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, 66 (13), 3917-30

Prolonged Expression of the BX1 Signature Enzyme Is Associated With a Recombination Hotspot in the Benzoxazinoid Gene Cluster in Zea Mays

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Prolonged Expression of the BX1 Signature Enzyme Is Associated With a Recombination Hotspot in the Benzoxazinoid Gene Cluster in Zea Mays

Linlin Zheng et al. J Exp Bot.

Abstract

Benzoxazinoids represent preformed protective and allelopathic compounds. The main benzoxazinoid in maize (Zea mays L.) is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to herbivores and microbes. Protective concentrations are found predominantly in young plantlets. We made use of the genetic diversity present in the maize nested association mapping (NAM) panel to identify lines with significant benzoxazinoid concentrations at later developmental stages. At 24 d after imbibition (dai), only three lines, including Mo17, showed effective DIMBOA concentrations of 1.5mM or more; B73, by contrast, had low a DIMBOA content. Mapping studies based on Mo17 and B73 were performed to reveal mechanisms that influence the DIMBOA level in 24 dai plants. A major quantitative trait locus mapped to the Bx gene cluster located on the short arm of chromosome 4, which encodes the DIMBOA biosynthetic genes. Mo17 was distinguished from all other NAM lines by high transcriptional expression of the Bx1 gene at later developmental stages. Bx1 encodes the signature enzyme of the pathway. In Mo17×B73 hybrids at 24 dai, only the Mo17 Bx1 allele transcript was detected. A 3.9kb cis-element, termed DICE (distal cis-element), that is located in the Bx gene cluster approximately 140 kb upstream of Bx1, was required for high Bx1 transcript levels during later developmental stages in Mo17. The DICE region was a hotspot of meiotic recombination. Genetic analysis revealed that high 24 dai DIMBOA concentrations were not strictly dependent on high Bx1 transcript levels. However, constitutive expression of Bx1 in transgenics increased DIMBOA levels at 24 dai, corroborating a correlation between DIMBOA content and Bx1 transcription.

Keywords: Allele-specific expression; DIMBOA; QTL mapping; biosynthetic cluster; cis-element; defence; secondary metabolites..

Figures

Fig. 1.
Fig. 1.
Schematic presentation of the Bx gene cluster on chromosome 4. Bx1 and Bx2 are separated by 2.5kb. DICE is a 3.9kb cis-element influencing transcriptional regulation of Bx1 and was detected in this study. It is located at position 3 113 226, about 0.5kb downstream of Bx5.
Fig. 2.
Fig. 2.
Analysis of 24 dai plants of the NAM founder line panel. (A) DIMBOA content. (B) Bx1 transcript level. (C) Bx4 transcript level. (D) Bx8 transcript level. Transcript levels were normalized to the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) mRNA level. Two sets of material were grown and at least one sample of pooled leaf material (three to four plants; results show means±standard deviation) was analysed.
Fig. 3.
Fig. 3.
Analysis of B73, Mo17, and hybrids in seedling shoots at 4 dai (A, C, E) and in 24 dai plants (B, D, F). (A, B), DIMBOA content. (C, D) Bx1 transcript levels. (E, F) Allele-specific analysis of Bx1 transcript levels; light grey shading indicates the transcript level of the B73 Bx1 allele and black indicates the transcript level of the Mo17 Bx1 allele.Transcript levels were normalized to GAPC. At least four samples of pooled leaf material (three to four plants) were analysed. Statistical analysis in (A–D) was by Kruskal–Wallis test and multiple comparison of treatments; identical letters above columns indicate no statistical difference (P>0.05). In (E) and (F), Student’s t-test was used. *P<0.05. Mean values and standard deviation are indicated. n.s., Not significant.
Fig. 4.
Fig. 4.
Analysis of recombinants. (A) Schematic presentation of recombination breakpoints. Marker positions are indicated in the header line and the distance of the marker to Bx1 is indicated by the number, e.g. M137 is located 137kb upstream of Bx1. The size of gaps between the markers demonstrating recombination in the neighbourhood of DICE is given in the light grey fields. The DICE region is delimited by grey lines. Grey, Mo17 genotype, white, B73 genotype, light grey: unknown genotype. (B, C) DIMBOA content at 24 dai (B) and Bx1 transcript level at 24 dai (C) in the respective groups of recombinants. All homozygous recombinants of group A–D were crossed with Mo17 and all recombinants of group E–G with B73, to yield heterozygous conformation for Bx1. B73 or Mo17 in (C) indicates whether the transcript level of the recombinant chromosome’s Bx1 gene is the lower (B73-type) or the higher (Mo17-type) one. Statistical analysis was by Kruskal–Wallis test and multiple comparison of treatments. Mean values and standard deviation are indicated. Identical letters above columns indicate no statistical difference (P>0.5).
Fig. 5.
Fig. 5.
Analysis and genotypes of selected lines. (A, B) DIMBOA content (A) and Bx1 mRNA level (B) in 24 dai plants. Statistical analysis was by Kruskal–Wallis test and multiple comparison of treatments. Mean values and standard deviation are indicated. Identical letters above columns indicate no statistical differences (P>0.5). (C) Schematic representation of the genotypes of these lines for chromosome 4 and the QTL regions on chromosomes 1 and 5. White, B73; black, Mo17; hatched, hybrid genotype. The positions of Bx1 and of QTLs are indicated. For genotyping, the markers listed in Supplementary material were used. The position of the QTL is given in Supplementary Table S2.
Fig. 6.
Fig. 6.
Analysis of NILs. (A) Scheme of the B73 isogenic line B183 and the Mo17 isogenic line M31. The Mo17 genotype is indicated in black and the B73 genotype in grey. The dashed line indicates the position of the clustered Bx genes (Bx1Bx5 and Bx8). (B, C) DIMBOA content (B) and Bx1 transcript levels (C) in 24 dai plants. Statistical analysis was by Kruskal–Wallis test and multiple comparison of treatments. Mean values and standard deviation are indicated. Identical letters above columns indicate no statistical differences (P>0.5).
Fig. 7.
Fig. 7.
Bx1 transcript levels in 24 dai hybrids of the NILs. (A) In the B184×Mo17 hybrid, Bx1 is homozygous for the Mo17 allele. (B) In the M31×Mo17 hybrid, the Mo17 and B73 alleles are present. The transcript levels were normalized to GAPC. Statistical analysis was by Kruskal–Wallis test and multiple comparison of treatments. Mean values and standard deviation are indicated. Identical letters above columns indicate no statistical differences (P>0.5).
Fig. 8.
Fig. 8.
DIMBOA content in 24 dai transgenic plants overexpressing Bx1. Statistical analysis was by Kruskal–Wallis test and multiple comparison of treatments. Mean values and standard deviation are indicated. Identical letters above columns indicate no statistical differences (P>0.5).

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