Identification, expression, and functional analysis of CLE genes in radish (Raphanus sativus L.) storage root.

BACKGROUND: Radish (Raphanus sativus L.) is a widespread agricultural plant forming storage root due to extensive secondary growth which involves cambium proliferation and differentiation of secondary conductive tissues. Closely related to the model object Arabidopsis thaliana, radish is a suitable model for studying processes of secondary growth and storage root development. CLE peptides are a group of peptide phytohormones which play important role in the regulation of primary meristems such as SAM, RAM, and procambium, as well as secondary meristems. However, the role of CLE peptides in lateral growth of root during storage root formation has not been studied to date. RESULTS: In present work we studied the role of CLE peptides in the development of storage root in radish. We have identified 18 CLE genes of radish (RsCLEs) and measured their expression in various plant organs and also at different stages of root development in R. sativus and Raphanus raphanistrum-its close relative which does not form storage root. We observed significant decline of expression levels for genes RsCLE1, 2, 11, 13, and 16, and also multifold increase of expression levels for genes RsCLE19, and 41 during secondary root growth in R. sativus but not in R. raphanistrum. Expression of RsCLE 2, 19, and 41 in R. sativus root was confined to certain types of tissues while RsCLE1, 11, 13, and 16 expressed throughout the root. Experiments on overexpression of RsCLE2, 19 and 41 or treatment of radish plants with synthetic CLE peptides revealed that CLE19 and CLE2 increase the number of xylem elements, and CLE41 induces the formation of extra cambium foci in secondary xylem. Expression levels of RsCLE2 and 19 strongly decrease in response to exogenous cytokinin, while auxin causes dramatic increase of RsCLE19 expression level and decrease of RsCLE41 expression. CONCLUSIONS: Our data allow us to hypothesize about the role of RsCLE2, 19 and 41 genes in the development of storage root of Raphanus sativus, e.g. RsCLE19 may play a role in auxin-dependent processes of xylem differentiation and RsCLE41 stimulates cambium activity.

The WOX Genes from the Intermediate Clade: Influence on the Somatic Embryogenesis in Medicago truncatula.

Transcription factors from the WOX family are well-known regulators of cell proliferation and differentiation in plants. Herein, we focused on several WOX genes from the intermediate clade and checked their impact on somatic embryogenesis using the model legume object Medicago truncatula. As a result, we show that MtWOX9-1 overexpression not only stimulates somatic embryogenesis in the embryogenic M. truncatula line, as it was shown previously, but can also induce somatic embryogenesis in the non-embryogenic line. Other intermediate clade WOX, including the close paralog of MtWOX9-1, as well as WOX11 homologs, did not have any significant impact on somatic embryogenesis in our in vitro cultivation system. Together, our results give new information about the diversity of the WOX family proteins and their specific functions. These data can be used for the search of new regeneration stimulators.

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.

MtCLE08, MtCLE16, and MtCLE18 Transcription Patterns and Their Possible Functions in the Embryogenic Calli of Medicago truncatula.

CLE peptides are well-known hormonal regulators of plant development, but their role in somatic embryogenesis remains undetermined. CLE genes are often regulated by WOX transcription factors and, in their turn, regulate the expression level of WOX genes. In this study, we used in vitro cultivation, as well as qPCR and transcriptomic analysis, to find CLE peptides which could regulate the MtWOX9-1 gene, stimulating somatic embryogenesis in Medicago truncatula. Three CLE peptides were found which could probably be such regulators, but none of them was found to influence MtWOX9-1 expression in the embryogenic calli. Nevertheless, overexpression of one of CLE genes under study, MtCLE16, decreased somatic embryogenesis intensity. Additionally, overexpression of MtCLE08 was found to suppress expression of MtWOX13a, a supposed antagonist of somatic embryo development. Our findings can be helpful for the search for new regeneration regulators which could be used for plant transformation.

MtWOX2 and MtWOX9-1 Effects on the Embryogenic Callus Transcriptome in Medicago truncatula.

WOX family transcription factors are well-known regulators of plant development, controlling cell proliferation and differentiation in diverse organs and tissues. Several WOX genes have been shown to participate in regeneration processes which take place in plant cell cultures in vitro, but the effects of most of them on tissue culture development have not been discovered yet. In this study, we evaluated the effects of MtWOX2 gene overexpression on the embryogenic callus development and transcriptomic state in Medicago truncatula. According to our results, overexpression of MtWOX2 leads to an increase in callus weight. Furthermore, transcriptomic changes in MtWOX2 overexpressing calli are, to a large extent, opposite to the changes caused by overexpression of MtWOX9-1, a somatic embryogenesis stimulator. These results add new information about the mechanisms of interaction between different WOX genes and can be useful for the search of new regeneration regulators.

Horizontal gene transfer from genus agrobacterium to the plant linaria in nature.

Genes can be transferred horizontally between prokaryotes and eukaryotes in nature. The best-studied examples occur between Agrobacterium rhizogenes and certain Nicotiana spp. To investigate possible additional cases of horizontal gene transfer in nature between Agrobacterium and plants, a real-time polymerase chain reaction-based approach was employed to screen 127 plant species, belonging to 38 families of Dicotyledones, for the presence of oncogenes homologous to the transfer DNA fragments (T-DNA) from both A. tumefaciens and A. rhizogenes. Among all of the analyzed plant species, we found that only Linaria vulgaris contained sequences homologous to the T-DNA of A. rhizogenes. All screened L. vulgaris plants from various parts of Russia contained the same homologous sequences, including rolB, rolC, ORF13, ORF14, and mis genes. The same opine gene is found in the species of Nicotiana which contain genes of A. rhizogenes. In L. vulgaris, there are two copies of T-DNA organized as a single tandem imperfect direct repeat. The plant DNA sequence of the site of integration shows similarity to a retrotransposon. This site is most likely silent, suggesting that the T-DNA is not expressed. Attempts to demonstrate expression of the T-DNA genes were negative. Our study indicates that the frequency of gene transfer and fixation in the germline from Agrobacterium to plant hosts is rare in the natural environment.

Selection Signatures in the First Exon of Paralogous Receptor Kinase Genes from the Sym2 Region of the Pisum sativum L. Genome.

During the initial step of the symbiosis between legumes (Fabaceae) and nitrogen-fixing bacteria (rhizobia), the bacterial signal molecule known as the Nod factor (nodulation factor) is recognized by plant LysM motif-containing receptor-like kinases (LysM-RLKs). The fifth chromosome of barrel medic (Medicago truncatula Gaertn.) contains a cluster of paralogous LysM-RLK genes, one of which is known to participate in symbiosis. In the syntenic region of the pea (Pisum sativum L.) genome, three genes have been identified: PsK1 and PsSym37, two symbiosis-related LysM-RLK genes with known sequences, and the unsequenced PsSym2 gene which presumably encodes a LysM-RLK and is associated with increased selectivity to certain Nod factors. In this work, we identified a new gene encoding a LysM-RLK, designated as PsLykX, within the Sym2 genomic region. We sequenced the first exons (corresponding to the protein receptor domain) of PsSym37, PsK1, and PsLykX from a large set of pea genotypes of diverse origin. The nucleotide diversity of these fragments was estimated and groups of haplotypes for each gene were revealed. Footprints of selection pressure were detected via comparative analyses of SNP distribution across the first exons of these genes and their homologs MtLYK2, MtLYK3, and MtLYK4 from M. truncatula retrieved from the Medicago Hapmap project. Despite the remarkable similarity among all the studied genes, they exhibited contrasting selection signatures, possibly pointing to diversification of their functions. Signatures of balancing selection were found in LysM1-encoding parts of PsSym37 and PsK1, suggesting that the diversity of these parts may be important for pea LysM-RLKs. The first exons of PsSym37 and PsK1 displayed signatures of purifying selection, as well as MtLYK2 of M. truncatula. Evidence of positive selection affecting primarily LysM domains was found in all three investigated M. truncatula genes, as well as in the pea gene PsLykX. The data suggested that PsLykX is a promising candidate for PsSym2, which has remained elusive for more than 30 years.

Horizontal gene transfer from Agrobacterium to plants.

Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.

Molecular markers of inbred radish (Raphanus sativus var. Radicola Pers.) lines.

An approach which could be used for quick searches for RAPD markers is described for groups of radish lines with certain morphological traits. The lines are characterized by various morpho-physiological abnormalities, including tumor formation (lines 12, 19, and 21) and non-terminal development of the flower meristem as a variant of tumor growth (line 6). We found four markers which differentiate tumor radish lines 12, 19, and 21 from the others, and two which differentiate line 6.