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

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.


INTRODUCTION
−5 BXDs can have cascading effects on food quality and human health, and their consumption is associated with anti-inflammatory and anticancer effects. 6,7Understanding the different molecular structures of BXDs is crucial for improving the reliability and robustness of analytical methods, for identifying their underlying biosynthesis genes, and ultimately, for comprehending their potential influence on human health. 8,9XDs include benzoxazolinones with a 1,3-benzoxazol-2one core structure and benzoxazinones, possessing a 1,4benzoxazin-3-one scaffold.In rye, BXD species with no methoxy group on the aromatic ring, such as DIBOA and DIBOA-Glc, are dominant, whereas in maize, methylated forms such as DIMBOA and DIMBOA-Glc are predominant.In wheat, both species can be found in similar amounts. 10−14 Consequently, the exact nature and configuration of the hexose units as well as the relative and absolute stereochemistry of multihexose BXDs have remained undefined.
BXDs can transition from plants to the human food supply, where they can impact human health.Food processing, such as watering/drying the seeds, or hydrothermal processing (HTP) of cereals were found to increase BXD levels, including dihexose BXDs, in processed products. 3,4,12For instance, rye-based products contain approximately 122 μg/g dry mass HBOA-Glc-Hex and 300 μg/g dry mass DIBOA-Glc-Hex. 4 The impact of multihexose BXDs on human health remains unexplored, hindered by several factors, including the unavailability of purified compounds (attributable to their limited presence in healthy plants), a lack of structural characterization, and their omission from analytical methodologies.
Here, we demonstrate that drought enhances the production of multihexose BXDs in maize root and shoot tissues.We conducted an extensive nuclear magnetic resonance (NMR) study to determine the nature and configuration of the multihexose chains.We then characterized their induction patterns and kinetics in different maize tissues.Understanding the structure of these multihexose BXDs is pivotal for enabling their inclusion in analytical methods, enhancing our ability to monitor their presence and impact on food quality and human nutrition.
2.2.Drought Induction.After planting, the soil moisture was either kept at 23% v/v (ambient conditions) or left to decrease to 16% v/v (drought conditions, reached at day 4 after planting).Ambient and drought conditions were then maintained by weighing the pots and adding the required amounts of water daily.The drought moisture conditions were determined based on the projected RCP8.5 climate scenario, using a correlation between decrease precipitation levels and soil moisture levels as in refs 17−19.A 16.6% v/v moisture led to moderate drought symptoms in maize plants.All plants were harvested 10 days after sowing.Roots and shoots were harvested separately, flash-frozen in liquid nitrogen, and ground to a fine powder using a mortar and a pestle in the presence of liquid nitrogen.
2.3.Benzoxazinoid Analyses.The BXDs analysis was adapted from a previously described protocol. 20Aliquots of powder samples (100 mg) were mixed with 1 mL of a 70:30 mixture of methanol (HPLC grade) and water (milli-Q) containing 0.1% formic acid (LC-MS grade).All extracts were vortexed for 20 s and centrifuged at 13000 rpm for 20 min at 10 °C, and the supernatants were collected for LC-MS analyses.Where necessary, samples were diluted 1:10 prior to analysis.
2.4.Crude Extract.Ten days after germination, root and shoot samples were gently washed in tap water, patted dry with paper, flash frozen in liquid nitrogen, and stored at −80 °C until further processing.The samples were ground manually with mortar and pestle in the presence of liquid nitrogen.Root and shoot samples were pooled to give 450 g of raw material.The extraction was performed in two equal batches (225 g each) as follows: the cold ground material was added to 1.5 L of MeOH and homogenized for 3 min with an immersion disperser (Polytron PT 10−35, Kinematica, CHE), followed by suction filtration through a P3 sintered glass filter equipped with two layers of filter paper.The filter cake was collected and suspended again in 1.5 L MeOH.After a second homogenization and a new filtration, the filtrates were combined and concentrated under reduced pressure on a rotary evaporator.Upon evaporation, a yellow-green sticky residue formed on the round-bottom flask walls.This residue was discarded, and the remaining aqueous phase was separated.The same procedure was repeated for the second batch of frozen ground material.The two aqueous residues obtained were pooled and lyophilized to provide a strong yellow crude dry extract.The presence of compounds 1−7 was verified by HPLC-MS prior to isolation.ppm) except for compounds 6 and 7.In this case, spectra were calibrated on H-2′ (3.25 ppm) and C2′ (73.1 ppm).Acquired spectra were processed using the Mnova NMR software package (v.14.2.0, MestReLab Research S.L., Spain).

Statistical Analysis.
All statistical analyses were conducted on Sigma Plot 14.5.The distribution and heteroscedasticity of the data residuals were assessed by using the Shapiro−Wilk and Brown−Forsythe tests.Two-way ANOVAs on ranks were conducted to assess the effects of drought and time on multihexose benzoxazinoid levels.Student t-tests and Mann−Whitney tests were performed to evaluate the impact of drought on individual compounds in maize.
While certain dihexose BXDs such as DIMBOA-dihexose (1) or HDMBOA/HM 2 BOA-dihexose (6) were produced in  fairly high concentrations as shown by their prominent peaks in the chromatogram, others were present in much lower amounts (e.g., HMBOA-trihexose (4), DIBOA-dihexose (7), Figures 1 and 2).Compound 8 was present in such low quantities that it could not be isolated from the plant material.To elucidate the structure of all detected di-and trihexose BXDs, we purified them from maize plants grown under drought for 10 days and fully characterized them by a combination of spectroscopic techniques.

Structural Elucidation of Multihexose Benzoxazinoids.
A methanol extract from maize roots was first fractionated by open column chromatography using a step gradient elution.The fraction (F9) containing dihexose and trihexose BXDs was eluted with a 1/1 mixture of ethyl acetate and methanol.This fraction was submitted to multiple steps of semipreparative fractionation to obtain compounds 1−7.All of the purification steps were controlled by LC-MS and the purity of each isolated compound was finally checked by NMR spectroscopy.Compounds 1 to 7 were identified as multihexose BXDs (Figure 3).
In the following section, the structural elucidation of compounds 1 to 7, based on NMR, HRMS and circular dichroism (CD) data, is briefly described.All 1 H NMR chemical shifts are displayed in Table 1 and a detailed report of all obtained data is provided in Supporting Information SI2−SI8.
The elemental composition of 2 was deduced to be C 27 H 39 NO 20 based on the HRESIMS and NMR data (Table 1, Supporting Information SI3).The aglycone part was identified as DIMBOA.Signals observed in the 1 H and 13   The elemental composition of 3 was deduced to be C 21 H 29 NO 14 based on the HRESIMS and NMR data (Table 1, Supporting Information SI4).Compound 1 possessed extra oxygen compared to 3. NMR data of 1 and 3 were closely related.The alcohol function located on the nitrogen induced a gamma-steric effect for compound 1.This gamma-steric effect of the alcohol function located on the nitrogen induced a variation of the 13 C chemical shift value of Δδ = −5.1 ppm on C-3 of 1 compared to 3 (Supporting Information SI2− SI4).NOESY correlations and the CD spectrum confirmed the structure of the dihexose unit as well as the (2R) configuration (Supporting Information SI4).Compound 3 was identified as HMBOA-β-gentiobiose (HMBOA-2Glc, Figure 3).
The elemental composition of 5 was deduced to be C 22 H 31 NO 15 based on the HRESIMS and NMR data (Table 1, Supporting Information SI6).Signals observed in the 1 H and 13 C NMR spectra of 5 exhibited resonances for two methoxy groups [δ C 56.2 (CH 3 ), δ H 3.89] and [δ C 61.6 (CH 3 ), δ H 3.92].Protons H-11 and H-12 located on each of the methoxy groups showed long-range HMBC cross-peaks, respectively, with the carbons C-8 and C-7.The aglycone unit was identified as HM 2 BOA.Chemical shifts of the two hexose units were closely related to both compounds 1 and 3 (Table 1).NOESY correlations and CD spectrum confirmed the structure of the dihexose unit as well as the (2R) configuration.Compound 5 was identified as HM 2 BOA-βgentiobiose (HM 2 BOA-2Glc, Figure 3).Spectral data of the second methoxy group [δ C 63.6 (CH 3 ), δ H 3.98] were characteristic of an N-OMe group.The aglycone unit was identified as HDMBOA.Chemical shifts of the two hexose units were closely related to compounds 1, 3, and 5 (Table 1).NOESY correlations and the CD spectrum confirmed the structure of the dihexose unit as well as the (2R) configuration.Compound 6 was identified as HDMBOA-β-gentiobiose (HDMBOA-2Glc, Figure 3).
However, low levels of HMBOA-2Glc could be detected in roots over 24 days of growth (Figure 5E,F).
Under drought conditions, maize seedlings transiently produced higher concentrations of DIMBOA-2Glc, DIM-BOA-3Glc, and HMBOA-2Glc in both shoot and root tissues (Figure 5).The production of the di-and trihexose BXDs was the highest in roots of 4-day-old seedlings and in shoots of 7-day-old seedlings (Figure 5).Whether these BXDs are produced in roots and subsequently transported to shoots remains to be investigated.
3.5.Drought-Induced Multihexose Benzoxazinoid Production Is Common in Maize.The drought-mediated induction of multihexose BXDs was found in leaves and roots of the four maize varieties, B73, CML277, Hp301, and Oh7B (Figure 6).Interestingly, the increase in multihexose BXDs was accompanied by a decrease in monoglucosylated BXDs, suggesting their use as precursors for multihexose BXDs (Figure 6).Some line-specific patterns were further observed, suggesting that genetic variability could be used to identify the biosynthesis pathways and glucosyl transferases involved in the production of multihexose BXDs.
3.6.Toward the Function(s) of Multihexose Benzoxazinoids.Overall, this study reports a method to produce maize extracts enriched in multihexose BXDs and provides the first characterization of the exact hexose nature and configuration.Glycosylation of a small organic compound can alter its solubility, stability, bioavailability, and bioactivity and modulate its storage, transport, and/or interactions with other organisms.−26 They include phenolic compounds such as eugenol triglycosides 26 and flavonoids, such as quercetin 3-O-gentiobiosides, gentiotriosides, and gentiotetrosides, 23 anthocyanidin 3-O-rutinosides, 24,25 or 3-O-2″-O-β-glucuronosyl-6″-O-malonylglucoside. 24Evidence suggests that the conversion of glycoside plant specialized metabolites into multihexoses can modulate plant volatile emissions 26 and flower coloration. 24he role of multihexose BXDs still remains to be elucidated.Several, nonexclusive, hypotheses can be formulated.First, multihexose BXDs may act as osmoprotectants through maintaining cell turgor pressure and osmotic balance.Second, these BXDs may form, through the sugar moieties, hydrogen bonds with water molecules and reduce the water loss associated with transpiration.Third, the multihexose compounds may act as stronger antioxidants than their precursors and prevent cellular damage associated with reactive oxygen species (ROS).Fourth, multihexoses may be used to reallocate and store sugars as an energy source to tolerate drought and decreased photosynthesis.Fifth, because BXDs modulate the plant interactions with pathogens and herbivores, multihexose BXDs may be involved in protecting the plant from biotic stress under challenging abiotic conditions.Alternatively, the sugar-rich compounds may be exuded in the rhizosphere and promote beneficial microorganisms.While our understanding of the biosynthesis and ecological functions of multihexose plant specialized metabolites is still in its infancy, they represent a fascinating aspect of phytochemical diversity, calling for future research.

2 . 5 .
Preparative Chromatography.The crude extract (1.8 g) was first fractionated by open column chromatography (CC) using 45 g of silica gel (63−200 μm, 60 Å) as the stationary phase.A step gradient elution consists of mixtures of dichloromethane-ethyl acetate, followed by ethyl acetate-methanol, and finally pure methanol yielded 11 fractions (F1−11).Compounds 1−7, which were all present in F9 (126.8 mg), were further purified by semipreparative HPLC using a LC-20AR Shimadzu pump (Japan) connected to a UV−vis detector (Shimadzu, model SPD-20A) and a fraction collector FRC-40 (Shimadzu).A detailed description of the semipreparative conditions for each molecule is provided in Supporting Information SI1.2.6.NMR Spectroscopy.NMR spectra were acquired on a Bruker Avance Neo Ascend 600 MHz (Bruker, Germany) in D 2 O. Standard pulse sequences were used to acquire 1D and 2D NMR analyses.HSQC and HMBC analyses were recorded in nonuniform sampling (NUS) mode with a sampling amount of 12.5%.Acetone was used as calibration standard (δ 1 H = 2.225 ppm; δ 13 C = 30.5

Figure 4 .
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).