Embryogenesis and plant regeneration of spinach (Spinacia oleracea L.) from hypocotyl segments

A system for somatic embryogenesis and plant regeneration of spinach from hypocotyl segments has been established. Callus was induced on solid media supplemented with 8.5–15.0 mg.l⁻¹ of indole-3-acetic acid and 3.46–34.64 mg.l⁻¹ gibberellic acid. Callus was then subcultured on different media (solid or liquid) with or without IAA, or continuously maintained on the initiating media. Somatic embryos were obtained in subcultures on IAA-containing media as well as in long-term cultures on initiating media. The best results were achieved in liquid subcultures. About 60% of plantlets survived after transplanting in pots.     

The role of the gas phase in the greening and growth of illuminated cell suspension cultures of spinach (Spinacia oleracea,L.)

Summary The gas phase developed above spinach suspension cultures critically affected their growth and greening. Ethylene accumulation inhibited greening; this effect of ethylene was antagonised when the culture gas phase was enriched with carbon dioxide. Greening was enhanced by reducing the partial pressure of oxygen below the air level; this effect was observed when oxygen supply did not restrict growth. One of the authors (C.C.D.) was supported by an S.R.C. studentship grant during this work.     

A comparison of low temperature growth vs low temperature shifts to induce resistance to photoinhibition in spinach (Spinacia oleracea)

Plants of Spinacia oleracea L. cv. Savoy grown under cold-hardening (5°C) and nonhardening (16°C) conditions were exposed to a photoinhibitory irradiance of 1300 μmol rrr^: m-² S-¹ 5°C for 12 h. Plants grown at 5°C exhibited a greater resistance to photoinhibition at low temperature in comparison to plants grown at 16°C as measured by the photochemical efficiency of photosyslem II. In contrast, tuily expanded leaves of plants grown at 16°C and then shifted to 5°C for 10 days did not exhibit increased resistance to photoinhibition. This was observed irrespective of the phoioperiod experienced during the shift to a lower temperature. Furthermore, spinach grown at 16°C and subsequently exposed to a stepped, daily decrease in temperature from 16 to 1°C over 10 days w ith a concomitant reduction in photoperiod. also did not exhibit any change in susceptibility to photoinhibition. Thus, a decrease in photoperiod accompanied by either an abrupt or stepped low temperature shift cannot induce increased resistance to photoinhibition. This confirms the hypothesis that growth and development at cold-hardening temperature are absolute requirements for the acquisition of resistance to photoinhibition at low temperature.     

Role of 5-aminolevulinic acid (ALA) on active oxygen-scavenging system in NaCl-treated spinach (Spinacia oleracea)

ALA is a key precursor in the biosynthesis of porphyrins such as chlorophyll and heme, and was found to induce temporary elevations in the photosynthesis rate, APX, and CAT; furthermore, treatment with ALA at a low concentration might be correlated to the increase of NaCl tolerance of spinach plants. The photosynthetic rate and the levels of active oxygen-scavenging system in the 3rd leaf of spinach (Spinacia oleracea) plants grown by foliar treatment with 0, 0.18, 0.60 and 1.80 mmol/L 5-aminolevulinic acid under 50 and 100 mmol/L NaCl were analyzed. Plants treated with 0.60 and 1.80 mmol/L ALA showed significant increases in the photosynthetic rate at 50 and 100 mmol/L NaCl, while that of 0.18 mmol/L ALA did not show any changes at 50 mmol/L NaCl and a gradual decrease at 100 mmol/L NaCl. In contrast, the rate with 0 mmol/L ALA showed reduction at both concentrations of NaCl. The increase of hydrogen peroxide content by treatment with 0.60 and 1.80 mmol/L ALA were more controlled than that of 0 mmol/L ALA under both NaCl conditions. These ALA-treated spinach leaves also exhibited a lower oxidized/reduced ascorbate acid ratio and a higher reduced/oxidized glutathione ratio than the 0 mmol/L-treated spinach leaves when grown at both NaCl conditions. With regard to the antioxidant enzyme activities in the leaves, ascorbate peroxidase, catalase, and glutathione reductase activities were enhanced remarkably, most notably at day 3, by treatment with 0.60 and 1.80 mmol/L ALA under both NaCl conditions in comparison to that of 0 and 0.18 mmol/L ALA. These data indicate that the protection against oxidative damage by higher levels of antioxidants and enzyme activities, and by a more active ascorbate-glutathione cycle related to the increase of the photosynthesis rate, could be involved in the increased salt tolerance observed in spinach by treatment with 0.60 to 1.80 mmol/L ALA with NaCl.     

Chalcone synthases from spinach (Spinacia oleracea L.)

The two chalcone-synthase forms from leaves ofSpinacia oleracea L. were purified to apparent homogeneity. Antibodies were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light.Abbreviations DEAE diethylaminoethyl - DTE 1,4-dithioerythritol - EDTA ethylenediaminetetraacetic acid - HPLC high-performance liquid chromatography - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Parts of the results were presented at the 14^th International Botanical Congress at Berlin in July 1987     

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang     

An efficient Agrobacterium tumefaciens-mediated transformation and regeneration system for cotyledons of spinach (Spinacia oleracea L.)

An efficient transformation and regeneration system was established for the production of transgenic spinach (Spinacia oleracea L.) plants. Cotyledon explants were infected with Agrobacterium tumefaciens strain LBA4404 carrying the selectable marker gene, neomycin phosphotransferase II (nptII), and the reporter gene smgfp, encoding soluble-modified green-fluorescent protein, driven by the cauliflower mosaic virus 35S promoter. The infected explants were cultured on Murashige and Skoog medium, containing 1 mg/l benzyladenine and 0.4 mg/l naphthaleneacetic acid. Shoots were regenerated on selection medium containing 50 mg/l kanamycin. Regenerated kanamycin-resistant shoots were rooted on medium containing 1 mg/l indolebutyric acid and subsequently grown in soil in the greenhouse. Southern blot analysis indicated that the smgfp gene had been integrated into the spinach genome. Northern and Western blots showed that the smgfp gene was expressed in progeny plants. Received: 31 March 1998 / Revision received: 27 September 1998 / Accepted: 10 Ocotber 1998     

Diurnal variations in growth rate and growth substrate levels of spinach (Spinacia oleracea L.) under nitrogen-limiting conditions

Spinach plants were grown in hydroponic culture provided with variable limiting amounts of N. During a complete diurnal cycle, growth of the root and shoot parts, as well as levels of soluble and insoluble sugars and of free amino acids, were monitored. No clear relationship could be detected between the level of N feeding and the levels of free sugars and amino acids. Analysis of variance revealed that the variances in the relative growth rates of plant root and shoot could be correlated with the levels of sugars and amino acids. Root amino acid concentration could be correlated with shoot amino acid concentration and root sugar concentration. No relationship was found between the variances in root and shoot free sugar concentrations.     

The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis

The complete nucleotide sequence of mulberry (Morus indica cv. K2) chloroplast genome (158,484 bp) has been determined using a combination of long PCR and shotgun-based approaches. This is the third angiosperm tree species whose plastome sequence has been completely deciphered. The circular double-stranded molecule comprises of two identical inverted repeats (25,678 bp each) separating a large and a small single-copy region of 87,386 bp and 19,742 bp, respectively. A total of 83 protein-coding genes including five genes duplicated in the inverted repeat regions, eight ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids, were assigned on the basis of homology to predicted genes from other chloroplast genomes. The mulberry plastome lacks the genes infA, sprA, and rpl21 and contains two pseudogenes ycf15 and ycf68. Comparative analysis, based on sequence similarity, both at the gene and genome level, indicates Morus to be closer to Cucumis and Lotus, phylogenetically. However, at genome level, inclusion of non-coding regions brings it closer to Eucalyptus, followed by Cucumis. This may reflect differential selection pressure operating on the genic and intergenic regions of the chloroplast genome.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Communicated by Y. Tsumura     

Purification of UDP-galactose: diacylglycerol galactosyltransferase from chloroplast envelopes of spinach (Spinacia oleracea L.)

Uridine 5-diphosphate(UDP)-galactose: 1,2-diacylglycerol 3-O--d-galactopyranosyltransferase (EC 2.4.1.46) is an integral protein of chloroplast envelope membranes from which it has been partially purified (Covès et al., 1986, FEBS Lett. 208, 401–406). We have worked out a purification procedure which after removal of peripheral membrane proteins, solubilization and two chromotographic steps allowed us to identify a 22-kDa protein as the galactosyltransferase. Enrichment of enzymatic activity was paralleled by an enrichment of this protein and its radioactive derivative obtained by photoaffinity labelling with [-^–32P]UDP which is a potent inhibitor of the enzyme. The purification factor of about 350 is substantially higher than achieved previously and indicates that the enzyme represents less than 0.3% of the envelope proteins. The purified enzyme has a Km of 87 M for UDP-galactose with dioleoylglycerol as acceptor and could not be activated by addition of other lipids.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate - DTE dithioerythritol - MGD monogalactosyl diacylglycerol - PMSF phenylmethanesulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by the Deutsche Forschungsgemeinschaft.     

Plasticity in Sex Expression of Spinach (Spinacia oleracea) Regenerated from Root Tissues

The sex expression of spinach (Spinacia oleracea L.) was modified by a procedure where plants were regenerated from root callus of spinach over three successive generations. Somatic embryos obtained from the root callus of female or male plants were grown in vitro under long day conditions until flowering. Although the regenerants derived from somatic embryos derived from female plants were all female at the first generation, few progenies produced male flower organs and expressed gynomonoecy in subsequent generations. Once the female plants regenerated into gynomonoecy, they never expressed complete femaleness at post generation. However, the male plants easily altered into andromonoecy at subsequent generations, and several andromonoecious plants showed the sex reversion at post generation, although this phenomenon did not occur in the female progenies. No absolute sex conversion was found in both female and male progenies. The plasticity of the sex expressions of spinach indicates that female and male individuals might essentially be capable of generating both flower organs.     

Molecular evidence that the genes for dioecism and monoecism in Spinacia oleracea L. are located at different loci in a chromosomal region

Spinach (Spinacia oleracea L.) is widely known to be dioecious. However, monoecious plants can also occur in this species. Sex expression in dioecious spinach plants is controlled by a single gene pair termed X and Y. Our previous study showed that a single, incompletely dominant gene, which controls the monoecious condition in spinach line 03–336, should be allelic or linked to X/Y. Here, we developed 19 AFLP markers closely linked to the monoecious gene. The AFLP markers were mapped to a 38.2-cM chromosomal region that included the monoecious gene, which is bracketed between flanking markers with a distance of 7.1 cM. The four AFLP markers developed in our studies were converted into sequence-characterized amplified region (SCAR) markers, which are linked to both the monoecious gene and Y and are common to both populations segregating for the genes. Linkage analysis using the SCAR markers suggested that the monoecious gene (M) and Y are located in different intervals, between different marker pairs. Analysis of populations segregating for both M and Y also directly demonstrates linkage of the genes at a distance of ∼12 cM. The data presented in this study may be useful for breeding dioecious and highly male monoecious lines utilized as the pollen parents for hybrid seed production, as well as for studies of the evolutionary history of sexual systems in this species, and can provide a molecular basis for positional cloning of the sex-determining genes.     

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.     

The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals

Summary The entire chloroplast genome of the monocot rice (Oryza sativa) has been sequenced and comprises 134525 bp. Predicted genes have been identified along with open reading frames (ORFs) conserved between rice and the previously sequenced chloroplast genomes, a dicot, tobacco (Nicotiana tabacum), and a liverwort (Marchantia polymorpha). The same complement of 30 tRNA and 4 rRNA genes has been conserved between rice and tobacco. Most ORFs extensively conserved betweenN. tabacum andM. polymorpha are also conserved intact in rice. However, several such ORFs are entirely absent in rice, or present only in severely truncated form. Structural changes are also apparent in the genome relative to tobacco. The inverted repeats, characteristic of chloroplast genome structure, have expanded outward to include several genes present only once per genome in tobacco and liverwort and the large single copy region has undergone a series of inversions which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes is proposed which accounts simultaneously for the origin of this pseudogene, the large inversion and the creation of repeated sequences near the inversion endpoints.     

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in Spinacia oleracea and other higher plants: extreme substrate ambiguity and other properties

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS-Co: EC 4.1.2.15) in Spinacia oleracea, Solanum tubersosum and many other higher plants was found to use a diversity of substrates. Diose (glycolaldehyde), triose (D-glyceraldehyde, L-glyceraldehyde and DL-glyceraldehyde 3-phosphate), tetrose (D-erythrose, L-erythrose, D-erythrose 4-phosphate, D-threose and L-threose), and pentose (D-ribose 5-phosphate and D-arabinose 5-phosphate) were utilized in combination with phosphoenolpyruvate (PEP) to make the corresponding 2-keto-3-deoxy sugar acids. Glyoxylate was also utilized by DS-Co. Glycoladehyde exhibited the highest reaction velocity when substrates were tested at 3 mM concentrations. Pentoses were poor substrates except when phsophorylated, an effect which is probably due to an increased fraction of the molecules being in the open-chain form. Little stereoselective discrimination exists since comparable velocities were demonstrated with the D and L isomers of glyceraldehyde, erythrose or threose. The enzyme is not a reversible aldolase since pyruvate failed to substitute for PEP. The use of D-erythrose 4-phsophate or glycolaldehyde resulted in K_m values of 1.95 mM and 8.60 mM, respectively. However, glycolaldehyde exhibited the largest V_maxK_m ratio, suggesting a greater catalytic efficiency for this substrate. Glycolaldehyde is an ideal substrate for inexpensive assays of DS-Co that are absolutely selective in the presence of two other plant enzymes which also utilize erythrose 4-phosphate and PEP. The spinach DS-Co enzymes required divalent metals for activity. The presence of 20 mM Mg²⁺, 1 mM Co²⁺ and 1 mM Mn²⁺ yielded relative activities of 100, 70 and 15, respectively. The pH optimum was 9.5 and temperature optimum for activity was 49°C. The molecular masses of DS-Co from spinach, tobacco and pea were all in the range of 400 kDa. The possible roles of DS-Co in biosynthesis of α-ketoglutarate and aromatic amino acids, in biosynthesis of components of cell wall and phytotoxin, and in acting as a sink for 2-and 3-carbon sugars are discussed.     

Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts

Using the expression vector gt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112–114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4–12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.