Anita Wiśniewska

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 heritable changes in metabolic profiles of plants regenerated in different types of in vitro culture

In this work we show how three types of cucumber in vitro cultures – leaf callus culture, cytokinin dependent cell suspension and liquid culture of meristematic clumps – influence the metabolite profiles of plants in the first generative progeny. Based on this study we conclude that there exists a specific and inheritable metabolic fingerprint reflecting the history of previous generations, probably related to specific stress factors accompanying the passage through different types of culture. The leaf callus culture generated the highest heritable differences in metabolite content and was the most distinctly separated cluster in PCA analysis. The smallest number of variable metabolites characterizes the plants regenerated from cytokinin dependent cell suspension whereas the liquid culture of meristematic clumps induced slightly more changes. Changes induced by these two culture types were not as pronounced as in the case of leaf callus culture. However the plants after these types of culture were well separated from the control on PCA diagram. The highest changes were over 2-fold increases in cystin and galactose-6-P and over 2-fold decreases in aspartate, myo-inositol, hydroxylamine, phosphate and putrescine. These changes concerned the plants, which were one generation after the leaf callus culture. The possible nature of observed heritable changes is discussed.

Assessment of the physiological responses to drought in different sugar beet genotypes in connection with their genetic distance.

Drought affects many physiological processes, which influences plant productivity. The aim of this study was to evaluate the degree of genotypic diversity in drought tolerance of sugar beet genotypes (Beta vulgaris L.) in connection with their genetic distance. Three hybrid genotypes produced by crossing double haploid genotype (P-pollinator) with cytoplasmic male-sterile female part (MS), as well as with two parent lines, were examined. Drought conditions were imposed by the cessation of watering at the 3-4 leaf stage for about three months, after which irrigation was resumed. Control plants were optimally irrigated throughout the entire vegetation period. Long-term drought significantly increased the wilting of leaves (Wilt.), specific leaf weight (SLW), the succulence index (Suc.I), leaf senescence and membrane damage (El-l). Simultaneously, the osmotic potential (ψs), leaf area index (LAI), absorption of photosynthetic active radiation (PAR) and the efficiency of the photosynthetic apparatus (Φ PSII) declined under water deficit conditions. The examined genotypes demonstrated a clear diversity in their physiological response to drought. Based on these findings, we suggest that traits that are strongly correlated with root and sugar yield, e.g. Φ PSII, LAI, PAR absorption and ψs, could be used as potential selection criteria in physiological-associated breeding strategies to improve drought tolerance in sugar beet. There was not a significant correlation between the genetic distance separating different sugar beet genotypes and the observed heterotic effect of root or sugar yields, with the exception of heterosis of root yield under optimal conditions, where the correlation was negative.

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

Somaclonal variation commonly occurs duringin 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.

Molecular characterization of SCARECROW (CsSCR) gene expressed during somatic embryo development and in root of cucumber (Cucumis sativus L.)

Somatic embryogenesis (SE) in plants can be used as a model for studying genes engaged in the embryogenic transition of somatic cells. The CsSCARECROW (CsSCR) gene was previously identified among a panel of genes upregulated after the induction of SE in cucumber (Cucumis sativus). The putative CsSCR protein contains conserved GRAS family domains and is extremely similar to AtSCR from Arabidopsis thaliana. SCR proteins are transcription factors involved in root radial patterning and are required for maintenance of the quiescent centre and differentiation of the endodermis. In comparison with other GRAS proteins from cucumber, phylogenetic analyses showed that CsSCR belongs to the SCR cluster. Increased CsSCR transcript accumulation was detected in somatic embryos and roots. Southern blot analysis and screening of the draft version of the cucumber genome confirmed the lack of close homologues in this species. CsSCR transcripts were localized by in situ hybridization in undifferentiated cells in the globular and heart stages of somatic embryogenesis, and in the endodermis of torpedo and cotyledonary stage somatic embryos, and developing primary and lateral roots. This localization was supported by the pattern of reporter gene activity driven by the CsSCR promoter in transgenic cucumber organs. These results suggest that CsSCR is likely to act in tissue radial organization during somatic embryogenesis and root development.

Analysis of tomato gene promoters activated in syncytia induced in tomato and potato hairy roots by Globodera rostochiensis

The potato cyst nematode (Globodera rostochiensis) induces feeding sites (syncytia) in tomato and potato roots. In a previous study, 135 tomato genes up-regulated during G. rostochiensis migration and syncytium development were identified. Five genes (CYP97A29, DFR, FLS, NIK and PMEI) were chosen for further study to examine their roles in plant–nematode interactions. The promoters of these genes were isolated and potential cis regulatory elements in their sequences were characterized using bioinformatics tools. Promoter fusions with the β-glucuronidase gene were constructed and introduced into tomato and potato genomes via transformation with Agrobacterium rhizogenes to produce hairy roots. The analysed promoters displayed different activity patterns in nematode-infected and uninfected transgenic hairy roots.

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGBs role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia.

Developmental expression of the cucumber Cs-XTH1 and Cs-XTH3 genes, encoding xyloglucan endotransglucosylase/hydrolases, can be influenced by mechanical stimuli

The expression of two genes encoding xyloglucan endotransglucosylase/hydrolases (XTHs), Cs-XTH1 and Cs-XTH3, was upregulated during the onset of cucumber somatic embryogenesis. As a means of characterising the developmental regulation of these genes, the activity of the respective upstream regulatory regions was investigated in seedlings and somatic embryos of Arabidopsis thaliana and Cucumis sativus. GUS assays revealed that both genes are under developmental control. In addition, elevated promoter activity was found in the tension-bearing regions of the plant and in response to touch and wounding, which is consistent with the existence of numerous stress-related cis elements in the 5′-regulatory regions. In vivo xyloglucan endotransglucosylase (XET) action assays were performed to gain an overview on the role of XTHs during somatic embryogenesis. The highest XET action was observed in the external cell layers of somatic embryos in the cotyledonary region and in the presumptive region of peg formation. Based on the results, we propose a dual mechanism (one developmental and the second adaptive) for the regulation of Cs-XTH1 and Cs-XTH3 activity wherein the developmental pattern can be modified by mechanical stimuli.