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.

A Homeotic Mutation Changes Legume Nodule Ontogeny into Actinorhizal-Type Ontogeny.

Some plants fix atmospheric nitrogen by hosting symbiotic diazotrophic rhizobia or Frankia bacteria in root organs known as nodules. Such nodule symbiosis occurs in 10 plant lineages in four taxonomic orders: Fabales, Fagales, Cucurbitales, and Rosales, which are collectively known as the nitrogen-fixing clade. Nodules are divided into two types based on differences in ontogeny and histology: legume-type and actinorhizal-type nodules. The evolutionary relationship between these nodule types has been a long-standing enigma for molecular and evolutionary biologists. Recent phylogenomic studies on nodulating and nonnodulating species in the nitrogen-fixing clade indicated that the nodulation trait has a shared evolutionary origin in all 10 lineages. However, this hypothesis faces a conundrum in that legume-type and actinorhizal-type nodules have been regarded as fundamentally different. Here, we analyzed the actinorhizal-type nodules formed by Parasponia andersonii (Rosales) and Alnus glutinosa (Fagales) and found that their ontogeny is more similar to that of legume-type nodules (Fabales) than generally assumed. We also show that in Medicago truncatula, a homeotic mutation in the co-transcriptional regulator gene NODULE ROOT1 (MtNOOT1) converts legume-type nodules into actinorhizal-type nodules. These experimental findings suggest that the two nodule types have a shared evolutionary origin.

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.

A tale of two lineages: how the strains of the earliest divergent symbiotic Frankia clade spread over the world.

It is currently assumed that around 100 million years ago, the common ancestor to the Fabales, Fagales, Rosales and Cucurbitales in Gondwana, developed a root nodule symbiosis with a nitrogen-fixing bacterium. The symbiotic trait evolved first in Frankia cluster-2; thus, strains belonging to this cluster are the best extant representatives of this original symbiont. Most cluster-2 strains could not be cultured to date, except for Frankia coriariae, and therefore many aspects of the symbiosis are still elusive. Based on phylogenetics of cluster-2 metagenome-assembled genomes (MAGs), it has been shown that the genomes of strains originating in Eurasia are highly conserved. These MAGs are more closely related to Frankia cluster-2 in North America than to the single genome available thus far from the southern hemisphere, i.e., from Papua New Guinea.To unravel more biodiversity within Frankia cluster-2 and predict routes of dispersal from Gondwana, we sequenced and analysed the MAGs of Frankia cluster-2 from Coriaria japonica and Coriaria intermedia growing in Japan, Taiwan and the Philippines. Phylogenetic analyses indicate there is a clear split within Frankia cluster-2, separating a continental from an island lineage. Presumably, these lineages already diverged in Gondwana.Based on fossil data on the host plants, we propose that these two lineages dispersed via at least two routes. While the continental lineage reached Eurasia together with their host plants via the Indian subcontinent, the island lineage spread towards Japan with an unknown host plant.

Streptomyces coriariae sp. nov., a novel streptomycete isolated from actinorhizal nodules of Coriaria intermedia.

An actinobacterial strain, CMB-FB, was isolated from surface-sterilized root nodules of a Coriaria intermedia plant growing along Halsema Highway in the province of Benguet (Luzon, Philippines). The 16S rRNA gene sequence of CMB-FB showed high sequence similarity to those of the type strains of Streptomyces rishiriensis (99.4 %), Streptomyces humidus (99.1 %), Streptomyces cacaoi subsp. asoensis (99.0 %), and Streptomyces phaeofaciens (98.6 %). The major menaquinones of CMB-FB were composed of MK-9(H4), MK-9(H6) and MK-9(H8), and there was a minor contribution of MK-9(H10). The polar lipid profile consisted of phosphatidylethanolamine, unidentified aminolipids and phospholipids, a glycophospholipid and four unidentified lipids. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The major fatty acids were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The results of physiological analysis indicated that CMB-FB was mesophilic. The results of phylogenetic, genome-genome distance calculation and average nucleotide identity analysis indicated that the isolated strain represents the type strain of a novel species. On the basis of these results, strain CMB-FB (=DSM 112754T=LMG 32457T) is proposed as the type strain of the novel species Streptomyces coriariae sp. nov.

Tomato Sterol 22-desaturase Gene CYP710A11: Its Roles in Meloidogyne incognita Infection and Plant Stigmasterol Alteration.

Sterols are isoprenoid-derived lipids that play essential structural and functional roles in eukaryotic cells. Plants produce a complex mixture of sterols, and changes in plant sterol profiles have been linked to plant-pathogen interactions. ß-Sitosterol and stigmasterol, in particular, have been associated with plant defense. As nematodes have lost the ability to synthesize sterols de novo, they require sterols from the host. Tomato (Solanum lycopersicum) plants infected by the plant parasitic nematode Meloidogyne incognita show a reduced level of stigmasterol and a repression of the gene CYP710A11, encoding the sterol C-22 desaturase that is responsible for the conversion of ß-sitosterol to stigmasterol. In this study, we investigated the role of the tomato sterol C-22 desaturase gene CYP710A11 in the response to infection by M. incognita. We explored the plant-nematode interaction over time by analyzing the plant sterol composition and CYP710A11 gene regulation in S. lycopersicum after M. incognita infection. The temporal gene expression analysis showed that 3 days after inoculation with M. incognita, the CYP710A11 expression was significantly suppressed in the tomato roots, while a significant decrease in the stigmasterol content was observed after 14 days. A cyp710a11 knockout mutant tomato line lacking stigmasterol was analyzed to better understand the role of CYP710A11 in nematode development. M. incognita grown in the mutant line showed reduced egg mass counts, presumably due to the impaired growth of the mutant. However, the nematodes developed as well as they did in the wild-type line. Thus, while the suppression of CYP710A11 expression during nematode development may be a defense response of the plant against the nematode, the lack of stigmasterol did not seem to affect the nematode. This study contributes to the understanding of the role of stigmasterol in the interaction between M. incognita and tomato plants and shows that the sterol C-22 desaturase is not essential for the success of M. incognita.

YABBY Genes in the Development and Evolution of Land Plants.

Mounting evidence from genomic and transcriptomic studies suggests that most genetic networks regulating the morphogenesis of land plant sporophytes were co-opted and modified from those already present in streptophyte algae and gametophytes of bryophytes sensu lato. However, thus far, no candidate genes have been identified that could be responsible for "planation", a conversion from a three-dimensional to a two-dimensional growth pattern. According to the telome theory, "planation" was required for the genesis of the leaf blade in the course of leaf evolution. The key transcription factors responsible for leaf blade development in angiosperms are YABBY proteins, which until recently were thought to be unique for seed plants. Yet, identification of a YABBY homologue in a green alga and the recent findings of YABBY homologues in lycophytes and hornworts suggest that YABBY proteins were already present in the last common ancestor of land plants. Thus, these transcriptional factors could have been involved in "planation", which fosters our understanding of the origin of leaves. Here, we summarise the current data on functions of YABBY proteins in the vegetative and reproductive development of diverse angiosperms and gymnosperms as well as in the development of lycophytes. Furthermore, we discuss a putative role of YABBY proteins in the genesis of multicellular shoot apical meristems and in the evolution of leaves in early divergent terrestrial plants.

Genomic Changes Associated with the Evolutionary Transitions of Nostoc to a Plant Symbiont.

Cyanobacteria belonging to the genus Nostoc comprise free-living strains and also facultative plant symbionts. Symbiotic strains can enter into symbiosis with taxonomically diverse range of host plants. Little is known about genomic changes associated with evolutionary transition of Nostoc from free-living to plant symbiont. Here, we compared the genomes derived from 11 symbiotic Nostoc strains isolated from different host plants and infer phylogenetic relationships between strains. Phylogenetic reconstructions of 89 Nostocales showed that symbiotic Nostoc strains with a broad host range, entering epiphytic and intracellular or extracellular endophytic interactions, form a monophyletic clade indicating a common evolutionary history. A polyphyletic origin was found for Nostoc strains which enter only extracellular symbioses, and inference of transfer events implied that this trait was likely acquired several times in the evolution of the Nostocales. Symbiotic Nostoc strains showed enriched functions in transport and metabolism of organic sulfur, chemotaxis and motility, as well as the uptake of phosphate, branched-chain amino acids, and ammonium. The genomes of the intracellular clade differ from that of other Nostoc strains, with a gain/enrichment of genes encoding proteins to generate l-methionine from sulfite and pathways for the degradation of the plant metabolites vanillin and vanillate, and of the macromolecule xylan present in plant cell walls. These compounds could function as C-sources for members of the intracellular clade. Molecular clock analysis indicated that the intracellular clade emerged ca. 600 Ma, suggesting that intracellular Nostoc symbioses predate the origin of land plants and the emergence of their extant hosts.

Comparative Proteomic Analysis of Nodulated and Non-Nodulated Casuarina glauca Sieb. ex Spreng. Grown under Salinity Conditions Using Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS).

Casuarina glauca displays high levels of salt tolerance, but very little is known about how this tree adapts to saline conditions. To understand the molecular basis of C. glauca response to salt stress, we have analyzed the proteome from branchlets of plants nodulated by nitrogen-fixing Frankia Thr bacteria (NOD+) and non-nodulated plants supplied with KNO3 (KNO3+), exposed to 0, 200, 400, and 600 mM NaCl. Proteins were identified by Short Gel, Long Gradient Liquid Chromatography coupled to Tandem Mass Spectrometry and quantified by Sequential Window Acquisition of All Theoretical Mass Spectra -Mass Spectrometry. 600 proteins were identified and 357 quantified. Differentially Expressed Proteins (DEPs) were multifunctional and mainly involved in Carbohydrate Metabolism, Cellular Processes, and Environmental Information Processing. The number of DEPs increased gradually with stress severity: (i) from 7 (200 mM NaCl) to 40 (600 mM NaCl) in KNO3+; and (ii) from 6 (200 mM NaCl) to 23 (600 mM NaCl) in NOD+. Protein-protein interaction analysis identified different interacting proteins involved in general metabolic pathways as well as in the biosynthesis of secondary metabolites with different response networks related to salt stress. Salt tolerance in C. glauca is related to a moderate impact on the photosynthetic machinery (one of the first and most important stress targets) as well as to an enhancement of the antioxidant status that maintains cellular homeostasis.

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.

Proposal of 'Candidatus Frankia californiensis', the uncultured symbiont in nitrogen-fixing root nodules of a phylogenetically broad group of hosts endemic to western North America.

The genus Frankia comprises a group of nitrogen-fixing actinobacteria that form root-nodule symbioses with perennial dicotyledonous plants in the nitrogen-fixing clade. These bacteria have been characterized phylogenetically and grouped into four clusters (clusters 1-4). Cluster 2 contains mostly uncultured strains that induce nodules on species of the genera Datisca (Datiscaceae), Coriaria (Coriariaceae), Ceanothus (Rhamnaceae) and several genera in the family Rosaceae (Cercocarpus, Chamaebatia, Dryas, Purshia), all of which except members of the genus Coriaria are present within the California Floristic Province (CFP) or neighbouring areas of western North America. Those strains occurring in western North America are genetically very closely related to one another, and genetically distinct from strains characterized from other locales. We hereby propose to create a 'Candidatus Frankia californiensis' species for those cluster 2 strains of the genus Frankia with both high genetic similarity and a geographical distribution in or near the CFP.

An assemblage of Frankia Cluster II strains from California contains the canonical nod genes and also the sulfotransferase gene nodH.

BACKGROUND: The ability to establish root nodule symbioses is restricted to four different plant orders. Soil actinobacteria of the genus Frankia can establish a symbiotic relationship with a diverse group of plants within eight different families from three different orders, the Cucurbitales, Fagales and Rosales. Phylogenetically, Frankia strains can be divided into four clusters, three of which (I, II, III) contain symbiotic strains. Members of Cluster II nodulate the broadest range of host plants with species from four families from two different orders, growing on six continents. Two Cluster II genomes were sequenced thus far, both from Asia. RESULTS: In this paper we present the first Frankia cluster II genome from North America (California), Dg2, which represents a metagenome of two major and one minor strains. A phylogenetic analysis of the core genomes of 16 Frankia strains shows that Cluster II the ancestral group in the genus, also ancestral to the non-symbiotic Cluster IV. Dg2 contains the canonical nod genes nodABC for the production of lipochitooligosaccharide Nod factors, but also two copies of the sulfotransferase gene nodH. In rhizobial systems, sulfation of Nod factors affects their host specificity and their stability. CONCLUSIONS: A comparison with the nod gene region of the previously sequenced Dg1 genome from a Cluster II strain from Pakistan shows that the common ancestor of both strains should have contained nodABC and nodH. Phylogenetically, Dg2 NodH proteins are sister to rhizobial NodH proteins. A glnA-based phylogenetic analysis of all Cluster II strains sampled thus far supports the hypothesis that Cluster II Frankia strains came to North America with Datisca glomerata following the Madrean-Tethyan pattern.

Genome analysis and biogeographic distribution of the earliest divergent Frankia clade in the southern hemisphere.

Coriariaceae are a small plant family of 14-17 species and subspecies that currently have a global but disjunct distribution. All species can form root nodules in symbiosis with diazotrophic Frankia cluster-2 strains, which form the earliest divergent symbiotic clade within this bacterial genus. Studies on Frankia cluster-2 mostly have focused on strains occurring in the northern hemisphere. Except for one strain from Papua New Guinea, namely Candidatus Frankia meridionalis Cppng1, no complete genome of Frankia associated with Coriaria occurring in the southern hemisphere has been published thus far, yet the majority of the Coriariaceae species occur here. We present field sampling data of novel Frankia cluster-2 strains, representing two novel species, which are associated with Coriaria arborea and Coriaria sarmentosa in New Zealand, and with Coriaria ruscifolia in Patagonia (Argentina), in addition to identifying Ca. F. meridionalis present in New Zealand. The novel Frankia species were found to be closely related to both Ca. F. meridionalis, and a Frankia species occurring in the Philippines, Taiwan, and Japan. Our data suggest that the different Frankia cluster-2 species diverged early after becoming symbiotic circa 100 million years ago.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

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.

3-Pentanol glycosides from root nodules of the actinorhizal plant Alnus cremastogyne.

Alnus cremastogyne Burkill (Betulaceae), an actinorhizal plant, can enter a mutualistic symbiosis with Frankia species that leads to the formation of nitrogen fixing root nodules. Some primary metabolites (carbohydrates, dicarboxylic acids, amino acids, citrulline and amides) involved in carbon and nitrogen metabolism in actinorhizal nodules have been identified, while specialized metabolites in A. cremastogyne root nodules are yet to be characterized. In this study, we isolated and identified three undescribed 3-pentanol glycosides, i.e., 3-pentyl α-l-arabinofuranosyl-(1''→6')-ß-d-glucopyranoside, 3-pentyl α-l-rhamnopyranosyl-(1''→6')-ß-d-glucopyranoside, and 3-pentyl 6'-(3-hydroxy3-methylglutaryl)-ß-d-glucopyranoside, as well as seventeen known compounds from A. cremastogyne root nodules. 3-Pentanol glycosides are abundantly distributed in root nodules, while they are distributed in stems, roots, leaves and fruits at low/zero levels. A. cremastogyne plants treated by root nodule suspension emit 3-pentanol. This study enriches the knowledge about specialized metabolites in the actinorhizal host, and provides preliminarily information on the signal exchange in the actinorhizal symbiosis between A. cremastogyne and Frankia.

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.

Silicate Inhibits the Cytosolic Influx of Chloride in Protoplasts of Wheat and Affects the Chloride Transporters, TaCLC1 and TaNPF2.4/2.5.

Chloride is an essential nutrient for plants, but high concentrations can be harmful. Silicon ameliorates both abiotic and biotic stresses in plants, but it is unknown if it can prevent cellular increase of chloride. Therefore, we investigated the influx of Cl- ions in two wheat cultivars different in salt sensitivity, by epifluorescence microscopy and a highly Cl--sensitive dye, MQAE, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide, in absence and presence of potassium silicate, K2SiO3. The Cl--influx was higher in the salt-sensitive cv. Vinjett, than in the salt-tolerant cv. S-24, and silicate pre-treatment of protoplasts inhibited the Cl--influx in both cultivars, but more in the sensitive cv. Vinjett. To investigate if the Cl--transporters TaCLC1 and TaNPF2.4/2.5 are affected by silicate, expression analyses by RT-qPCR were undertaken of TaCLC1 and TaNPF 2.4/2.5 transcripts in the absence and presence of 100 mM NaCl, with and without the presence of K2SiO3. The results show that both transporter genes were expressed in roots and shoots of wheat seedlings, but their expressions were differently affected by silicate. The TaNPF2.4/2.5 expression in leaves was markedly depressed by silicate. These findings demonstrate that less chloride accumulates in the cytosol of leaf mesophyll by Si treatment and increases salt tolerance.

Legume NCRs and nodule-specific defensins of actinorhizal plants-Do they share a common origin?

The actinorhizal plant Datisca glomerata (Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbiotic Frankia clade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed in D. glomerata nodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodule-specific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels, DgDef1, was characterized. Promoter-GUS studies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity. In vitro studies with E. coli and Sinorhizobium meliloti 1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality for S. meliloti 1021 at 20.8 µM. Analysis of the S. meliloti 1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages.