Organelle DNA variation in parental<Emphasis Type="Italic"> Solanum</Emphasis> spp. genotypes and nuclear-cytoplasmic interactions in<Emphasis Type="Italic"> Solanum tuberosum</Emphasis> (+)<Emphasis Type="Italic"> S. commersonii</Emphasis> somatic hybrid-backcross progeny

Nuclear-cytoplasmic interactions can influence fertility and agronomic performance of interspecific hybrids in potato as well as other species. With the aim of assessing the potential value of a novel recombinant cytoplasm derived by interspecific somatic hybridization, backcross progeny were produced by crossing a somatic hybrid between Solanum tuberosum (tbr) and the wild incongruous species S. commersonii (cmm) with various potato clones. BC₁ clones were evaluated for male fertility and other agronomic traits. Male fertility clearly depended on the cross direction and the cytoplasm source. Genotypes with cytoplasms sensitive to nuclear genes derived from Solanum commersonii and inducing male sterility showed identical mtDNA composition, as based on mtDNA analyses with various PCR-based and RFLP markers. On the other hand, genotypes with cytoplasms not inducing male sterility in the presence of the cmm nuclear genes showed a different mtDNA organisation. Analysis of cpDNA confirmed similarity of cytoplasmic composition in CMS-inducing genotypes and clear differences with the others. Genotypes with recombinant cytoplasm induced by somatic hybridization generally showed similar agronomic performances in reciprocal hybrids with tbr cytoplasm, suggesting that the novel cytoplasm can be used in potato breeding.Contribution no. 24 from the Institute of Plant Genetics, Research Division of Portici     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

Overexpression of the <Emphasis Type="Italic">plg</Emphasis>1 gene encoding pectin lyase in <Emphasis Type="Italic">Penicillium griseoroseum</Emphasis>

The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml⁻¹ respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.     

Regeneration of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.) from root explants

Summary Eggplant (Solanum melongena L.) was efficiently regenerated from cultured roots of 15-d-old seedlings on Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron and 13.3 μM 6-benzyladenine. Within 28d of culture initiation, induction of organogenic calluses and subsequent differentiation into shoot buds were observed. Shoot buds upon subculture to MS basal medium elongated into healthy shoots. Excised shoots (2–4 cm) were rooted on Soilrite^? irrigated with water either in vitro or in vivo. Plants with well-developed root systems were established under field conditions after hardening in the glasshouse, where they developed into flowering plants and produced mature fruits with viable seeds.     

Interspecific somatic hybrids <Emphasis Type="Italic">Solanum bulbocastanum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis> H-8105

Interspecific somatic hybrids between a dihaploid potato clone H-8105 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuberizing species Solanum bulbocastanum were generated and analysed. Only ten regenerants displaying the intermediate morphology with dominating characteristics of the wild parent (simple leaves, anthocyanin pigmentation) were produced in 15 weeks after a single PEG-mediated fusion event. The RAPD patterns confirmed the hybridity of all of them. The hybrids rooted poorly and grew slowly in vitro. The cytological analysis revealed a high degree of aneuploidy in the hybrids with morphological and growth anomalies in vitro, while the morphologically normal hybrids were tetraploids. All the S. bulbocastanum (+) H-8105 hybrids were unstable in culture and three of them were consequently lost during three years of propagation in vitro. The possible reasons for instability of somatic hybrids between the distantly related species are discussed.     

Activity of an atypical <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> pectin methylesterase

An Arabidopsis thaliana pectin methylesterase that was not predicted to contain any signaling sequence was produced in E. coli and purified using a His tag added at its N-terminus. The enzyme demethylesterified Citrus pectin with a Km of 0.86 mg/ml. The enzyme did not require salt for activity and was found to be relatively temperature-sensitive. The precipitation of enzyme-treated pectin by CaCl2 suggested that the enzyme had a blockwise mode of pectin demethylesterification. A purified kiwi (Actinidia chinensis) pectin methylesterase inhibitor had no effect on the activity of the enzyme whereas it strongly inhibited a flax pectin methylesterase. A model of the protein structure revealed that an extra amino acid sequence in this particular Arabidopsis pectin methylesterase could form a ss-strand outside the core structure, which might be preventing the inhibitor from binding the protein.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.     

Toxic effect of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether on growth of soil isolate <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> T1/1

The toxic and growth inhibiting effects of methyl tert-butyl ether (MTBE) on the hydrocarbon-degrading Pseudomonas veronii T1/1 strain (isolated from gasoline contaminated soil) were studied. In our experiments, the MIC of MTBE was found to be 60 mM and the EC₅₀ was 51.7 mM. In the concentration range 0–30 mM, MTBE did not significantly influence the growth parameters of this bacterium, but at concentrations over 30 mM MTBE exerted a significant growth inhibiting effect. In the presence of 70 mM MTBE, the specific growth rate dropped from 0.4731 to 0.1201 h⁻¹, while the length of the lag period increased from 5.41 to 17.01 h and the yield coefficient declined from 0.2652 to 0.0718 g g⁻¹. MTBE at 100 mM inhibited the growth of this strain completely. These findings may have important environmental implications, as high concentrations of MTBE could influence the efficiency of soil and groundwater bioremediation processes significantly.     

Growth,photosynthesis, and ion distribution in hydroponically cultured <Emphasis Type="Italic">Populus alba</Emphasis> L. cuttings grown under various salinity concentrations

Growth, photosynthesis, and Na⁺, K⁺, and Ca²⁺ distributions were investigated in 2-year-old hydroponically cultured Populus alba L. cuttings exposed to salt stresses (0, 0.85, 8.5, 17, and 85 mM NaCl in experiment 1 and 0, 50, 100, 150, and 200 mM in experiment 2) for 4 weeks in 2/5 Hoagland solution. Salt did not markedly inhibit height growth and diameter increment in 150 and 100 mM NaCl, respectively. The 85 mM NaCl treatment increased the dry weights of roots and total dry weight of plants, while 150 mM NaCl significantly reduced the dry weights of leaves, stems, and total plant weight. The decline in the photosynthetic rate lagged 2 weeks behind that of stomatal conductance in the 50 and 100 mM salt solutions. Different ions exhibited different distributions in different parts of the plant. Most Na⁺ ions were excluded and/or compartmentalized in roots at low and moderate salt stress (≤50 mM). K⁺ content in leaves increased with the increase in the salt concentration in the growth solutions.     

Lovastatin inhibits its own synthesis in<Emphasis Type="Italic"> Aspergillus terreus</Emphasis>

Lovastatin suppresses its own synthesis in the microfungus Aspergillus terreus. The inhibitory effect was documented by spiking identical batch cultures with pure lovastatin (0, 50, 100 and 250 mg/l) 24 h after initiation from spores.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at     

Interspecific potato somatic hybrids between <Emphasis Type="Italic">Solanum berthaultii</Emphasis> and <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. showed recombinant plastome and improved tolerance to salinity

In this study three somatic hybrid lines originating from protoplast fusion between Solanum tuberosum cv. BF15 and Solanum berthaultii were subjected to a detailed molecular analysis using the I-SSR-PCR technique based on 5′-anchored microsatellite primers. The data obtained revealed a polymorphism between the different lines, suggesting that they correspond to symmetric hybrids. The analysis of chloroplast genome of these hybrids showed that they are resulting from a recombination between parental plastomes. When transferred to a greenhouse, these hybrid lines displayed an improved vigour compared to the cultivated potato BF15 parent. Indeed, an important growth rate and high tuber yield and weight were obtained for these hybrids compared to the parent. Some of these hybrids showed also an improved ion homeostasis control and they seem to display a better tolerance to salt stress compared to the potato BF15 parent.     

Inhibition of <Emphasis Type="Italic">Zizania latifolia</Emphasis> growth by <Emphasis Type="Italic">Phragmites australis</Emphasis>: an experimental study

The influence of Phragmites australis on the growth of Zizania latifolia, and the morphological characteristics of both species, were investigated at two nutrient levels. The experiment was carried out over three growing seasons between May 2003 and December 2005. The experimental plot was longitudinally divided into two equal halves, one of which was planted with Zizania latifolia (Zizania zone) and the other one with Phragmites australis (Phragmites zone). Four weeks after transplantation, the plot was again divided horizontally, perpendicular to the previous division, and subject to low (LN) and high nutrient (HN) treatments. Measured growth indices of the two species were more developed in the HN than in the LN treatments. Both species were observed to invade each other’s zone, but Phragmites latifolia seemed to out-compete Zizania latifolia individuals. This was evident from the decrease in below ground biomass of Zizania latifolia in the third growing season and deterioration in above ground organs with time. It was concluded that Phragmites australis out-competes Zizania latifolia due to better developed root and rhizome system.     

Molecular cloning and characterization of nitrate reductase from<Emphasis Type="Italic"> Ricinus communis</Emphasis> L. heterologously expressed in<Emphasis Type="Italic"> Pichia pastoris</Emphasis>

In a previous paper, we showed that nitrate reductase (NR; EC 1.6.6.1) from leaves of Ricinus communis L. differed from most other higher-plant NRs by an unusually strong Mg2+-sensitivity, a different pH-activity profile and only little ATP-dependent inactivation [A. Kandlbinder et al. (2000) J Exp Bot 51:1099-1105]. In order to elucidate these deviating properties in more detail, the NR gene from R. communis was cloned, expressed heterologously and characterized. The deduced protein sequence showed that Ricinus NR has a serine phosphorylation site and a 14-3-3 binding motif, a common characteristic of NRs. Functional Ricinus NR protein was expressed in the yeast Pichia pastoris and compared with the features of Arabidopsis thaliana NR2 synthesized by the same expression system (AtNR2). The recombinant Ricinus NR (RcNR) itself was not inactivated by incubation with MgATP. As yeast extracts might lack factors required for NR regulation, desalted leaf extracts containing NR kinases and 14-3-3 proteins were prepared from 4-day-darkened (and therefore NR-free) leaves of Ricinus, and added to the assay of RcNR to check for ATP-dependent inactivation and Mg2+-sensitivity. When RcNR was combined with the NR-free extracts described above, its unusually high Mg2+-sensitivity was restored, but it remained unresponsive to ATP. In contrast, AtNR2 became inactive when incubated with the protein mixture and ATP. Thus, insensitivity to ATP appears to be an inherent property of Ricinus NR, whereas the high Mg2+-sensitivity depends on one or several factors in Ricinus leaves. This as yet unknown factor(s) was boiling-sensitive and appeared to interact specifically with recombinant Ricinus NR to provide the Mg2+-sensitivity of the authentic leaf enzyme.     

Use of gain-of-function study to delineate the roles of<Emphasis Type="Italic">crumbs</Emphasis> in<Emphasis Type="Italic">Drosophila</Emphasis> eye development

The cell polarity gene,crumbs (crb), has been shown to participate in the development and degeneration of theDrosophila retina. Mutations inCRB1, the human homologue ofDrosophila crb, also result in retinitis pigmentosa and Leber congential amaurosis. In this study, we used the gain-of-function approach to delineate the roles ofcrb in developingDrosophila eye. In the third-instar larval stage, eye development is initiated with photoreceptor differentiation and positioning of photoreceptor nuclei in the apical cellular compartment of retinal epithelium. In the pupal stage, differentiated photoreceptors begin to form the photosensitive structures, the rhabdomeres, at their apical surface. UsingGMR-Gal4 to drive overexpression of the Crb protein at the third-instar eye disc, we found that differentiation of photoreceptors was disrupted and the nuclei of differentiated photoreceptors failed to occupy the apical compartment. Usinghs-Gal4 to drive Crb overexpression in pupal eyes resulted in interference with extension of the adherens junctions and construction of the rhabdomeres, and these defects were stage-dependent. This gain-of-function study has enabled us to delineate the roles of Crb at selective stages of eye development inDrosophila.     

Wax and suberin development of native and wound periderm of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) and its relation to peridermal transpiration

Native and wound periderm was isolated enzymatically from potato (Solanum tuberosum L. cv. Desirée) tubers at different time intervals between 0 days up to 4 weeks after harvesting. Wound periderm formation was induced by carefully removing native periderm from freshly harvested tubers before storage. The chemical composition of lipids (waxes) obtained by chloroform extraction, as well as the monomeric composition of native and wound suberin polymer after transesterification by boron trifluoride/methanol, was analyzed using gas chromatography and mass spectrometry. Both types of periderm contained up to 20% extractable lipids. Besides linear long-chain aliphatic wax compounds, alkyl ferulates were detected as significant constituents. In wound periderm they amounted to more than 60% of the total extracts. Within 1 month of storage, suberin amounts in the polymer increased 2-fold in native periderm (180 g cm^–2), whereas in wound periderm about 75.0 g cm^–2 suberin polymer was newly synthesized. Native potato tuber periderm developed a very efficient transport barrier for water with permeances decreasing from 6.4×10^–10 m s^–1 to 5.5×10^–11 m s^–1 within 1 month of storage. However, the water permeability of wound periderm was on average 100 times higher with permeances decreasing from 4.7×10^–8 m s^–1 after 3 days to only 5.4×10^–9 m s^–1 after 1 month of storage, although suberin and wax amounts in wound periderm amounted to about 60% of native periderm. From this result it must be concluded that the occurrence of suberin with wax depositions in cell walls does not necessarily allow us to conclude that these cell walls must be nearly perfect barriers to water transport. In addition to the occurrence of the lipophilic biopolymer suberin and associated waxes, the still unknown molecular arrangement and precisely localized deposition of suberin within the cell wall must contribute to the efficiency of suberin as a barrier to water transport.