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

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.This study was supported by two grants from the Swedish Research Council Vetenskapsrådet (VR 2007-17840-52674-16 and VR 2012-03061) and by a grant from Carl Tryggers Stiftelse för Vetenskaplig Forskning (CTS 13:354) to KP, by a grant from the Russian Science Foundation (analyses of auxin response pattern, grant no. 16-16-00089) to KND, and by a grant from the Ministry of Education, Youth and Sports of CR within the National Sustainability Program I (NPUI, grant number LO1415) to TR. UM was supported by the Australian Research Council (DP150102002)

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 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 et al., 2013). Calreticulin (CRT), a highly conserved Ca2+-binding protein found in all multi-cellular 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 et al., 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

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

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699In 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. 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 -glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively. 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. 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

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

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

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

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

WUSCHEL-RELATED HOMEOBOX5 gene expression and interaction of CLE peptides with components of the systemic control add two pieces to the puzzle of autoregulation of nodulation

In legumes, the symbiotic nodules are formed as a result of dedifferentiation and reactivation of cortical root cells. A shoot-acting receptor complex, similar to the Arabidopsis (Arabidopsis thaliana) CLAVATA1 (CLV1)/CLV2 receptor, regulating development of the shoot apical meristem, is involved in autoregulation of nodulation (AON), a mechanism that systemically controls nodule number. The targets of CLV1/CLV2 in the shoot apical meristem, the WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family transcription factors, have been proposed to be important regulators of apical meristem maintenance and to be expressed in apical meristem "organizers." Here, we focus on the role of the WOX5 transcription factor upon nodulation in Medicago truncatula and pea (Pisum sativum) that form indeterminate nodules. Analysis of temporal WOX5 expression during nodulation with quantitative reverse transcription-polymerase chain reaction and promoter-reporter fusion revealed that the WOX5 gene was expressed during nodule organogenesis, suggesting that WOX genes are common regulators of cell proliferation in different systems. Furthermore, in nodules of supernodulating mutants, defective in AON, WOX5 expression was higher than that in wild-type nodules. Hence, a conserved WUS/WOX-CLV regulatory system might control cell proliferation and differentiation not only in the root and shoot apical meristems but also in nodule meristems. In addition, the link between nodule-derived CLE peptides activating AON in different legumes and components of the AON system was investigated. We demonstrate that the identified AON component, NODULATION3 of pea, might act downstream from or beside the CLE peptides during AON