Genome-wide analysis indicates diverse physiological roles of the turnip (Brassica rapa var. rapa) oligopeptide transporters gene family

Oligopeptide transporters (OPTs) encode integral membrane-localized proteins and have a broad range of substrate transport capabilities.Here,28 BrrOPT genes were identified in the turnip.Phylogenetic analyses revealed two well-supported clades in the OPT family,containing 15 BrrOPTs and 13 BrrYSLs. The exon/intron structure of OPT clade are conserved but the yellow stripe-like (YSL) clade was different. The exon/intron of the YSL clade possesses structural differences,whereas the YSL class motifs structure are conserved.The OPT genes are distributed unevenly among the chromosomes of the turnip genome. Phylogenetic and chromosomal distribution analyses revealed that the expansion of the OPT gene family is mainly attributable to segmental duplication.For the expression profiles at different developmental stages,a comprehensive analysis provided insights into the possible functional divergence among members of the paralog OPT gene family.Different expression levels under a variety of ion deficiencies also indicated that the OPT family underwent functional divergence during long-term evolution. Furthermore,BrrOPT8.1,BrrYSL1.2,BrrYSL1.3,BrrYSL6 and BrrYSL9 responded to Fe (Ⅱ) treatments and BrrYSL7 responded to calcium treatments,BrrYSL6 responded to multiple treatments in root,suggesting that turnip OPTs may be involved in mediating cross-talk among different ion deficiencies.Our data provide important information for further functional dissection of BrrOPTs,especially in transporting metal ions and nutrient deficiency stress adaptation.     

Translocation and multiplication ofSpiroplasma citri in turnip (Brassica rapa)

Following inoculation of designated leaves of turnip plants withSpiroplasma citri byCirculifer tenellus, spiroplasmas were cultured first from roots (four days) and then from youngest leaves (eight days), but almost never from oldest leaves. In experiments using enzyme-linked immunosorbent assay to monitor changes in titer in turnip leaves during the course of plant infection,S. citri was detected seven days after inoculation and reached peak titers of 10¹⁰–10¹¹ colony-forming units/g 12–20 days after inoculation, declining thereafter. Spiroplasmas were detected 5–9 days before symptoms appeared.     

Impact of treated municipal wastewater irrigation on turnip (Brassica rapa)

The effect of treated municipal wastewater on the roots and the leaves of turnip was studied to compare the 50% and 100% wastewater of 34 ml/d Sewage Treatment Plant (STP) with different doses of potassic fertilizers. Turnip (Brassica rapa) was used as a test plant. A pot experiment was conducted, using a factorial randomized block design to investigate the growth and translocation of heavy metals to the leaves and the roots of turnip. The concentration of heavy metal in wastewater used for irrigation was within the limits. However, the concentration in the plant parts showed a significant rise due to continuous use of wastewater. The concentration of heavy metals in leaves and roots was at excessive levels at 40 and 55 days after sowing (DAS), while at 70 DAS, metal concentration was comparatively low. The range of heavy metals in wastewater irrigated plants was Cd = 1–16.3, Ni = 0–136, Fe = 263–1197, Cu = 0–18, Mn = 37–125, and Zn = 42–141 mg/kg. Concentration of heavy metals in plants was found in the order of Fe>Zn>Ni>Mn>Cu>Cd.     

Genetic analysis of novel intra-species unilateral incompatibility in Brassica rapa (syn. campestris) L.

Plants have evolved many systems to prevent inappropriate fertilization. Among them, incompatibility is a well-organized system in which pollen germination or pollen-tube growth is inhibited in pistils. Self-incompatibility (SI), rejecting self-pollen, promotes outbreeding in flowering plants. On the other hand, inter-species incompatibility, preventing gene flow among species to restrict outbreeding, usually occurs unilaterally, and is known as unilateral incompatibility (UI). In Brassicaceae, little is known about the molecular mechanism of UI, although S-locus genes involved in recognition of self-pollen have been characterized in the SI system. In the present study, we characterized novel UI observed between members of the same species, Brassica rapa; pollen of Turkish SI lines was specifically rejected by pistils of the Japanese commercial SI variety Osome. The incompatible phenotype of this intra-species UI closely resembled that of SI. Segregation analysis revealed that the pollen factor of this UI was not linked to the S-locus.The revised version was published online in December 2004 with corrections to figure 1.     

Sink development, sucrose metabolising enzymes and carbohydrate status in turnip (Brassica rapa L.)

Accumulation of 60–70 % of biomass in turnip root takes place between 49–56 days after sowing. To understand the phenomenon of rapid sink filling, the activities of sucrose metabolising enzymes and carbohydrate composition in leaf blades, petiole and root of turnip from 42–66 days of growth were determined. An increase (2–3 folds) in glucose and fructose contents of roots accompanied by an increase in activities of acid and alkaline invertases was observed during rapid biomass accumulating phase of roots. The observed decrease in the activities of acid and alkaline invertases along with sucrose synthase (cleavage) in petiole during this period could facilitate unrestricted transport of sucrose from leaves to the roots. During active root filling period, a decrease in sucrose synthase (cleavage) and alkaline invertase activities was also observed in leaf blades. A rapid decline in the starch content of leaf blades was observed during the phase of rapid sink filling. These metabolic changes in the turnip plant led to increase in hexose content (35–37 %) of total dry biomass of roots at maturity. High hexose content of the roots appears to be due to high acid invertase activity of the root.     

Ultraviolet A-specific induction of anthocyanin biosynthesis in the swollen hypocotyls of turnip (Brassica rapa)

Ultraviolet A (UV-A)-mediated regulation of anthocyanin biosynthesis was investigated in swollen hypocotyls of the red turnip 'Tsuda'. The shaded swollen hypocotyls which contained negligible anthocyanin were exposed to artificial light sources including low fluence UV-B, UV-A, blue, red, far-red, red plus UV-A, far-red plus UV-A, and blue plus red. Among these lights, only UV-A induced anthocyanin biosynthesis and co-irradiation of red or far-red with UV-A did not affect the extent of UV-A-induced anthocyanin accumulation. The expression of phenylalanine ammonia lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), and anthocyanidin synthase (ANS; EC 1.14.11.19) genes was increased with time during a 24 h exposure to UV-A. In contrast, irradiation with red, blue, UV-B, and a combination of blue with red failed to induce CHS expression. Microarray analysis showed that only a few genes, including CHS and F3H, were induced significantly by UV-A, while a separate set of many genes was induced by low fluence UV-B. The UV-A-specific induction of anthocyanin biosynthesis and the unique gene expression profile upon UV-A irradiation as compared with blue and UV-B demonstrated that the observed induction of anthocyanin biosynthesis in red turnips was mediated by a distinct UV-A-specific photoreceptor, but not by phytochromes, UV-A/blue photoreceptors, or UV-B photoreceptors.     

Transfer of resistance to clubroot (Plasmodiophora brassicae) to swedes {Brassica napus L. var. napobrassica Peterm) from B. rapa

In 1975, tests with UK populations of Plasmodiophora brassicae not only revealed a lack of effective clubroot resistance in swedes (Brassica napus), but also the outstanding resistance of the European Clubroot Differential (ECD)04 (B. rapa). It was, therefore, decided to transfer the resistance genes from ECD04 to swedes, using the most pathogenic UK population of clubroot (C56) available for screening purposes. An autotetraploid form of ECD04 was crossed with tetraploid kale (B. oleracea) using the latter as female parent. One of the euploid, 2n = 38, hybrids secured by embryo rescue in 1976 was crossed to the swede cultivars Marian and Ruta Øtofte. Three further backcrosses of clubroot resistant plants to lines derived from modern swede cultivars were made over the period 1980 to 1982. Selfing commenced in 1983 to produce F₂ populations. From F₃ to F₅ there was family selection for yield and agronomic characters, as well as single plant selection for clubroot resistance. In 1991, the six most promising F₅ families were multiplied for subsequent evaluation in replicated yield trials in Dundee. The most promising family entered official trials at the beginning of 1993 and, 2 years later, was added to the National List as cv. Invitation and granted Plant Breeders' Rights. The first certified seed was sold in 1996, 20 years after the original synthetic B. napus was produced. The breeding programme provided evidence for only one of the three postulated dominant genes in ECD04 being required for resistance to C56 and also good evidence of differential resistance from tests with other clubroot populations. Hence, whilst the differential resistance in cv. Invitation should prove useful in the UK in the immediate future, it may not be durable in the longer term. It is, therefore, argued that the next and more difficult goal to achieve should be to introduce high levels of non-differential resistance from B. oleracea.     

Genetic transformation of cauliflower (Brassica oleracea L. var. Botrytis) by Agrobacterium rhizogenes

Cauliflower plantlets were inoculated with different Agrobacterium rhizogenes strains. Numerous hairy roots were induced on cauliflower hypocotyls by agropine-type strains. Fewer roots were obtained with mannopine-type strains. In vitro cultures were established both from normal and hairy roots. Plant regeneration occured spontaneously from normal and transformed roots. Regenerated plants contained the same opines (if present) as root cultures. Some mannopine-positive regenerants displayed a modified phenotype. Relationships between phenotype, opine content, T-DNA content and/or expression are discussed.     

Variation of glucosinolates on position orders of flower buds in turnip rape (Brassica rapa)

To glucosinolate (GSL) contents on flower buds depending on their position orders in turnip rape (Brassica rapa), three Japanese ‘Nabana’ cultivars such as cv. No. 21 (Brassica rapa, early type), cv. Husanohana (B. rapa, late type) and cv. Norin No. 20 (B. napus) were investigated using HPLC analysis. Ten GSLs including glucoraphanin, sinigrin, glucoalyssin, napoleiferin, gluconapin, 4-hydroxyglucobrassicin, glucobrassicanapin, glucobrassicin, and gluconasturtiin were detected. Differences in individual and total GSL contents were found between two plant varieties, which are also depending on various developmental stages. Among the GSLs, gluconapin (mean 23.11 μmole/g dry weight (DW) and glucobrassicanapin (mean 13.41 μmole/g DW) documented the most abundant compounds and contributed average 39 and 27% of the total GSLs, but indolyl and aromatic GSLs together accounted >10% of the total GSLs. The presence of significant quantities of gluconapin in the cultivars should be studied more extensively, since the GSL is mainly responsible for the bitter taste.     

Insect pollinators diversity and abundance in Eruca sativa Mill. (Arugula) and Brassica rapa L. (Field mustard) crops

Studies on the insect pollinators diversity and their relative abundance in Eruca sativa Mill. (Arugula) and Brassica rapa L. (field mustard) was carried out during spring season from February to April consecutively during all the three years of 2016–18. Insect pollinators observed belonged to four orders i.e. Hymenoptera, Diptera, Lepidoptera, and Coleoptera. A total of 20 major species of insect pollinators were recorded. The highest abundance of pollinator species belonged to Hymenoptera. The most prominent insect pollinator species were Apis mellifera followed by other three honey bee species of A. cerana, A. florea, and A. dorsata respectively. Some species of solitary bees were also recorded. From Diptera, four species of syrphid fly and one species from Muscidae family were also recorded. Insect pollinators recorded from order Lepidoptera were Pieris brassicae, Vanessa cardui, and Papilio demoleus. Lady bird beetle Coccinella septempunctata was recorded from Coleoptera order as occasional visitor. It was noticed that E. sativa attracted more insect pollinators than B. rapa which may be attributed to different amount and chemical properties of nectar, with number of pollen grains, and flower canopy of both crops. Further studies are needed to confirm the reasons for higher pollinator visitation to E. sativa than B. rapa through chemical analysis of nectar, amount of pollens, flower physiology and phenology of both crops.     

Mapping of a QTL for oleic acid concentration in spring turnip rape (Brassica rapa ssp. oleifera)

Bulk segregant analysis was used to search for RAPD (random amplified polymorphic DNA) markers linked to gene(s) affecting oleic acid concentration in an F₂ population from the Brassica rapa ssp. oleifera cross Jo4002 x a high oleic acid individual from line Jo4072. Eight primers (=8 markers) out of 104 discriminated the high and low bulks consisting of extreme individuals from the oleic acid distribution. These markers were analysed throughout the entire F₂ population, and their association with oleic acid was studied using both interval mapping and ANOVA analysis. Six of the markers mapped to one linkage group. A quantitative trait locus (QTL) affecting oleic acid concentration was found to reside within this linkage group with a LOD score >15. The most suitable marker for oleic acid content is OPH-17, a codominant marker close (<4cM) to the QTL. The mean seed oleic acid content in the F₂ individuals carrying the larger allele of this marker was 80.14±9.76%; in individuals with the smaller allele, 54.53±6.83%; in the heterozygotes, 65.47±8.15%. To increase reproducibility, the RAPD marker was converted into a SCAR (sequence characterized amplied region) marker with specific primers. Marker OPH-17 can be used to select spring turnip rape individuals with the desired oleic acid content.     

Anthocyanin formation in turnip seedlings (Brassica rapa L.): Evidence for two light steps in the biosynthetic pathway

Summary In turnip seedlings, anthocyanin synthesis can be induced with light as soon as water uptake enables the seed coat to be removed. In very young seedlings the main site of production is in the cotyledons but this moves to the hypocotyl when the period of dark growth, before transfer to the light, is increased. The total amount of anthocyanin formed decreases as the seedlings become older. It is suggested that a substance stored in the cotyledons is needed for anthocyanin synthesis and that this substance disappears during growth in the dark. It cannot be replaced by known anthocyanin precursors such as phenylalanine, acetate, shikimic acid and sugars.Anthocyanin synthesis in the hypocotyl is almost completely prevented when the cotyledons are excised, or covered: no anthocyanin is formed in the hypocotyl when the cotyledons alone are irradiated. Cotyledons that have been excised from the hypocotyl synthesize about as much anthocyanin as is formed in the whole intact seedling, but covering the hypocotyl does not increase the amount formed in the cotyledons. These results suggest that pigment synthesis begins in the cotyledons, where a light reaction is needed for the formation of a precursor; the precursor is translocated to the hypocotyl where a second photochemical reaction is required for anthocyanin synthesis. If translocation to the hypocotyl is prevented, anthocyanin is formed in the cotyledons. The nature of the transported precursor is not yet known.

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Zusammenfassung In Keimlingen vonBrassica rapa kann Anthocyansynthese durch Licht induziert werden, sobald es möglich ist, die Samenschale zu entfernen. In den jüngsten Keimlingen sind die Kotyledonen der Ort stärkster Anthocyanbildung, in älteren Keimlingen das Hypokotyl. Die Gesamtmenge an gebildetem Anthocyan nimmt mit zunehmendem Alter der Keimlinge ab. Es wird vermutet, daß eine für Anthocyansynthese notwendige Substanz in den Kotyledonen gespeichert ist und während des Wachstums im Dunkeln abnimmt. Diese Substanz konnte durch bekannte Anthocyanvorstufen wie Phenylalanin, Acetat, Shikimisäure und Zucker nicht ersetzt werden.Anthocyansynthese ist im Hypokotyl fast vollständig unterdrückt, wenn die Kotyledonen entfernt oder verdunkelt werden: Kein Anthocyan wird im Hypokotyl gebildet, wenn die Kotyledonen allein belichtet werden. Isolierte Kotyledonen synthetisieren ungefähr die gleiche Menge Anthocyan wie intakte Keimlinge, aber eine Verdunkelung des Hypokotyls bewirkt keine Steigerung der Anthocyanbildung in den Kotyledonen. Diese Ergebnisse lassen vermuten, daß die Synthese von Anthocyan in den Kotyledonen beginnt, wo eine lichtabhängige Reaktion zur Bildung einer Zwischenstufe notwendig ist; diese Zwischenstufe wird in das Hypokotyl transportiert, wo eine zweite photochemische Reaktion für Anthocyansynthese erforderlich ist. Wird der Transport in das Hypokotyl verhindert, findet Anthocyansynthese in den Kotyledonen statt. Über die Natur dieser Zwischenstufe ist jedoch noch nichts bekannt.

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With 9 Figures in the Text     

Aphid responses to volatile cues from turnip plants (Brassica rapa) infested with phloem-feeding and chewing herbivores

Herbivore-induced plant volatiles provide foraging cues for herbivores and for herbivores’ natural enemies. Aphids induce plant volatile emissions and also utilize plant-derived olfactory volatile cues, but the chemical ecology of aphids and other phloem-feeding insects is less extensively documented than that of chewing insects. Here, we characterize the volatile cues emitted by turnip plants (Brassica rapa) under attack by an aphid (Myzus persicae) or by the chewing lepidopteran larva Heliothis virescens. We also tested the behavioral responses of M. persicae individuals to the odors of undamaged and herbivore-damaged plants presented singly or in combination, as well as to the odor of crushed conspecifics (simulating predation). Gas chromatographic analysis of the volatile blend of infested turnips revealed distinct profiles for both aphid- and caterpillar-induced plants, with induced compounds including green-leaf alcohols, esters, and isothiocyanates. In behavioral trials, aphids exhibited increased activity in the presence of plant odors and positive attraction to undamaged turnip plants. However, aphids exhibited a strong preference for the odors of healthy versus plants subjected to herbivore damage, and neither aphid- or caterpillar-damaged plants were attractive compared to clean-air controls. Reduced aphid attraction to herbivore-infested plants may be mediated by changes in the volatile blend constituent composition, including large amounts of isothiocyanates and green-leaf volatiles or, in the case of aphid-infested plants, of the aphid alarm pheromone, (E)-β-farnesene.     

Bioaffinity-based an inexpensive and high yield procedure for the immobilization of turnip (Brassica rapa) peroxidase

This study demonstrates the immobilization of carbohydrate containing turnip peroxidase on an inexpensive bioaffinity adsorbent, Concanavalin A-cellulose support. The bioaffinity support was prepared simply by incubating cellulose powder with jack bean extract at 4 degrees C. Cellulose powder adsorbed 30 mg concanavalin A/g of the matrix. Concanavalin A adsorbed cellulose has been employed for the simultaneous purification and immobilization of glycoenzymes directly from ammonium sulphate fractionated proteins of turnip. The obtained bioaffinity support was quite effective in high yield immobilization of peroxidase from turnip and it retained 672 U/g. Turnip peroxidase immobilized on concanavalin A-cellulose support retained 80% of the initial activity. Immobilized turnip peroxidase preparation was quite resistant against the denaturation mediated by pH, heat, urea, guanidinium-HCl, Surf Excel, cetyltrimethylammonium bromide and water-miscible organic solvents; dimethyl formamide, dioxane and n-propanol. Low concentration of detergents like Surf Excel and cetyltrimethylammonium bromide enhanced the activity of soluble and immobilized turnip peroxidase.     

Metallo-proteinase from the seedlings of kale (Brassica oleracea L. var. sabellica):

Metallo-proteinase from 8-d-old seedlings of kale was isolated. The enzyme was extracted with 1% NaCl, concentrated by ammonium sulfate and finally purified by high-performance liquid chromatography. The isolated enzyme had a molecular weight of 22.4 kDa and showed a maximum activity at pH 9.0 using casein as a substrate. Proteolytic activity of proteinase was inhibited by chelators. The inhibition by ethylenediaminetetraacetate (EDTA) was abolished by some divalent metals ions, especially by Zn²⁺. The enzyme showed activity against the synthetic peptides Suc-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Phe-pNA, and hydrolized the following peptide bonds in the oxidized insulin B-chain: Leu6-Cya7, Leu15-Tyr16, Leu17—Val18 and Phe25-Tyr26.Abbreviations EDTA ethylenediaminotetraacetic acid - HPLC high-performance liquid chromatography - NEM N-ethylmaleimide - PCMB p-mecuribenzoic acid - PMSF phenylmethylsulfonyl fluoride This work was supported by the University Science Programme, Ministry of National Education, and Polish Academy of Science, Warsaw, Poland.     

Behavioural and chemical ecology underlying the success of turnip rape (Brassica rapa) trap crops in protecting oilseed rape (Brassica napus) from the pollen beetle (Meligethes aeneus)

There is increasing interest in the use of trap crops as components of integrated pest management (IPM) strategies. Understanding the mechanisms underlying host plant preferences of herbivorous pests can lead to improved effectiveness and reliability of the trap crop. We investigated the behavioural and chemical ecology underlying the success of turnip rape, Brassica rapa, trap crops in protecting oilseed rape, Brassica napus, from the pollen beetle, Meligethes aeneus, which feeds in the flowers and lays its eggs in the buds causing yield loss. Using a semi-field arena bioassay, plant growth stage was found to be a major factor in the preference of this pest for B. rapa over B. napus. Plants at early-flowering growth stages were preferred over plants in the bud stage, irrespective of species. No preference was found when both species were flowering. As B. rapa develops faster than B. napus in the field, this could explain part of the mechanism of its success as a trap crop. However, B. rapa was preferred over B. napus when both species were in the bud stage, indicating some inherent preferences for B. rapa. Responses of M. aeneus in olfactometer tests to the odours of B. napus and B. rapa at the bud and flowering growth stages, reflected those of the semi-field arena bioassay. These behavioural responses can be explained by volatile compounds associated with the flowering stage. Phenylacetaldehyde, indole and (E,E)-α-farnesene were found to be present in air entrainment samples of both plant species at the flowering growth stage, but only in those of B. rapa at the bud stage. The former two compounds were behaviourally-active in olfactometer tests. These compounds are likely to be involved in host location by M. aeneus, and, at least partially, responsible for the attractiveness of B. rapa and its success as a trap crop to protect B. napus from this pest.     

Genetic transformation of Brassica campestris var. rapa protoplasts with an engineered cauliflower mosaic virus genome

Summary A hybrid Cauliflower Mosaic Virus (CaMV) genome containing a selectable marker gene was constructed by replacing the gene VI coding region with the aminoglycoside (neomycin) phosphotransferase type II [APH(3)II] gene from Tn5. This modified viral genome was tested for its infectivity both in planta and in a protoplast transformation system of Brassica campestris var. rapa. Stable, genetically transformed cell lines of B. campestris var. rapa were obtained after transformation. DNA of the hybrid CaMV genome was found to be integrated into high molecular weight plant genomic DNA. Transformation was achieved only when the hybrid genome was supplied together with wild type viral DNA. A possible complementation of the modified CaMV genome with the wild type viral DNA as a helper molecule in planta and in the protoplast system is discussed.