Rearrangement of the genes for the biosynthesis of benzoxazinones in the evolution of Triticeae species

Gramineous plants, including the major agricultural crops wheat (Triticum aestivum L.), rye (Secale cereale L.) and maize (Zea mays L.), accumulate benzoxazinones (Bxs) as defensive compounds. Previously, we isolated cDNAs of the Bx biosynthetic genes in wheat, TaBx2- TaBx5, that encode the enzymes catalyzing the sequential hydroxylation of indole to Bxs. In this study we isolated a cDNA of TaBx1, which encodes the first enzyme of the Bx pathway of wheat. The level of identity (80%) in deduced amino-acid sequence between TaBx1 and the corresponding maize gene Bx1 was as high as those shown between TaBx2- TaBx5 and the corresponding maize genes Bx2- Bx5, respectively. Southern blot analysis using the TaBx1- TaBx5 cDNAs as probes was conducted with aneuploid lines of hexaploid wheat in order to determine sub-chromosomal locations of the five Bx biosynthetic genes in Triticeae species. In wheat, TaBx1 and TaBx2 co-existed in specific regions of chromosomes 4A, 4B and 4D, and TaBx3- TaBx5 were localized together in the distal regions of the short arms of chromosomes 5A, 5B and 5D. TaBx3 and TaBx5 were found to have duplicated loci in the long arm and the short arm of chromosome 5B, respectively. In rye, homoeoloci of TaBx1 and TaBx2 were located on chromosome 7R and those for TaBx3- TaBx5 were located on chromosome 5R. In barley, no Southern blot band was detected with any of the probes under the highly stringent hybridization conditions, suggesting that the non-Bx phenotype of barley is attributable to the loss of Bx biosynthetic genes.     

Near-isogenic lines for measuring phenotypic effects of DIMBOA-Glc methyltransferase activity in maize

Three O-methyltransferases (BX10a, b, c) catalyze the conversion of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) to 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) in maize (Zea mays). Variation in benzoxazinoid accumulation and resistance to Rhopalosiphum maidis (corn leaf aphid) was attributed to a natural CACTA family transposon insertion that inactivates Bx10c. Whereas maize inbred line B73 has this transposon insertion, line CML277 does not. To characterize the phenotypic effects of DIMBOA-Glc methyltransferase activity, we created near-isogenic lines derived from B73 and CML277 that do or do not contain the transposon insertion. Bx10c inactivation causes high DIMBOA-Glc, low HDMBOA-Glc, and decreased aphid reproduction relative to near-isogenic lines that have a functional Bx10c gene. These results confirm the importance of this locus in maize aphid resistance. The availability of Bx10c near-isogenic lines will facilitate further research on the function of different benzoxazinoids and DIMBOA-Glc methyltransferase activity in maize defense against herbivores and pathogens.     

叶片涂施茉莉酸对玉米幼苗防御反应的时间和浓度效应

茉莉酸是环境胁迫下植物产生防御反应的重要信号物质, 但它发挥生理作用的时间和浓度效应以及该效应在叶片和根系中差异性并不清楚。该文以‘高油115’玉米(Zea mays)为材料, 采用4种浓度(1、2.5、5和10 mmol·L^-1)的外源茉莉酸溶液涂施玉米幼苗叶片, 在3~48 h的不同时间内跟踪测定叶片和根系中的直接防御物质(丁布(DIMBOA)和总酚)含量及其合成调控基因(Bx1、Bx9和PAL)、直接防御蛋白调控基因(PR-1、PR-2a和MPI)和间接防御物质挥发物调控基因(FPS和TPS)表达的动态变化。结果表明, 外源茉莉酸处理对玉米叶和根系的化学防御反应具有显著的时间和浓度效应。茉莉酸处理玉米叶片后3~6 h就能诱导叶片中Bx9和PAL基因的表达, 使得丁布和总酚的含量显著增加, 且与处理浓度有呈正比的趋势, 随后诱导作用逐渐减弱; 茉莉酸处理还能明显诱导叶片中PR-2a和MPI基因的表达, 诱导作用分别持续到24和48 h; 在处理后3~6 h内, 高浓度茉莉酸处理对挥发物调控基因FPS表达起诱导作用, 而低浓度茉莉酸则对TPS基因的表达起诱导作用。此外, 茉莉酸处理玉米叶片还能间接影响到根系的防御反应, 但大部分检测指标表明间接诱导作用主要出现在处理后期(24~48 h)。例如, 在处理后48 h, 茉莉酸能系统增加根系中直接防御物质丁布和总酚的含量, 增强根系中防御相关基因PR-2a、MPI、FPS和TPS的表达, 并有随茉莉酸处理浓度的增加而增强的趋势。可见, 外源茉莉酸叶片涂施玉米幼苗对根系的间接诱导作用不如对叶片的直接诱导作用强; 叶片启动防御反应的时间较根系早; 随着处理浓度的增加, 茉莉酸对叶片和根系中防御反应的诱导作用有增强的趋势。     

Natural Variation in Maize Aphid Resistance Is Associated with 2,4-Dihydroxy-7-Methoxy-1,4-Benzoxazin-3-One Glucoside Methyltransferase Activity

Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.     

Induction of HDMBOA-Glc accumulation and DIMBOA-Glc 4-O-methyltransferase by jasmonic acid in poaceous plants

Induction of the accumulation of 2-(2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (HDMBOA-Glc) by jasmonic acid (JA) was investigated in wheat, Job's tears (Coix lacryma-jobi), and rye. An increase in HDMBOA-Glc and a corresponding decrease in 2-(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (DIMBOA-Glc) were found in wheat and Job's tears, whereas no such changes were observed in rye. The activity of S-adenosyl-L-methionine:DIMBOA-Glc 4-O-methyltransferase which catalyzes the conversion of DIMBOA-Glc to HDMBOA-Glc was detected in wheat leaves treated with 50 micro M JA. The activity started to increase 3 h after treatment with JA, reached a maximum after 9 h, and then decreased gradually. This mode of induction was well correlated with that for the accumulation of HDMBOA-Glc, indicating the induction of enzyme activity was responsible for the accumulation of HDMBOA-Glc. The enzyme was purified from JA-treated wheat leaves by three steps of chromatography, resulting in 95-fold purification. The enzyme showed strict substrate specificity for DIMBOA-Glc with a K(m) value of 0.12 mM. DIBOA-Glc was also accepted as substrate, but the K(m) value was 10 times larger than that for DIMBOA-Glc. The aglycones, DIMBOA and DIBOA, were not methylated by the enzyme. The K(m) value for S-adenosyl-L-methionine was 0.06 mM. The optimum pH and temperature were 7.5 and 35 degrees C, respectively. The activity was slightly enhanced by the presence of 1 mM EDTA, while heavy metal ions at 5 mM completely inhibited the activity.     

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.     

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.     

Selenium nano-particle induced alterations in expression patterns of heat shock factor A4A (HSFA4A), and high molecular weight glutenin subunit 1Bx (Glu-1Bx) and enhanced nitrate reductase activity in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Taking account of heat shock factor A4A (HSFA4A) as a hydrogen peroxide sensor, anti-apoptosis agent, and crosslink component with critical signaling cascades, the current study was carried out to monitor possible changes in expression of this gene as well as some other important characteristics in wheat plants exposed to selenium nano-particle (nSe). Wheat seedlings were treated with nSe (0, 5, 10, and 50 mgl^?1). In germinating stage, plant fresh weights were reduced in nSe-treated seedlings, among which the nSe of 50 provoked roots turned brown. The nSe triggered the increases in the expressions of HSFA4A, in the plate. In the pot condition, shoot fresh weights in nSe-supplemented seedlings were decreased by approximately 22%. The nSe of 5 and 10 mgl^?1, respectively induced the expression of HSFA4A by 3.4- and 9.15-folds, contrasted with nSe50. Increasing times of sprays caused the dramatic reductions of the expression of HSFA4A in the nSe-supplemented groups. Moreover, the treatment of nSe stimulated expression of high molecular weight glutenin subunit 1Bx (Glu-1Bx) by fourfold, over the control. While the dramatic decrease in the expression of Glu-1Bx was recorded with increasing times of spray. Nitrate reductase activities were significantly improved by approximately 47% in nSe-fortified seedlings. Also, the foliar supplementation of nSe of 5 mgl^?1 provoked the significant inductions in peroxidase activity by 8%, whereas two other nSe treatments declined it. It may be stated that the nSe may modify the expression of HSFA4A, thereby triggering specific signaling and altering metabolism.     

Conservation in wheat high-molecular-weight glutenin gene promoter sequences: comparisons among loci and among alleles of the GLU-B1-1 locus

 The high-molecular-weight glutenin (HMW) genes and encoded subunits are known to be critical for wheat quality characteristics and are among the best-studied cereal research subjects. Two lines of experiments were undertaken to further understand the structure and high expression levels of the HMW-glutenin gene promoters. Cross hybridizations of clones of the paralogous x-type and y-type HMW-glutenin genes to a complete set of six genes from a single cultivar showed that each type hybridizes best within that type. The extent of hybridization was relatively restricted to the coding and immediate flanking DNA sequences. Additional DNA sequences were determined for four published members of the HMW-glutenin gene family (encoding subunits Ax2^*, Bx7, Dx5, and Dy10) and showed that the flanking DNA of the examined genes diverge at approximately −1200 bp 5′ to the start codon and 200–400 bp 3′ to the stop codon. These divergence sites may indicate the boundaries of sequences important in gene expression. In addition, promoter sequences were determined for alleles of the Bx gene (Glu-B1-1), a gene reported to show higher levels of expression than other HMW-glutenin genes and with variation among cultivars. The sequences of Bx promoters from three cultivars and one wild tetraploid wheat indicated that all Bx alleles had few differences and contained a duplicated portion of the promoter sequence “cereal-box” previously suspected as a factor in higher levels of expression. Thus, the “cereal-box” duplication preceeded the origin of hexaploid wheat, and provides no evidence to explain the variations in Bx subunit synthesis levels. One active Bx allele contained a 185-bp insertion that evidently resulted from a transposition event. Received: 5 August 1997 / Accepted: 6 November 1997     

Structure,variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence

In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2554 bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> − 700 bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5′-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.     

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.     

Induced accumulation of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) in maize leaves

Accumulation of 2-(2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (HDMBOA-Glc) was induced in maize leaves by treatment with CuCl2, chitopentaose, penta-N-acetylchitopentaose, or jasmonic acid (JA). The accumulation of HDMBOA-Glc was accompanied by a decrease in level of 2-(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (DIMBOA-Glc). When the leaf segments were treated with JA in the presence of [Me-2H3]L-methionine, the label was efficiently incorporated into HDMBOA-Glc, while no incorporation into DIMBOA-Glc or HMBOA-Glc was detected, suggesting the conversion of constitutive DIMBOA-Glc to HDMBOA-Glc by methylation at the 4-position. Levels of endogenous JA and its leucine conjugate transiently increased prior to the accumulation of HDMBOA-Glc in leaf segments treated with CuCl2 and chitopentaose. The lipoxygenase inhibitor ibuprofen suppressed the accumulation of HDMBOA-Glc induced by CuCl2 treatment, and the reduced accumulation of HDMBOA-Glc was recovered by addition of JA. These findings suggested that JA functions as a signal transducer in the induction of HDMBOA-Glc accumulation.     

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.