Molecular characterization of high molecular weight glutenin subunit allele 1Bx23 in common wheat introduced from hexaploid triticale

High molecular weight glutenin subunit (HMW-GS) 1Bx23, an x-type subset encoded by Glu-B1p, which is only distributed in Triticum turgidum, was successfully transferred from hexaploid triticale to common wheat line SY95-71. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that subunit 1Bx23 has a faster mobility than subunit 1Bx7 and 1Bx20, but slower than 1Bx17. Primers designed from the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of SY95-71. Total nucleotide sequences of 3426 bp including an open reading frame of 2385 bp and upstream sequence of 1038 bp were obtained. Compared with the reported gene sequences of Glu-B1-1 alleles, including 1Bx7, 1Bx14, 1Bx20 and 1Bx17, the promoter region of the 1Bx23 was displayed close to 1Bx7 and 1Bx17. The deduced amino acid sequence of coding region of 1Bx23 exhibited 34, 30, 20 and 22 amino acid substitutions from that of 1Bx14, 1Bx20, 1Bx7 and 1Bx17, respectively. A phylogenetic tree based on the nucleotide sequence alignment of the Glu-1Bx alleles shows that the 1Bx23 are apparently clustered with 1Bx7 and 1Bx17, and more ancient than 1Bx14 and 1Bx20, suggesting that the evolution speeds are different among Glu-1Bx genes. Additionally, the potential use of wheat line SY95-71 to further screen the quality contribution of unique subunit 1Bx23 is also discussed.     

高分子量麦谷蛋白1Bx14亚基AS-PCR标记

根据已发表的1Bx14亚基的基因序列在不同位点设计了10对特异引物,从中筛选出1对引物,对HMW-GS在Glu-1Bx位点已知的10个小麦品种进行了PCR扩增.结果表明,具有1Bx14亚基的4个品种都能扩增出1条1 256 bp左右的特异带.用这一特异标记对山东省种植面积较大的40个品种进行PCR扩增(即等位专一PCR,AS-PCR),发现仅有5个品种携带1Bx14亚基.该AS-PCR标记可用于检测小麦品种在该位点的亚基组成,与SDS-PAGE相比,可显著提高检测的准确性和效率,可为种质鉴定和育种工作提供参考.     

Elucidation of the final reactions of DIMBOA-glucoside biosynthesis in maize: characterization of Bx6 and Bx7

Benzoxazinoids were identified in the early 1960s as secondary metabolites of the grasses that function as natural pesticides and exhibit allelopathic properties. Benzoxazinoids are synthesized in seedlings and stored as glucosides (glcs); the main aglucone moieties are 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA). The genes of DIBOA-glc biosynthesis have previously been isolated and the enzymatic functions characterized. Here, the enzymes for conversion of DIBOA-glc to DIMBOA-glc are identified. DIBOA-glc is the substrate of the dioxygenase BENZOXAZINLESS6 (BX6) and the produced 2,4,7-trihydroxy-2H-1,4-benzoxazin-3-(4H)-one-glc is metabolized by the methyltransferase BX7 to yield DIMBOA-glc. Both enzymes exhibit moderate K(m) values (below 0.4 mm) and k(cat) values of 2.10 s(-1) and 0.25 s(-1), respectively. Although BX6 uses a glucosylated substrate, our localization studies indicate a cytoplasmic localization of the dioxygenase. Bx6 and Bx7 are highest expressed in seedling tissue, a feature shared with the other Bx genes. At present, Bx6 and Bx7 have no close relatives among the members of their respective gene families. Bx6 and Bx7 map to the cluster of Bx genes on the short arm of chromosome 4.     

Isolation and molecular characterization of high molecular weight glutenin subunit genes 1Bx13 and 1By16 from hexaploid wheat

The high molecular weight glutenin subunit （HMW-GS） pair 1Bx13＋1By16 are recognized to positively correlate with bread-making quality; however, their molecular data remain unknown. In order to reveal the mechanism by which 1By16 and 1Bx13 creates high quality, their open reading frames （ORFs） were amplified from common wheat Atlas66 and Jimai 20 using primers that were designed based on published sequences of HMW glutenin genes. The ORF of 1By16 was 2220bp, deduced into 738 amino acid residues with seven cysteines including 59 hexapeptides and 22 nanopeptides motifs. The ORF of 1Bx13 was 2385bp, deduced into 795 amino acid residues with four cysteines including 68 hexapeptides, 25 nanopeptides and six tripepUdes motifs. We found that 1By16 was the largest y-type HMW glutenin gene described to date in common wheat. The 1By16 had 36 amino acid residues inserted in the central repetitive domain compared with 1By15. Expression in bacteria and western-blot tests confirmed that the sequence cloned was the ORF of HMW-GS 1By16, and that 1Bx13 was one of the largest 1Bx genes that have been described so far in common wheat, exhibiting a hexapeptide （PGQGQQ） insertion in the end of central repetitive domain compared with 1Bx7. A phylogenetic tree based on the deduced full-length amino acid sequence alignment of the published HMW-GS genes showed that the 1By16 was clustered with Glu-1B-2, and that the 1Bx13 was clustered with Glu-1B-1 alleles.     

Overexpression of the Bx7 high molecular weight glutenin subunit on the <Emphasis Type="Italic">Glu</Emphasis>-<Emphasis Type="Italic">B1</Emphasis> locus in a Korean wheat landrace

The Glu-B1al (Bx7^OE + By8) allele is important for bread-making quality. The allele was found in a Korean wheat landrace using specific DNA markers. Molecular analyses were conducted to identify the overexpressed Bx7 (Bx7^OE) subunit of the allele. The Korean wheat landrace (accession ID: IT166460) showed a similar protein expression level of Bx7 subunit, i.e., overexpression of Bx7 subunit towards cv. Glenlea, Canadian Western Red Spring wheat, which harbors Bx7^OE subunit of Glu-B1al as detected on SDS–PAGE (sodium dodecyl sulfate poly-acrylamide gel electrophoresis). In addition, 2-DE (two-dimensional electrophoresis) analysis revealed similar protein expression patterns of the Bx7 subunit regions of IT166460 and Glenlea. The proportion of Bx7 to total HMW-GSs (high molecular weight glutenin subunits) in IT166460 (56.17 ± 0.22%) was higher than that of Chinese Spring (34.75 ± 1.03%) and even that of Glenlea (46.25 ± 1.76%) as assessed by RP-HPLC (reverse-phase high-performance liquid chromatography). Overexpression of Bx7 subunit was caused by gene duplication and indels of the promoter region of the Bx7 gene. IT166460 attained the 43 bp indel of the promoter region, as did Glenlea, i.e., the amplicon size of IT166460 was the same as that of Glenlea. In addition, the nucleotides present in the duplicated gene in IT166460 were the same as those in Glenlea. Bx7^OE subunit is critical for dough strength. However, most wheat accessions harboring the subunit are distributed in America. Furthermore, most Korean wheats have little genetic variation in glutenin composition and are associated with inferior bread quality. Hence, IT166460 could be used to improve bread-making quality in the Korean wheat breeding program.     

High-frequency plant regeneration by <Emphasis Type="Italic">in vitro</Emphasis> shoot proliferation in cotyledonary node explants of grasspea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Summary Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl⁻¹ (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl⁻¹ (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.     

The effects of hydrocarbon pollution from a two‐stroke outboard engine on the feeding behaviour of Lythrypnus dalli (Perciformes: Gobidae)

Feeding behaviour (time of attack on flake fish food) in Lythrypnus dalli was measured under pollution concentrations of 2.1 and 4.01mgl^?1 total extractable material produced by a two‐stroke outboard engine. The variance of the times increased in pollution versus control treatments for both concentrations. No significant difference was found between the mean times of the control and 2.1 mgl^?1 pollution treatments. A significant difference was found between the mean times of the control and pollution treatments for two different tests at 4.01 mgl^?1. The combination of the increase in mean time of food attack and the increase of variance indicates that pollution concentrations lower than LD50 levels change feeding behaviour, and thus may indirectly effect the survival of the organism.     

小麦HMW-GS 1Bx14基因特异标记体系的建立

比较1Bx14及其它已知HMW-GS基因的启动子和编码区,根据其不同点设计出1Bx14基因特异扩增引物。以8种已知HMW-GS组成的小麦DNA为模板进行PCR扩增。结果表明：具有1Bx14亚基的品种扩增出1条400bp左朽特异条带。结合该特异标记和已报道的1Dx5特异标记对2个F2杂交群体进行检测,从184个F2单株中筛选出111个同时含有1Bx14和1Dx5基因的单株。该研究结果可为种质鉴定和亚基整合育种提供参考。     

Inducible RNA interference uncovers the Drosophila protein Bx42 as an essential nuclear cofactor involved in Notch signal transduction

We used the UAS/GAL4 two component system to induce mRNA interference (mRNAi) during Drosophila development. In the adult eye the expression from white transgenes or the resident white locus is significantly repressed by the induction of UAS-wRNAi using different GAL4 expressing strains. By induced RNAi we demonstrate that the conserved nuclear protein Bx42 is essential for the development of many tissues. Phenotypically the effects of Bx42 RNAi resemble those obtained for certain classes of Notch mutants, pointing to an involvement of Bx42 in the Notch signal transduction pathway. The wing phenotype following overexpression of Suppressor of Hairless is strongly enhanced by simultaneous Bx42 RNAi induction in the same tissue. Target genes of Notch signaling like cut and Enhancer of split m8 were suppressed by induction of Bx42 RNAi. Our results demonstrate that inducible RNAi is a powerful tool to study the role of essential genes throughout development.     

Molecular characterization of HMW glutenin subunit allele <Emphasis Type="Italic">1Bx14</Emphasis>: further insights into the evolution of <Emphasis Type="Italic">Glu-B1-1</Emphasis> alleles in wheat and related species

1Bx14 is a member of the high molecular weight (HMW) glutenin subunits specified by wheat Glu-B1-1 alleles. In this work, we found that the full-length amino acid sequence of 1Bx14 derived from cloned coding region was similar, but not identical, to that of 1Bx20. In the N-terminal domains of 1Bx14 and 1Bx20, the last two of the three cysteine residues, which are conserved in 1Bx7, 1Bx17 and homoeologous 1Ax and 1Dx subunits, were replaced by tyrosine residues. In the 5 flanking regions (–900 to –1,200 bp relative to the start codon), a novel miniature inverted-repeat transposable element insertion was present in 1Bx14 and 1Bx20 but not 1Bx7 and 1Bx17. 1Bx14 and 1Bx20 like alleles were readily found in tetraploid wheat subspecies but not several S genome containing Aegilops species. Phylogenetic analysis showed that the four molecularly characterized Glu-B1-1 alleles (1Bx7, 1Bx14, 1Bx17, 1Bx20) could be divided into two allelic lineages. The lineage represented by 1Bx7 and 1Bx17 was more ancient than the one represented by 1Bx14 and 1Bx20. Combined, our data establish that 1Bx14 and 1Bx20 represent a novel subclass of Glu-B1-1 alleles. Based on current knowledge, potential mechanism involved in the differentiation of two Glu-B1-1 lineages is discussed.Electronic Supplementary Material Supplementary material is available for this article at     

Dissemination of the highly expressed Bx7 glutenin subunit (<Emphasis Type="Italic">Glu-B1al</Emphasis> allele) in wheat as revealed by novel PCR markers and RP-HPLC

Increased expression of the high molecular weight glutenin subunit (HMW-GS) Bx7 is associated with improved dough strength of wheat (Triticum aestivum L.) flour. Several cultivars and landraces of widely different genetic backgrounds from around the world have now been found to contain this so-called over-expressing allelic form of the Bx7 subunit encoded by Glu-B1al. Using three methods of identification, SDS-PAGE, RP-HPLC and PCR marker analysis, as well as pedigree information, we have traced the distribution and source of this allele from a Uruguayan landrace, Americano 44D, in the mid-nineteenth century. Results are supported by knowledge of the movement of wheat lines with migrants. All cultivars possessing the Glu-B1al allele can be identified by the following attributes: (1) the elution of the By sub-unit peak before the Dx sub-unit peak by RP-HPLC, (2) high expression levels of Bx7 (>39% Mol% Bx), (3) a 43 bp insertion in the matrix-attachment region (MAR) upstream of the gene promoter relative to Bx7 and an 18 bp nucleotide duplication in the coding region of the gene. Evidence is presented indicating that these 18 and 43 bp sequence insertions are not causal for the high expression levels of Bx7 as they were also found to be present in a small number of hexaploid species, including Chinese Spring, and species expressing Glu-B1ak and Glu-B1a alleles. In addition, these sequence inserts were found in different isolates of the tetraploid wheat, T. turgidum, indicating that these insertion/deletion events occurred prior to hexaploidization.     

Cold plasma relieved toxicity signs of nano zinc oxide in <Emphasis Type="Italic">Capsicum annuum</Emphasis> cayenne via modifying growth,differentiation, and physiology

Taking functional scientific devices and metal-based nanoparticles into account, the present research was carried out to evaluate the plant (Capsicum annuum) responses to cold plasma and zinc oxide nanoparticle (nZnO) in in vitro and pot conditions. Seeds were exposed to plasma (0.84 W/cm² surface power densities) with three exposure times (0, 60, and 120 s) and/or two concentrations of nZnO (0 and 100 mgl^??1). The treated seeds were cultured in hormone-free MS medium (MS) or supplemented with 2 mgl^??1 BA and 0.5 mgl^??1 IAA (MSH). The seed pre-treatment with plasma enhanced a germination process and plant early growth, in contrast with the nZnO treatment. The treatment of nZnO significantly decreased the total fresh mass and leaf area in the seedlings grown in both culture media, while its growth-delaying impact was mitigated by the plasma treatment. The chlorophyll a and carotenoid were increased to 39.35 and 32% for the plasma-treated seedlings, respectively, than the control. The plasma and/or nZnO treatments acted as effective elicitors to induce the peroxidase activities in both culture media. Similarly, the activities of phenylalanine ammonia-lyase and soluble phenols were found to be significantly higher in the plasma and/or nZnO groups in both roots and leaves. Interestingly, inhibiting effects of nZnO on xylem differentiation was alleviated by the plasma treatments. In the pot condition, soaking seeds before the plasma treatment was the most effective method to affect plant growth. This is a first report reflecting the potential benefits of the cold plasma treatment to improve plant growth and resistance to the nanoparticle.     

Protoplasts from <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed cell line of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. regenerated to hairy roots

Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×10⁶ per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented with 2 mgl⁻¹ (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl⁻¹ (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl⁻¹ casein hydrolyzate at a plating density of 1.0×10⁵ per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators. Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast culture system would be valuable for further somatic hybridization in forage legumes.     

Effect of Putrescine, 4-PU-30, and Abscisic Acid on Maize Plants Grown under Normal, Drought, and Rewatering Conditions

The experiments were carried out with maize (Zea mays L.) seedlings, hybrid Kneja 530, grown hydroponically in a growth chamber. Twelve-day-old plants were foliar treated with putrescine, N¹-(2-chloro-4-pyridyl)-N²-phenylurea (4-PU-30), and abscisic acid (ABA) at concentrations of 10⁻⁵ m. Twenty-four hours later the plants were subjected to a water deficit program, induced by 15% polyethylene glycol (PEG; molecular weight, 6,000). Three days after drought stress half of the plants were transferred to nutrient solution for the next 3 days. The effects of the water shortage, rewatering, and plant growth regulator (PGR) treatment on the fresh and dry weights, leaf pigment content, proline level, relative water content (RWC), transpiration rate, activities of catalase and guaiacol peroxidase, hydrogen peroxide content, and level of the products of lipid peroxidation were studied. It was established that the application of PGRs alleviated to some extent the plant damage provoked by PEG stress. At the end of the water shortage program the plants treated with these PGRs possessed higher fresh weight than drought-subjected control seedlings. It was found also that putrescine increased the dry weight of plants. Under drought, the RWC and transpiration rate of seedlings declined, but PGR treatment reduced these effects. The accumulation of free proline, malondialdehyde, and hydrogen peroxide was prevented in PGR-treated plants compared with the water stress control. The results provided further information about the influence of putrescine, 4-PU-30, and ABA on maize plants grown under normal, drought, and rewatering conditions. Received September 25, 1997; accepted August 10, 1998     

Effects of benthivorous bream (Abramis brama) and carp (Cyprinus carpio) on sediment resuspension and concentrations of nutrients and chlorophyll a

1. The effect of benthivorous bream and carp on sediment resuspension and the concentrations of nutrients and chlorophyll a were studied in sixteen experimental ponds (mean depth 1m, mean area 0.1 ha, sandy clay/clay sediment), stocked with bream or carp at densities varying from 0 to 500 kg ha^?1. Planktivorous perch (Perca fluviatilis L.) were added to some ponds to suppress zooplankton. 2. Suspended sediment concentrations increased linearly with biomass of benthivorous fish. Bream caused an increase of 46 g sediment m^?2 day^?1 per 100kg bream ha^?1 and a reduction of 0.38m^?1 in reciprocal Secchi disc depth, corresponding to an increase in the extinction coefficient of 0.34m^?1. 3. No relationship was found between size of fish and amount of resuspension, but the effect of bream was twice as great as that of carp. Benthivorous feeding was reduced in May because alternative food (zooplankton) was available. 4. Assuming a linear relationship, chlorophyll a level increased by 9.0 μgI^?1, total P by 0.03mgl^?1 and Kjeldahl-N by 0.48mgl^?1 per 100kg bream ha^?1. Silicate, chlorophyll a, total P and total N were all positively correlated with fish biomass, but orthophosphate showed no correlation.