Isolation and Structure Determination of Drought-Induced Multihexose Benzoxazinoids from Maize (Zea mays)

Abstract

Benzoxazinoids (BXDs) are plant specialized metabolites exerting a pivotal role in plant nutrition, allelopathy, and defenses. Multihexose benzoxazinoids were previously observed in cereal-based food products such as whole-grain bread. However, their production in plants and exact structure have not been fully elucidated. In this study, we showed that drought induced the production of di-, tri-, and even tetrahexose BXDs in maize roots and leaves. We performed an extensive nuclear magnetic resonance study and elucidated the nature and linkage of the sugar units, which were identified as gentiobiose units β-linked (1″ → 6') for the dihexoses and (1″ → 6')/(1‴ → 6″) for the trihexoses. Drought induced the production of DIMBOA-2Glc, DIMBOA-3Glc, HMBOA-2Glc, HMBOA-3Glc, and HDMBOA-2Glc. The induction was common among several maize lines and the strongest in seven-day-old seedlings. This work provides ground to further characterize the BXD synthetic pathway, its relevance in maize-environment interactions, and its impact on human health.

Keywords: DIMBOA dihexose; DIMBOA trihexose; NMR; drought; maize; multihexose benzoxazinoids.

Figure 1

Drought induced putative multihexose benzoxazinoids (BXDs) in maize leaves. Base peak intensity (BPI) chromatograms of control (A) and drought-stressed (B) maize leaves. Putative multihexose benzoxazinoids (BXDs) are labeled as numbers in the chromatograms. (1) Putative DIMBOA-dihexose (m/z 534.1459), (2) putative DIMBOA-trihexose (m/z 696.1987), (3) putative HMBOA-dihexose (m/z 518.1510), (4) putative HMBOA-trihexose (m/z 680.2038), and (6) putative HDMBOA-dihexose (m/z 594.1670). m/z: mass-to-charge ratio.

Figure 2

Extracted ion chromatograms of multihexose benzoxazinoids detected in control (dashed line) and drought-stressed (solid line) maize leaves. (1) Putative DIMBOA-dihexose (m/z 534.1459), (2) putative DIMBOA-trihexose (m/z 696.1987), (3) putative HMBOA-dihexose (m/z 518.1510), (4) putative HMBOA-trihexose (m/z 680.2038), (5) putative HM₂BOA-dihexose (m/z 548.1615), (6) putative HDMBOA-dihexose (m/z 594.1670), (7) putative DIBOA-dihexose (m/z 504.1353), and (8) putative DIMBOA-tetrahexose (m/z 858.2515). cps: count per second. m/z: mass-to-charge ratio.

Figure 3

Structure of compounds 1 to 7.

Figure 4

Drought induced di- and trihexose benzoxazinoids (BXDs) in maize roots and shoots. BXD peak heights (Mean ± sem) in B73 maize leaves (A) and roots (B) (n = 4–5 per treatment and tissue). Drought was established 4 days after sowing and all plants were harvested 6 days later. Gray bars: ambient conditions, black bars: drought conditions. cps: count per second. Student t tests and Mann–Whitney Rank Sum tests were conducted. Dots and stars indicate trends and significant differences respectively (.: p < 0.10, * p < 0.05).

Figure 5

Drought-mediated induction of double and triple hexoses was transient. DIMBOA-2Glc concentrations in maize shoots (A) and roots (B) under control and drought conditions. DIMBOA-3Glc concentrations in maize shoots (C) and roots (D) under control and drought conditions. HMBOA-2Glc concentrations in maize shoots (E) and roots (F) under control and drought conditions (n = 8 per treatment and time point). Control (green lines): 23% soil moisture (v/v); Drought (yellow lines): 16.6% soil moisture (v/v). tmt: treatment (control or drought). FW: fresh weight. Mean ± SEM are shown. Two-way ANOVAs on ranks were conducted, followed by posthoc Holm–Sidak tests when relevant. Stars indicate significant differences within time points (.: p < 0.10; *: p < 0.05; **: p < 0.01; ***: p < 0.001).

Figure 6

Multihexose benzoxazinoids were induced by drought in several maize varieties. Heatmap of benzoxazinoid (BXD) levels in drought-stressed plants (leaves and roots) compared to control plants in B73, CML277, Hp301, and Oh7B maize varieties (n = 2–5 per treatment and variety). The log fold (log10) changes between drought-stressed and control plants are shown. Green indicates a decrease in BXD levels upon drought. Red indicates an increase in BXD levels upon drought. Student t tests and Mann–Whitney Sum Rank tests were conducted. Dots and stars indicate trends and significant differences respectively (.: p < 0.10, *: p < 0.05, **: p < 0.01, ***: p < 0.001).