the cytoskeleton in cells of root tips of A. cepa and C. arvense, respectively. Finally, STA was supposed to inhibit the intracellular vesicular traffic from the endoplasmic reticulum to the Golgi apparatus, thus interfering with mitosis. HBI is likely to have another main MOA, probably inhibiting the biosynthesis of carotenoids.

OMICs, Epigenetics, and Genome Editing Techniques for Food and Nutritional Security.

The incredible success of crop breeding and agricultural innovation in the last century greatly contributed to the Green Revolution, which significantly increased yields and ensures food security, despite the population explosion. However, new challenges such as rapid climate change, deteriorating soil, and the accumulation of pollutants require much faster responses and more effective solutions that cannot be achieved through traditional breeding. Further prospects for increasing the efficiency of agriculture are undoubtedly associated with the inclusion in the breeding strategy of new knowledge obtained using high-throughput technologies and new tools in the future to ensure the design of new plant genomes and predict the desired phenotype. This article provides an overview of the current state of research in these areas, as well as the study of soil and plant microbiomes, and the prospective use of their potential in a new field of microbiome engineering. In terms of genomic and phenomic predictions, we also propose an integrated approach that combines high-density genotyping and high-throughput phenotyping techniques, which can improve the prediction accuracy of quantitative traits in crop species.

Leaf Epidermis: The Ambiguous Symplastic Domain.

The ability to develop secondary (post-cytokinetic) plasmodesmata (PD) is an important evolutionary advantage that helps in creating symplastic domains within the plant body. Developmental regulation of secondary PD formation is not completely understood. In flowering plants, secondary PD occur exclusively between cells from different lineages, e.g., at the L1/L2 interface within shoot apices, or between leaf epidermis (L1-derivative), and mesophyll (L2-derivative). However, the highest numbers of secondary PD occur in the minor veins of leaf between bundle sheath cells and phloem companion cells in a group of plant species designated "symplastic" phloem loaders, as opposed to "apoplastic" loaders. This poses a question of whether secondary PD formation is upregulated in general in symplastic loaders. Distribution of PD in leaves and in shoot apices of two symplastic phloem loaders, Alonsoa meridionalis and Asarina barclaiana, was compared with that in two apoplastic loaders, Solanum tuberosum (potato) and Hordeum vulgare (barley), using immunolabeling of the PD-specific proteins and transmission electron microscopy (TEM), respectively. Single-cell sampling was performed to correlate sugar allocation between leaf epidermis and mesophyll to PD abundance. Although the distribution of PD in the leaf lamina (except within the vascular tissues) and in the meristem layers was similar in all species examined, far fewer PD were found at the epidermis/epidermis and mesophyll/epidermis boundaries in apoplastic loaders compared to symplastic loaders. In the latter, the leaf epidermis accumulated sugar, suggesting sugar import from the mesophyll via PD. Thus, leaf epidermis and mesophyll might represent a single symplastic domain in Alonsoa meridionalis and Asarina barclaiana.

Cappable-Seq Reveals Specific Patterns of Metabolism and Virulence for Salmonella Typhimurium Intracellular Survival within Acanthamoeba castellanii.

The bacterial pathogen Salmonella enterica, which causes enteritis, has a broad host range and extensive environmental longevity. In water and soil, Salmonella interacts with protozoa and multiplies inside their phagosomes. Although this relationship resembles that between Salmonella and mammalian phagocytes, the interaction mechanisms and bacterial genes involved are unclear. Here, we characterized global gene expression patterns of S. enterica serovar Typhimurium within Acanthamoeba castellanii at the early stage of infection by Cappable-Seq. Gene expression features of S. Typhimurium within A. castellanii were presented with downregulation of glycolysis-related, and upregulation of glyoxylate cycle-related genes. Expression of Salmonella Pathogenicity Island-1 (SPI-1), chemotaxis system, and flagellar apparatus genes was upregulated. Furthermore, expression of genes mediating oxidative stress response and iron uptake was upregulated within A. castellanii as well as within mammalian phagocytes. Hence, global S. Typhimurium gene expression patterns within A. castellanii help better understand the molecular mechanisms of Salmonella adaptation to an amoeba cell and intracellular persistence in protozoa inhabiting water and soil ecosystems.

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

Mutational analysis indicates that abnormalities in rhizobial infection and subsequent plant cell and bacteroid differentiation in pea (Pisum sativum) nodules coincide with abnormal cytokinin responses and localization.

BACKGROUND AND AIMS: Recent findings indicate that Nod factor signalling is tightly interconnected with phytohormonal regulation that affects the development of nodules. Since the mechanisms of this interaction are still far from understood, here the distribution of cytokinin and auxin in pea (Pisum sativum) nodules was investigated. In addition, the effect of certain mutations blocking rhizobial infection and subsequent plant cell and bacteroid differentiation on cytokinin distribution in nodules was analysed. METHODS: Patterns of cytokinin and auxin in pea nodules were profiled using both responsive genetic constructs and antibodies. KEY RESULTS: In wild-type nodules, cytokinins were found in the meristem, infection zone and apical part of the nitrogen fixation zone, whereas auxin localization was restricted to the meristem and peripheral tissues. We found significantly altered cytokinin distribution in sym33 and sym40 pea mutants defective in IPD3/CYCLOPS and EFD transcription factors, respectively. In the sym33 mutants impaired in bacterial accommodation and subsequent nodule differentiation, cytokinin localization was mostly limited to the meristem. In addition, we found significantly decreased expression of LOG1 and A-type RR11 as well as KNOX3 and NIN genes in the sym33 mutants, which correlated with low cellular cytokinin levels. In the sym40 mutant, cytokinins were detected in the nodule infection zone but, in contrast to the wild type, they were absent in infection droplets. CONCLUSIONS: In conclusion, our findings suggest that enhanced cytokinin accumulation during the late stages of symbiosis development may be associated with bacterial penetration into the plant cells and subsequent plant cell and bacteroid differentiation.

Accumulation of and Response to Auxins in Roots and Nodules of the Actinorhizal Plant Datisca glomerata Compared to the Model Legume Medicago truncatula.

Actinorhizal nodules are structurally different from legume nodules and show a greater similarity to lateral roots. Because of the important role of auxins in lateral root and nodule formation, auxin profiles were examined in roots and nodules of the actinorhizal species Datisca glomerata and the model legume Medicago truncatula. The auxin response in roots and nodules of both species was analyzed in transgenic root systems expressing a beta-glucuronidase gene under control of the synthetic auxin-responsive promoter DR5. The effects of two different auxin on root development were compared for both species. The auxin present in nodules at the highest levels was phenylacetic acid (PAA). No differences were found between the concentrations of active auxins of roots vs. nodules, while levels of the auxin conjugate indole-3-acetic acid-alanine were increased in nodules compared to roots of both species. Because auxins typically act in concert with cytokinins, cytokinins were also quantified. Concentrations of cis-zeatin and some glycosylated cytokinins were dramatically increased in nodules compared to roots of D. glomerata, but not of M. truncatula. The ratio of active auxins to cytokinins remained similar in nodules compared to roots in both species. The auxin response, as shown by the activation of the DR5 promoter, seemed significantly reduced in nodules compared to roots of both species, suggesting the accumulation of auxins in cell types that do not express the signal transduction pathway leading to DR5 activation. Effects on root development were analyzed for the synthetic auxin naphthaleneacetic acid (NAA) and PAA, the dominant auxin in nodules. Both auxins had similar effects, except that the sensitivity of roots to PAA was lower than to NAA. However, while the effects of both auxins on primary root growth were similar for both species, effects on root branching were different: both auxins had the classical positive effect on root branching in M. truncatula, but a negative effect in D. glomerata. Such a negative effect of exogenous auxin on root branching has previously been found for a cucurbit that forms lateral root primordia in the meristem of the parental root; however, root branching in D. glomerata does not follow that pattern.

Frankia-Enriched Metagenomes from the Earliest Diverging Symbiotic Frankia Cluster: They Come in Teams.

Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

Lateral Root Initiation in the Parental Root Meristem of Cucurbits: Old Players in a New Position.

While in most higher plants, including the model system Arabidopsis thaliana, the formation of lateral root primordia is induced in the elongation zone of the parental root, in seven plant families, including Cucurbitaceae, an alternative root branching mechanism is established such that lateral roots are initiated directly in the apical meristem of the parental root. In Arabidopsis, the transcription factor GATA23 and MEMBRANE-ASSOCIATED KINASE REGULATOR4 (MAKR4) are involved in the gene regulatory network of lateral root initiation. Among all marker genes examined, these are the earliest known marker genes up-regulated by auxin during lateral root initiation. In this study, putative functional orthologs of Arabidopsis GATA23 and MAKR4 were identified in cucumber (Cucumis sativus) and squash (Cucurbita pepo). Both cucurbits contained 26 genes encoding GATA family transcription factors and only one MAKR4 gene. Phylogenetic and transcriptional analysis of up-regulation by auxin led to the identification of GATA23 putative functional orthologs in Cucurbitaceae - CpGATA24 and CsGATA24. In squash, CpMAKR4 was up-regulated by naphthylacetic acid (NAA) and, similar to MAKR4 in Arabidopsis, indole-3-butyric acid (IBA). A detailed analysis of the expression pattern of CpGATA24 and CpMAKR4 in squash roots from founder cell specification until emergence of lateral root primordia was carried out using promoter-fluorescent reporter gene fusions and confocal microscopy. Their expression was induced in the protoxylem, and then expanded to founder cells in the pericycle. Thus, while the overall expression pattern of these genes was significantly different from that in Arabidopsis, in founder cells their expression was induced in the same order as in Arabidopsis. Altogether, these findings suggest that in Cucurbitaceae the putative functional orthologs of GATA23 and MAKR4 might play a role in founder cell specification and primordium positioning during lateral root initiation. The role of the protoxylem in auxin transport as a trigger of founder cells specification and lateral root initiation is discussed.

Lateral root initiation and formation within the parental root meristem of Cucurbita pepo: is auxin a key player?

Background and Aims: In some plant families, including Cucurbitaceae, initiation and development of lateral roots (LRs) occur in the parental root apical meristem. The objective of this study was to identify the general mechanisms underlying LR initiation (LRI). Therefore, the first cellular events leading to LRI as well as the role of auxin in this process were studied in the Cucurbita pepo root apical meristem. Methods: Transgenic hairy roots harbouring the auxin-responsive promoter DR5 fused to different reporter genes were used for visualizing of cellular auxin response maxima (ARMs) via confocal laser scanning microscopy and 3-D imaging. The effects of exogenous auxin and auxin transport inhibitors on root branching were analysed. Key Results: The earliest LRI event involved a group of symmetric anticlinal divisions in pericycle cell files at a distance of 250-350 µm from the initial cells. The visualization of the ARMs enabled the precise detection of cells involved in determining the site of LR primordium formation. A local ARM appeared in sister cells of the pericycle and endodermis files before the first division. Cortical cells contributed to LR development after the anticlinal divisions in the pericycle via the formation of an ARM. Exogenous auxins did not increase the total number of LRs and did not affect the LRI index. Although exogenous auxin transport inhibitors acted in different ways, they all reduced the number of LRs formed. Conclusions: Literature data, as well as results obtained in this study, suggest that the formation of a local ARM before the first anticlinal formative divisions is the common mechanism underlying LRI in flowering plants. We propose that the mechanisms of the regulation of root branching are independent of the position of the LRI site relative to the parental root tip.

Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules.

Jasmonic acid (JA), its derivatives and its precursor cis-12-oxo phytodienoic acid (OPDA) form a group of phytohormones, the jasmonates, representing signal molecules involved in plant stress responses, in the defense against pathogens as well as in development. Elevated levels of JA have been shown to play a role in arbuscular mycorrhiza and in the induction of nitrogen-fixing root nodules. In this study, the gene families of two committed enzymes of the JA biosynthetic pathway, allene oxide synthase (AOS) and allene oxide cyclase (AOC), were characterized in the determinate nodule-forming model legume Lotus japonicus JA levels were to be analysed in the course of nodulation. Since in all L. japonicus organs examined, JA levels increased upon mechanical disturbance and wounding, an aeroponic culture system was established to allow for a quick harvest, followed by the analysis of JA levels in whole root and shoot systems. Nodulated plants were compared with non-nodulated plants grown on nitrate or ammonium as N source, respectively, over a five week-period. JA levels turned out to be more or less stable independently of the growth conditions. However, L. japonicus nodules formed on aeroponically grown plants often showed patches of cells with reduced bacteroid density, presumably a stress symptom. Immunolocalization using a heterologous antibody showed that the vascular systems of these nodules also seemed to contain less AOC protein than those of nodules of plants grown in perlite/vermiculite. Hence, aeroponically grown L. japonicus plants are likely to be habituated to stress which could have affected JA levels.

Comparative analysis of the tubulin cytoskeleton organization in nodules of Medicago truncatula and Pisum sativum: bacterial release and bacteroid positioning correlate with characteristic microtubule rearrangements.

In this study we analyzed and compared the organization of the tubulin cytoskeleton in nodules of Medicago truncatula and Pisum sativum. We combined antibody labeling and green fluorescent protein tagging with laser confocal microscopy to observe microtubules (MTs) in nodules of both wild-type (WT) plants and symbiotic plant mutants blocked at different steps of nodule development. The 3D MT organization of each histological nodule zone in both M. truncatula and P. sativum is correlated to specific developmental processes. Endoplasmic MTs appear to support infection thread growth, infection droplet formation and bacterial release into the host cytoplasm in nodules of both species. No differences in the organization of the MT cytoskeleton between WT and bacterial release mutants were apparent, suggesting both that the phenotype is not linked to a defect in MT organization and that the growth of hypertrophied infection threads is supported by MTs. Strikingly, bacterial release coincides with a change in the organization of cortical MTs from parallel arrays into an irregular, crisscross arrangement. After release, the organization of endoplasmic MTs is linked to the distribution of symbiosomes. The 3D MT organization of each nodule histological zone in M. truncatula and P. sativum was analyzed and linked to specific developmental processes.

Initiation of spontaneous tumors in radish (Raphanus sativus): Cellular, molecular and physiological events.

In plant meristems, the balance of cell proliferation and differentiation is maintained by phytohormones, specifically auxin and cytokinin, as well as transcription factors. Changing of the cytokinin/auxin balance in plants may lead to developmental abnormalities, and in particular, to the formation of tumors. The examples of spontaneous tumor formation in plants include tumors formed on the roots of radish (Raphanus sativus) inbred lines. Previously, it was found that the cytokinin/auxin ratio is altered in radish tumors. In this study, a detailed histological analysis of spontaneous radish tumors was performed, revealing a possible mechanism of tumor formation, namely abnormal cambial activity. The analysis of cell proliferation patterns revealed meristematic foci in radish tumors. By using a fusion of an auxin-responsive promoter (DR5) and a reporter gene, the involvement of auxin in developmental processes in tumors was shown. In addition, the expression of the root meristem-specific WUSCHEL-related homeobox 5 (WOX5) gene was observed in cells adjacent to meristematic foci. Taken together, the results of the present study show that tumor tissues share some characteristics with root apical meristems, including the presence of auxin-response maxima in meristematic foci with adjacent cells expressing WOX5.

Plasmodesmata without callose and calreticulin in higher plants - open channels for fast symplastic transport?

Plasmodesmata (PD) represent membrane-lined channels that link adjacent plant cells across the cell wall. PD of higher plants contain a central tube of endoplasmic reticulum (ER) called desmotubule. Membrane and lumen proteins seem to be able to move through the desmotubule, but most transport processes through PD occur through the cytoplasmic annulus (Brunkard etal., 2013). Calreticulin (CRT), a highly conserved Ca(2+)-binding protein found in all multicellular eukaryotes, predominantly located in the ER, was shown to localize to PD, though not all PD accumulate CRT. In nitrogen-fixing actinorhizal root nodules of the Australian tree Casuarina glauca, the primary walls of infected cells containing the microsymbiont become lignified upon infection. TEM analysis of these nodules showed that during the differentiation of infected cells, PD connecting infected cells, and connecting infected and adjacent uninfected cells, were reduced in number as well as diameter (Schubert etal., 2013). In contrast with PD connecting young infected cells, and most PD connecting mature infected and adjacent uninfected cells, PD connecting mature infected cells did not accumulate CRT. Furthermore, as shown here, these PD were not associated with callose, and based on their diameter, they probably had lost their desmotubules. We speculate that either this is a slow path to PD degradation, or that the loss of callose accumulation and presumably also desmotubules leads to the PD becoming open channels and improves metabolite exchange between cells.

Lignification of cell walls of infected cells in Casuarina glauca nodules that depend on symplastic sugar supply is accompanied by reduction of plasmodesmata number and narrowing of plasmodesmata.

The oxygen protection system for the bacterial nitrogen-fixing enzyme complex nitrogenase in actinorhizal nodules of Casuarina glauca resembles that of legume nodules: infected cells contain large amounts of the oxygen-binding protein hemoglobin and are surrounded by an oxygen diffusion barrier. However, while in legume nodules infected cells are located in the central tissue, actinorhizal nodules are composed of modified lateral roots with infected cells in the expanded cortex. Since an oxygen diffusion barrier around the entire cortex would also block oxygen access to the central vascular system where it is required to provide energy for transport processes, here each individual infected cell is surrounded with an oxygen diffusion barrier. In order to assess the effect of these oxygen diffusion barriers on oxygen supply for energy production for transport processes, apoplastic and symplastic sugar transport pathways in C. glauca nodules were examined. The results support the idea that sugar transport to and within the nodule cortex relies to a large extent on the less energy-demanding symplastic mechanism. This is in line with the assumption that oxygen access to the nodule vascular system is substantially restricted. In spite of this dependence on symplastic transport processes to supply sugars to infected cells, plasmodesmal connections between infected cells, and to a lesser degree with uninfected cells, were reduced during the differentiation of infected cells.

Composite Cucurbita pepo plants with transgenic roots as a tool to study root development.

BACKGROUND AND AIMS: In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash. METHODS: The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2'-deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or ß-glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively. KEY RESULTS: Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots. CONCLUSIONS: The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.

The diversity of actinorhizal symbiosis.

Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.