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.

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.

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.

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.

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.