Persistence and Protection of Mitochondrial DNA in the Generative Cell of Cucumber is Consistent with its Paternal Transmission.

Plants predominantly show maternal transmission of mitochondrial DNA (mtDNA). One known exception is cucumber, in which the mtDNA is paternally inherited. However, the mechanisms regulating this unique mode of transmission are unclear. Here we monitored the amounts of mtDNA throughout the development of cucumber microspores into pollen and observed that mtDNA decreases in the vegetative cell, but persists in the generative cell that ultimately produces the sperm cells. We characterized the cucumber homolog (CsDPD1) of the Arabidopsis gene defective in pollen organelle DNA degradation 1 (AtDPD1), which plays a direct role in mtDNA degradation. CsDPD1 rescued an Arabidopsis AtDPD1 mutant, indicating the same function in both plants. Expression of CsDPD1 coincided with the decrease of mtDNA in pollen, except in the generative cell where both the expression of CsDPD1 and mtDNA levels remained high. Our cytological results confirmed that the persistence of mtDNA in the cucumber generative cell is consistent with its paternal transmission. Our molecular analyses suggest that protection of mtDNA in the generative cell may be the critical factor for paternal mtDNA transmission, rather than mtDNA degradation mediated by CsDPD1. Taken together, these findings indicate that a mechanism may protect paternal mtDNA from degradation and is likely to be the genetic basis of paternal mtDNA transmission.

Molecular Cytogenetic Analysis of Cucumis Wild Species Distributed in Southern Africa: Physical Mapping of 5S and 45S rDNA with DAPI.

Wild Cucumis species have been divided into Australian/Asian and African groups using morphological and phylogenetic characteristics, and new species have been described recently. No molecular cytogenetic information is available for most of these species. The crossability between 5 southern African Cucumis species (C. africanus, C. anguria, C. myriocarpus, C. zeyheri, and C. heptadactylus) has been reported; however, the evolutionary relationship among them is still unclear. Here, a molecular cytogenetic analysis using FISH with 5S and 45 S ribosomal DNA (rDNA) was used to investigate these Cucumis species based on sets of rDNA-bearing chromosomes (rch) types I, II and III. The molecular cytogenetic and phylogenetic results suggested that at least 2 steps of chromosomal rearrangements may have occurred during the evolution of tetraploid C. heptadactylus. In step 1, an additional 45 S rDNA site was observed in the chromosome (type III). In particular, C. myriocarpus had a variety of rch sets. Our results suggest that chromosomal rearrangements may have occurred in the 45 S rDNA sites. We propose that polyploid evolution occurred in step 2. This study provides insights into the chromosomal characteristics of African Cucumis species and contributes to the understanding of chromosomal evolution in this genus.

Karyotype analysis and chromosomal distribution of repetitive DNA sequences of cucumis metuliferus using fluorescence in situ hybridization.

Cucumis metuliferus (2n = 24) is a cultivated species of the Cucumis genus which is a potential genetic resource for Cucumis crops. Although some cytogenetic research has been reported, there is no study of karyotyping in this species. Here, we used 4',6-diamidino-2-phenylindole and chromomycin A3 staining to identify 12 pairs of chromosomes in early-metaphase cells. Fluorescence in situ hybridization revealed the chromosomal distribution patterns of the 5S and 45S ribosomal DNA (rDNA) genes, telomeres, and 3 different satellite repeats. The 2 major signals of the 45S rDNA were located on the satellite of chromosome 11, and the 2 signals of the 5S rDNA and 2 minor signals of the 45S rDNA were located on chromosome 12. The telomere probes hybridized to the ends of all chromosomes. The 3 satellite DNAs were localized at the ends of chromosomes 1, 2, 4-10, and at the end of the short arm of chromosome 3. In summary, we reported the identification of all chromosomes of C. metuliferus. We also depicted the location of 5S and 45S rDNA, the telomere motif sequence, CmetSat1, CmetSatT2, and CmetmSat1 in an ideogram.

Transgenic crops: the present state and new ways of genetic modification.

Transgenic crops were first commercialised almost 20 years ago, which makes it a good opportunity to reflect on this technology. In this review, we compare its status with the predictions included in Vasil's forecast published in 2002. Our analysis shows that science has provided a wide range of possibilities to modify different traits in plants, yet the economy benefits from that range to very different extents. We also point out the most important constituents of the technology development involving methodology improvement and novel traits expressed in varieties introduced into agriculture. Using native genes (or their elements) in transgenes, accumulating previously produced transgenes to cascade resistance and using herbicide resistance as a selectable marker have been considered typical of novel genetically modified (GM) plant varieties. A vast portion of the novelties in stacked varieties is doubtful in terms of EU regulations. Attention has also been directed to completely novel methodology solutions that hold out the prospect of a more comprehensive use of genetic modification in agriculture as a whole, and, particularly, make its use possible in the EU and even in sustainable agriculture.

Identification of genes up-regulated during somatic embryogenesis of cucumber.

Somatic embryogenesis is a method of plant regeneration, but it can also be used as a model to study plant development. A normalized library of cDNA fragments representing genes up-regulated after the induction of somatic embryogenesis in cucumber suspension cultures was constructed using the suppression subtractive hybridization technique. Candidate cDNA fragments (119) were classified according to their similarity to genes encoding known proteins and the presence of potential functional domains. Of the translation products with homology to known proteins, about 23% were possibly involved in metabolism, 13% represented proteins with a probable role in cellular communication and signal transduction, about 12% were likely to participate in protein synthesis, while around 10% were potential transcription factors. The genes corresponding to four of the cDNAs were subsequently analyzed in more detail: CsSEF2, CsSEM1 and CsSESTK1 encoding putative transcription factors or co-activators, and CsSECAD1 encoding cinnamyl alcohol dehydrogenase. Full-length cDNAs were isolated and analyzed. RT-PCR confirmed the up-regulation of these genes after the induction of somatic embryogenesis and showed the presence of their transcripts in other tissues. The in situ localization of transcripts of the CsSEF2 and CsSEM1 genes demonstrated that signalling in somatic embryo tissues involving these factors is concentrated in the cotyledon primordia and roots.

The genome sequence of the North-European cucumber (Cucumis sativus L.) unravels evolutionary adaptation mechanisms in plants.

Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas and secondary domestication regions includes highly divergent climate conditions e.g. temperate and subtropical. We wanted to uncover adaptive genome differences between the cucumber cultivars and what sort of evolutionary molecular mechanisms regulate genetic adaptation of plants to different ecosystems and organism biodiversity. Here we present the draft genome sequence of the Cucumis sativus genome of the North-European Borszczagowski cultivar (line B10) and comparative genomics studies with the known genomes of: C. sativus (Chinese cultivar--Chinese Long (line 9930)), Arabidopsis thaliana, Populus trichocarpa and Oryza sativa. Cucumber genomes show extensive chromosomal rearrangements, distinct differences in quantity of the particular genes (e.g. involved in photosynthesis, respiration, sugar metabolism, chlorophyll degradation, regulation of gene expression, photooxidative stress tolerance, higher non-optimal temperatures tolerance and ammonium ion assimilation) as well as in distributions of abscisic acid-, dehydration- and ethylene-responsive cis-regulatory elements (CREs) in promoters of orthologous group of genes, which lead to the specific adaptation features. Abscisic acid treatment of non-acclimated Arabidopsis and C. sativus seedlings induced moderate freezing tolerance in Arabidopsis but not in C. sativus. This experiment together with analysis of abscisic acid-specific CRE distributions give a clue why C. sativus is much more susceptible to moderate freezing stresses than A. thaliana. Comparative analysis of all the five genomes showed that, each species and/or cultivars has a specific profile of CRE content in promoters of orthologous genes. Our results constitute the substantial and original resource for the basic and applied research on environmental adaptations of plants, which could facilitate creation of new crops with improved growth and yield in divergent conditions.

Characterization of CsSEF1 gene encoding putative CCCH-type zinc finger protein expressed during cucumber somatic embryogenesis.

Somatic embryos obtained in vitro are a form of vegetative reproduction that can be used in artificial seed technology, as well as a model to study the principles of plant development. In order to isolate the genes involved in somatic embryogenesis of the cucumber (Cucumis sativus L.), we utilized the suppression subtractive hybridization (SSH). One of the obtained sequences was the CsSEF1 clone (Cucumis sativus Somatic Embryogenesis Zinc Finger 1), with a level of expression that sharply increased with the induction of embryogenesis. The full length cDNA of CsSEF1 encodes the putative 307 amino acid long protein containing three zinc finger motifs, two with CCCH and one with the atypical CHCH pattern. The CsSEF1 protein shows significant similarity to other proteins from plants, in which the zinc fingers arrangement and patterns are very similar. Transcripts of CsSEF1 were localized in the apical part of somatic embryos, starting as early as the polarity was visible and in later developmental stages marking the cotyledon primordia and procambium tissues. As a result of transferring an antisense fragment of CsSEF1 into Arabidopsis thaliana abnormalities in zygotic embryos and also in cotyledons and root development were observed.

The complete structure of the cucumber (Cucumis sativus L.) chloroplast genome: its composition and comparative analysis.

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.

The selection of mosaic (MSC) phenotype after passage of cucumber (Cucumis sativus L.) through cell culture - a method to obtain plant mitochondrial mutants.

Mosaic (MSC) mutants of cucumber (Cucumis sativus L.) appear after passage through cell cultures. The MSC phenotype shows paternal transmission and is associated with mitochondrial DNA rearrangements. This review describes the origins and phenotypes of independently produced MSC mutants of cucumber, including current knowledge on their mitochondrial DNA rearrangements, and similarities of MSC with other plant mitochondrial mutants. Finally we propose that passage of cucumber through cell culture can be used as a unique and efficient method to generate mitochondrial mutants of a higher plant in a highly homozygous nuclear background.

Unintended consequences of plant transformation: a molecular insight.

Plant genomes are dynamic structures having both the system to maintain and accurately reproduce the information encoded therein and the ability to accept more or less random changes, which is one of the foundations of evolution. Crop improvement and various uncontrolled stress factors can induce unintended genetic and epigenetic variations. In this review it is attempted to summarize factors causing such changes and the molecular nature of these variations in transgenic plants. Unintended effects in transgenic plants can be divided into three main groups: first, pleiotropic effects of integrated DNA on the host plant genome; second, the influence of the integration site and transgene architecture on transgene expression level and stability; and third, the effect of various stresses related to tissue handling, regeneration and clonal propagation. Many of these factors are recently being redefined due to new researches, which apply modern highly sensitive analytical techniques and sequenced model organisms. The ability to inspect large portions of genomes clearly shows that tissue culture contributes to a vast majority of observed genetic and epigenetic changes. Nevertheless, monitoring of thousands transcripts, proteins and metabolites reveals that unintended variation most often falls in the range of natural differences between landraces or varieties. We expect that an increasing amount of evidence on many important crop species will support these observations in the nearest future.

Cucumber (Cucumis sativus L.).

We describe two novel Agrobacterium tumefaciens-based methods of cucumber transformation. The first involves direct regeneration from leaf microexplants selected on kanamycin-containing medium. The second involves regeneration from a long-term established embryogenic suspension culture emitting green autofluorescence (GAF) and selection on medium containing hygromycin. In the latter method, GAF was used as a reporter, thereby allowing a simple and reliable identification of transgenic cells with a high regeneration capacity. (No false positives were observed.) The transformation efficiency in the leaf microexplants fluctuated from 0.8 to 6.5% of the primary explants, whereas in the embryogenic suspension-cultured cells it varied from 6.4 to 17.9% of the aggregates. In the GAF method, the step involving the elimination of the Agrobacterium cells by antibiotics could be omitted; however, this reduced the transformation efficiency to about 3%. The time required from inoculation to regenerated plant in the greenhouse was the same for both methods, but the GAF method required more preinoculation time than the leaf microexplant method.

The DefH9-iaaM-containing construct efficiently induces parthenocarpy in cucumber.

Parthenocarpy (seedless fruits) is a desirable trait that has been achieved in many plant cultivars. We generated parthenocarpic cucumber fruits by introducing the chimeric DefH9-iaaM construct into the cucumber genome using an Agrobacterium tumefaciens-mediated protocol. The construct consists of the DefH9 promoter from Antirrhinum majus and the iaaM coding sequence from Pseudomonas syringae. Transgenic plants were obtained from nine independent transformation events: half of these were tetraploid and did not produce seeds following self-pollination, while the remaining half were capable of displaying parthenocarpy in the subsequent reproductive generation. Of the fruits produced by the transgenic lines, 70-90% were parthenocarpic. The segregation of the marker gene in the transgenic T(1) progeny indicated single gene inheritance. The seed set in the transgenic lines and their F(1) hybrids was lower than in the non-transgenic control plants. Some of the methodological details and the practical significance of the results are discussed.

Tissue-culture-responsive and autotetraploidy-responsive changes in metabolic profiles of cucumber (Cucumis sativus L.).

Somaclonal variation commonly occurs during in vitro plant regeneration and may introduce unintended changes in numerous plant characters. In order to assess the range of tissue-culture-responsive changes on the biochemical level, the metabolic profiles of diploid and tetraploid cucumber R1 plants regenerated from leaf-derived callus were determined. Gas chromatography and mass spectrometry were used for monitoring of 48 metabolites and many significant changes were found in metabolic profiles of these plants as compared to a seed-derived control. Most of the changes were common to diploids and tetraploids and were effects of tissue culture. However, tetraploids showed quantitative changes in 14 metabolites, as compared to regenerated diploids. These changes include increases in serine, glucose-6P, fructose-6P, oleic acid and shikimic acid levels. Basing on this study we conclude that the variation in metabolic profiles does not correlate directly with the range of genome changes in tetraploids.

The metabolic profiles of transgenic cucumber lines vary with different chromosomal locations of the transgene.

The metabolic profiles of five transgenic cucumber lines were compared taking into consideration their transgene integration sites. The plants analyzed were homozygous and contained transgenes integrated in a single locus on chromosomes I, II, III or IV. The transgenes were preferentially located in the euchromatic regions. Each of these locations possessed a specific metabolic profile. The number of altered compounds in the transgenic lines varied between 9 and 23 of the 47 metabolites identified. These alterations seem to be specific for each independent transgene integration. However, some changes are common: a decrease in the levels of phenylalanine, aspartate, ethanolamine and pipecolate, and an increase in the level of benzoic acid. The observed effects of transgene introduction are discussed in this paper.

Differences in the inheritance stability of kanamycin resistance between transgenic cucumbers (Cucumis sativus L.) containing two constructs.

A phenotypic segregation of kanamycin resistance conferred by nosnptII was investigated in two groups of transgenic cucumber lines one containing the PR-2duidA-nosnptII construct and the other with the thaumatinII-nosnptII construct to the third and fifth generation, respectively. Expression of the nptII gene was more stable and predictable in the progeny of the PR-2d lines than that of thaumatin lines. In T2 and T3 generations representing seven PR-2d primary transformants, 78% of the hemizygous progenies exhibited a segregation ratio consistent with Mendelian inheritance; non-Mendelian ratio occurred in the remaining 22%. Mendelian segregation was observed in 46% of the hemizygous progenies derived from 11 thaumatin primary transformants. The segregation ratio for two and three independent loci appeared in single PR-2d and thaumatin lines, respectively.

Tobacco PR-2d promoter is induced in transgenic cucumber in response to biotic and abiotic stimuli.

The PR-2d promoter/uidA (GUS) gene construct was introduced into the cucumber (Cucumis sativus L.) genome and several transgenic lines were produced. Activation of the PR-2d promoter was investigated in these plants in response to inoculation with fungal pathogens and after salicylic acid (SA) or cold treatments. Treatment with exogenous SA increased GUS activity 2 to 11 fold over that of the control. Endogenous SA and its conjugate salicylic acid glucoside (SAG) rose in parallel after inoculation with the fungal pathogen Pseudoperonospora cubensis, with SAG becoming the predominant form. The free SA levels increased 15 fold above the basal level at 5 dpi and preceded the induction of the PR-2d promoter by five days, which occurred at 10 dpi with a 12 fold increase over the control. Inoculation with another fungal pathogen, Erysiphe polyphage, increased GUS activity 4 to 44 fold over that of the control. During normal development of flowers in the cucumber, the PR-2d/uidA gene expressed in the floral organs was similar to that of the primary host. In addition, we present the first evidence that the PR-2d promoter was induced (624 fold) under cold stress. We demonstrate that in the heterologous state the gene construct was expressed according to the signalling pattern of the native species and was stably transmitted to progeny over four generations.

Transgene inheritance in plants.

The patterns of transgene inheritance in plants and the possible explanations for non-Mendelian transmission are reviewed. The non-Mendelian inheritance of a transgene has been recorded with a frequency between 10% and 50% in transgenic plants produced either by Agrobacterium-mediated transformation or through particle bombardment. Different effects such as deletion, duplication, rearrangement, repeated sequence recombination as well as gene interaction have been observed for transgenic loci. The nature of the recipient genome, nature of the transgene and the interactions between them seem to contribute to the non-Mendelian segregation of transgenes.

Xyloglucan endotransglucosylase/hydrolase genes in cucumber (Cucumis sativus) - differential expression during somatic embryogenesis.

Defined changes in the cell wall directed by many proteins accompany every morphogenetic process in plants. Xyloglucan endotransglucosylase/hydrolase proteins (XTH; EC 2.4.1.207) have the potential to modify the hemicellulose matrix within the cell wall. Cs-XTH1 and Cs-XTH3 genes, which encode XTH proteins, were found among numerous genes that are differentially expressed after the induction of cucumber somatic embryogenesis. The expression of these genes increased during somatic embryogenesis. The Cs-XTH1 gene was localized on the second chromosome near the centromere region, whereas Cs-XTH3 was found in the middle of the fifth chromosome's longer arm. Northern blot hybridization showed that both genes were preferentially expressed in roots. We also observed higher accumulation of both transcripts in somatic embryos than in the proembryogenic mass. The localization of mRNA by in situ hybridization revealed that the Cs-XTH1 transcripts were largely accumulated in the presumptive cotyledon primordia of somatic embryos. The XTH gene family consists of a number of genes with a high degree of structural similarity. Screening a cucumber genomic library has identified other members of this gene family. The intron/exon structure, sequence similarities and the close chromosomal distance between some members suggest their common evolutionary origin. The involvement of XTH-related genes in somatic embryo formation is discussed.

The cloning of sequences differentially transcribed during the induction of somatic embryogenesis in cucumber (Cucumis sativus L.).

Somatic embryogenesis in cucumber cell suspension culture is a convenient tool to study differential gene expression, particularly during the early stages of this process. In this study, we used the cucumber somatic embryogenesis system to detect genes that were differentially transcribed during the induction of embryo development. We identified and cloned 120 candidate cDNA fragments from differential display gels. The selected cDNAs were confirmed by reverse northern, and 83 were sequenced. The obtained sequences represent 64 independent transcripts. The search for similarities in the databases gave a significant result in 16 cases. The potential involvement of these sequences in somatic embryogenesis is discussed.

Transcriptional expression of a Solanum sogarandium pGT::Dhn10 gene fusion in cucumber, and its correlation with chilling tolerance in transgenic seedlings.

The expression pattern of a Solanum sogarandinum pGT::Dhn10 gene fusion encoding a dehydrin DHN10 protein and the potential role of that protein in cold tolerance in cucumber were analysed in three T1transgenic lines. An accumulation of Dhn10 mRNA was detected in the leaves, cotyledons, hypocotyls and roots of the transgenic seedlings both under the control conditions and after a cold treatment at 6 degrees C for 24 h. This was confirmed by RT-PCR. However, no DHN10 protein was detected by the alkaline phosphatase-conjugated antibody. The transgenic lines exhibited different levels of chilling tolerance. The TCC5/1 line showed a significant increase in its chilling tolerance compared to the non-transgenic line. No chilling injury was observed when the cold hardened (6 degrees C, 24 h) TCC5/1 plants were subsequently exposed to a temperature of 2 degrees C for 6 h. The other two transgenic lines, TCC2/1 and TCC3/2, exhibited a comparable level of chilling tolerance to that of the non-transgenic control. The transgenic lines showed similar or significantly decreased freezing tolerance compared to the non-transgenic control, as evaluated by an electrolyte leakage test. We concluded that the S. sogarandnium GT promoter is functional in the chilling sensitive species Cucumis sativus L., and that the pGT::Dhn10 gene fusion is expressed at the transcriptional level.