Purification and characterization of peroxidase from cauliflower (Brassica oleracea L. var. botrytis) buds

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes. In this study, peroxidase, a primer antioxidant enzyme, was purified with 19.3 fold and 0.2% efficiency from cauliflower (Brassica oleracea L.) by ammonium sulphate precipitation, dialysis, CM-Sephadex ion-exchange chromatography and Sephadex G-25 purification steps. The substrate specificity of peroxidase was investigated using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol), 1,2,3-trihyidroxybenzene (pyrogallol) and 4-methylcatechol. Also, optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature, thermal inactivation conditions were determined for guaiacol/H(2)O(2), pyrogallol/H(2)O(2), ABTS/H(2)O(2), catechol/H(2)O(2) and 4-methyl catechol/H(2)O(2) substrate patterns. The molecular weight (M(w)) of this enzyme was found to be 44 kDa by gel filtration chromatography method. Native polyacrylamide gel electrophoresis (PAGE) was performed for isoenzyme determination and a single band was observed. K(m) and V(max) values were calculated from Lineweaver-Burk graph for each substrate patterns.     

The chromoplasts of Or mutants of cauliflower (Brassica oleracea L. var. botrytis)

Summary. The Or mutation in cauliflower (Brassica oleracea L. var. botrytis) leads to abnormal accumulations of -carotene in orange chromoplasts, in tissues in which leucoplasts are characteristic of wild-type plants. Or chromoplasts were investigated by light microscopy of fresh materials and electron microscopy of glutaraldehyde- and potassium permanganate-fixed materials. Carotenoid inclusions in Or chromoplasts resemble those found in carrot root chromoplasts in their optical activity and angular shape. Electron microscopy revealed that the inclusions are made up of parallel, membrane-bound compartments. These stacks of membranes are variously rolled and folded into three-dimensional objects. We classify Or chromoplasts as membranous chromoplasts. The Or mutation also limits plastid replication so that a single chromoplast constitutes the plastidome in most of the affected cells. There are one to two chromoplasts in each cell of a shoot apex. The ability of differentiated chromoplasts to divide in the apical meristems of Or mutant plants resembles the ability of proplastids to maintain plastid continuity from cell to cell in meristems of Arabidopsis thaliana mutants in which plastid replication is drastically limited. The findings are used to discuss the number of levels of regulation involved in plastid replication.     

Transformation of cauliflower (Brassica oleracea L. var. botrytis) — an experimental survey

The paper compares different approaches for the genetic transformation of cauliflower (Agrobacterium-mediated, PEG-mediated and/or electroporation). Transient expression of the neomycin phosphotransferase II (NPTII) gene could be detected after direct gene transfer. Stable transformation was achieved using both Agrobacterium-mediated and direct gene transfer. Expression as well as incorporation of the NPTII sequence could be demonstrated.     

Temporal and spatial root development of cauliflower (Brassica oleracea L. var. botrytis L.)

Row crops are often inefficient in utilizing soil resources. One reason for this appears to be inefficient rooting of the available soil volume. Five experiments were performed to study the temporal and spatial root development of cauliflower (cv. Plana). The crop was grown with 60 cm between rows, and root development was followed in minirhizotrons placed under the crop rows, 15 cm, and 30 cm from the crop rows. Soil was sampled and analyzed for nitrate content at the final harvest and once during growth. In two of the experiments N fertilizer rate was varied and in two of the other experiments two cultivars were compared (cv. Plana and Siria).The rooting depth of cauliflower was found to be linearly related to temperature sum, with a growth rate of 1.02 mm day^-1 °C^-1. Depending on duration of growth this leads to rooting depths at harvest of 85–115 cm. Soil analysis showed that the cauliflower was able to utilize soil nitrogen down to at least 100 cm.With Plana differences in root growth between row and interrow soil were only observed during early growth, but with Siria this difference was maintained until harvest. However, at harvest both cultivars had depleted row and interrow soil nitrate equally efficient. Nitrogen fertilizer did not affect overall root development significantly.The branching frequency of actively branching roots was increased in all soil layers from about 6 to 10 branches cm^-1 by increasing N fertilizer additions from 130 to 290 kg N ha^-1. Increasing N supply increased the number of actively branching roots in the topsoil and reduced it in the subsoil.The average growth rate of the roots was always highest in the newly rooted soil layers, but fell during time. At 74 days after planting very few roots were growing in the upper 60 cm of the soil whereas 70% of the root tips observed in the 80–100 cm soil layer were actively growing. Within each soil layer there was a large variation in growth rate of individual root tips.     

An ATP-inhibited endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence: purification and characterization

In our studies on the role of enzymes in plant DNA replication, recombination, and repair, we isolated from cauliflower (Brassica oleracea L. var. botrytis) inflorescences a single-stranded DNA-specific endonuclease that was inhibited by ATP. The endonuclease, designated cauliflower nuclease II, was purified to near homogeneity through six successive column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl sulfate-polyacry amide gel electrophoresis, activity gel, and gel-filtration column chromatography. The enzyme can cleave a linear or a circular single-stranded DNA but cannot cut or nick a double-stranded DNA. The mode of activity of the nuclease is endonucleolytic and non-processive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus far unclear. While ATPγS and GTP can also inhibit the activity, other ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ionic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca²⁺, which cannot be replaced by Mg²⁺ or Mn²⁺. The features of the enzyme and its relation to plant DNA metabolism are discussed. Received: 26 March 1998 / Accepted: 4 June 1998     

A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence.

A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.     

Plant regeneration from cotyledonary protoplasts of cauliflower (Brassica oleracea L. var.botrytis L.)

Summary Protoplasts isolated enzymatically from precultured cotyledonary leaves ofB. oleracea var.botrytis and cultured in KM8p medium (Kao andMichayluk 1975) underwent sustained divisions in about 0.1% population to eventually produce callus, whereas mesophyll protoplasts from either field grown orin vitro raised plants failed to divide. The callus readily differentiated on Murashige-Skoog medium as modified for shoot culture (Binding 1974) to give rise to shoot and roots.     

Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.     

Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. var. botrytis) stems

Pectic substances are a major component of cell walls in vegetable plants and have an important influence on plant food texture. Cauliflower (Brassica oleracea L. var. botrytis) stem sections at different regions of the mature plant stem have been monitored for tissue-related changes in the native pectic polysaccharides. Chemical analysis detected appreciable differences in the degree of methyl-esterification (ME) of pectic polysaccharides. About 65% of galacturonic acid (GalpA) residues were methyl-esterified in floret tissues. Relative ME showed a basipetal decrease, from 94% in the upper stem to 51% in the lower-stem vascular tissues. The decrease was not related to a basipetal increase in glucuronic acid (GlcpA) residues. The monoclonal antibodies, JIM 5 and JIM 7, produced distinct labelling patterns for the relatively low-methyl-esterified and high-methyl-esterified pectin epitopes, respectively. Labelling was related to cell type and tissue location in the stem. Floret cell walls contained epitopes for both JIM 5 and JIM 7 throughout the wall. Stem vascular tissues labelled more strongly with JIM 5. Whereas pith parenchyma in the upper stem labelled more strongly with JIM 7, in the lower-stem pith parenchyma, JIM 5 labelling predominated. Localization of pectic polysaccharide epitopes in cell walls provides an insight into how structural modifications might relate to the textural and nutritional properties of cell walls. Received: 16 August 1997 / Accepted: 20 December 1997     

A study of factors affecting anther culture of cauliflower (Brassica oleracea var. botrytis)

Experiments on three autumn-heading cauliflower genotypes (2 hybrids and a genotype selected from a population) were conducted to study different factors affecting anther culture. Culture conditions of the donor plants proved to be important: the best results were obtained during spring in a greenhouse where the temperature was maintained between 10 and 20°C. Overall winter and spring seemed more suitable than summer and early autumn for culture establishment. The optimal bud development stage depended on the genotype: for the hybrid 702, the greatest number of embryos for 100 plated anthers was obtained at the uninucleate pollen stage of the microspores; for V23.2 and 703, the optimal stage of the buds corresponded to the first mitotic division. Sucrose proved to be the best carbon supply for embryogenesis with an optimal concentration of 140 g l^-1. The addition of a cytokinin (BAP) in the medium led to lower embryo production, and this negative effect increased when the hormone concentration in the medium increased. The use of liquid medium and a dark incubation period immediately after the high temperature treatment were favourable for embryogenesis.     

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l^?1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.     

Effects of temperature and irradiance on vegetative growth of cauliflower (Brassica oleracea L. botrytis) and broccoli (Brassica oleracea L. italica)

Cauliflower (Brassica oleracea L. botrytis) and broccoli (Brassicaoleracea L. italica) plants were grown in large pots in growthchambers for a range of temperatures (mean air temperaturesfrom 7.0-25.3 C) and irradi-ances (from 9.3-50.8 mol m^–2d^–1 or 4.7-25.4 MJ m^–2 d^–1). The extinctioncoefficient for PAR decreased with plant size reaching a valueof 0.55 in cauliflower and 0.45 in broccoli at plant leaf areasof 0.235 m² and 0.227 m², respectively. The leaf area expansionrate was unaffected by irradiance when compared at identicalleaf surface temperatures. The response of expansion rate tosurface temperature was fitted to a broken stick model witha base temperature of –0.7C and an optimum temperatureof 21.0C. The radiation conversion coefficient increased withair temperature below 13.8C and remained constant above this.The estimated radiation conversion coefficient above 13.8Cand for a PPFD of 20 mol m^–2 d^–1 was 0.77 g mol^–1in cauliflower and 0.87 g mol^–1 in broccoli. The radiationconversion coefficient declined with increasing irradiance levelfrom a maximum of 1.89 g mol^–1 at near nil irradiancein cauliflower. Key words: Leaf area, dry matter, radiation use efficiency, extinction coefficient     

The use of a novel stabilizing substrate for the in vitro cultivation of cauliflower (Brassica oleracea L. var. botrytis)

A new stabilizing substrate derived from cellulose crystallite aggregates (CCA) is presented in its use for tissue culture and regeneration of cauliflower (Brassica oleracea L.), particularly at the stage of root formation. The material is suitable instead of agar for the large scale propagation of cultured plants. Frequently growth promoting effects are being observed with the medium.     

Characteristics of cytosine methylation status and methyltransferase genes in the early development stage of cauliflower (Brassica oleracea L. var. botrytis)

DNA methylation is one of the most important epigenetic modifications involved in the development and differentiation in plants. Hypocotyl and cotyledon are the two major tissues of cauliflower (Brassica oleracea L. var. botrytis) seedlings. Both tissues show significantly different tissue specificity and regenerative abilities in vitro. However, the characteristics of DNA methylation modification and its roles in regulating the organ development in cauliflower remain largely unknown. In the present study, the DNA methylation status between the hypocotyl and cotyledon of cauliflower seedlings were analyzed. The results indicated that although the hypocotyl and cotyledon of cauliflower seedlings share the same genome, the genomic DNA methylation levels and patterns at CCGG sites were different. Compared with the cotyledon, the hypocotyl showed higher DNA methylation level, and more loci showing methylation pattern adjustments were also discovered. Twelve loci with changes of DNA methylation patterns were further explored. The quantitative expression analysis indicated that eight out of twelve sequenced fragments showed differential expression between the hypocotyl and cotyledon, of which the expression of six sequences was identified to be negative correlation with their DNA methylation status. In addition, three main DNA methyltransferase genes MET1, CMT3 and DRM were first explored in cauliflower. The results indicated that the expression of these three genes was closely associated with the different DNA methylation status in the hypocotyl and cotyledon. These findings provided more information to further explore the roles of DNA methylation modification in tissue differentiation and development of cauliflower.     

Genetic transformation of cauliflower (Brassica oleracea var. botrytis) by direct DNA uptake into mesophyll protoplasts

Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the β -glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants.     

Molecular mapping of Or, a gene inducing beta-carotene accumulation in cauliflower (Brassica oleracea L. var. botrytis).

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a semi-dominant, single-locus mutation that induces the accumulation of high levels of beta-carotene in various tissues of the plant, turning them orange. As part of a map-based cloning strategy, molecular mapping of the Or gene in the cauliflower genome was undertaken in a mapping population consisting of 195 F2 individuals. By using amplified fragment length polymorphism (AFLP) in conjunction with bulked segregant analysis, we identified 10 AFLP markers closely linked to the Or gene. Four of the most closely linked flanking markers were converted into restriction fragment length polymorphism (RFLP) markers. Mapping of these markers in the mapping population placed two of them at 0.5 cM from the Or locus on one side, while another marker flanked the Or gene at 1.6 cM on the other side. Three of these markers were also successfully converted into sequence-characterized amplified region (SCAR) markers. These PCR-based markers will be useful for a large-scale application in facilitating the positional cloning of the Or gene.     

In vitro propagation of cauliflower,Brassica oleracea var. botrytis for hybrid seed production

Methods for obtaining heterotic F₁ and maintaining purebred lines for breeding of Brassica oleracea are limited by absence of male sterile lines and occurrence of inbreeding depression, respectively. The use of vegetative (stem, petiole, leaf, leaf rib) and floral (peduncle, pedicel, flower bud, curd) explants of cauliflower to regenerate purebred lines for crossing were examined. Of four growth regulator treatments and explant types used, best results were obtained with curd explants on MS medium with 6-benzyladenine (cytokinin) and gibberellic acid. Although 6-benzyladenine alone promoted formation of shoots in floral explants, both 6-benzyladenine and α-napthaleneacetic acid were required for vegetative explants. Use of α-napthaleneacetic acid, however, often increased callus formation. These culture techniques to maintain purebred regenerated plants will complement newly-derived nuclear-based male sterile lines obtained by the introduction of antisense copies of the gene BcpI, which is required for pollen fertility. This revised version was published online in June 2006 with corrections to the Cover Date.     

Root growth of cauliflower (Brassica oleracea L. botrytis) under unstressed conditions: Measurement and modelling

Plant and Soil - Root observations were carried out on cauliflower using the minirhizotron and the soil core method in two years on two locations with different soil types, a loess loam and a humic...     

The pattern of frost-hardening of leaves of winter cauliflower (Brassica oleracea var. botrytis cv. Clause 30)

The suitability of an electrical conductivity assay of leaf material for determining frost hardiness in winter cauliflower was established. The assay was used to monitor the changes in hardiness of field-grown plants during two seasons at two sites. Levels of frost hardiness were related to mean air temperature over the 7-day period prior to sampling (r = 0.6008, P< 0.001). There was no clear disjunction of any temperature to indicate a threshold temperature of hardening. Dehardening occurred in spring and during mild spells in winter. In the mild regions where this crop is grown commercially it is likely that plants are not fully hardened for much of the winter. This may facilitate curd growth but may put the crop at risk from frost damage.