Virginia Polytechnic Institute and State University

DIMBOA-glu concentration in different taxa of teosinte
--Shahid, M, Esen, A

Hydroxamic acids, Hxs (4-hydroxy-1,4-benzoxazin-3-ones), including 2, 4-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), play an important role in the defense of certain cereals against bacteria, fungi and insects (Niemeyer, Phytochemistry 27:3349-3358, 1988). They have been reported in rye, wheat and maize, where their levels have been shown to have a direct correlation with resistance against different pests (Niemeyer, Phytochemistry 27:3349-3358, 1988). DIMBOA and other Hxs occur in plants as 2-b-O-D-glucoside; in this form they are not toxic. DIMBOA-glu and b-glucosidase in intact plant tissues do not interact due to their presence in different subcompartments of the cell. Upon plant tissue disruption, b-glucosidase and DIMBOA-glu come into contact; the enzyme acts on DIMBOA-glu releasing DIMBOA, which is toxic to the organisms that attack the plant.

DIMBOA-glu is found in all parts of maize seedlings; its concentration in leaves is higher than in any other part/organ. In vitro assays show that DIMBOA-glu is a substrate of maize b-glucosidase (Babcock and Esen, Plant Sci. 101:31-39, 1994; Cuevas et al., Phytochemistry 31:2609-2612, 1992). Thus, the enzyme has an important role in the activation of plant defense against various pests. In order to investigate whether or not teosinte, the postulated progenitor of maize, has DIMBOA-glu in its leaves, four of its taxa viz., Z. mays ssp. parviglumis; Z. mays ssp. mexicana; Z. mays ssp. huehuetenangensis; and Z. diploperennis were analyzed. The same material was also analyzed for ?-glucosidase activity.

For DIMBOA-glu extraction, three leaves from three different plants of each taxon were ground to a powder in a chilled mortar and suspended in methanol (weight in gram : volume in ml ratio = 1:3). The homogenate was centrifuged at 14,000 rpm for 5 min in a Beckman microcentrifuge, and the supernatant was used for DIMBOA-glu analysis by HPLC (Waters 450). Ten µl of the supernatant was fractionated on a C18 column (particle size 5 µM; dimensions 150 x 4.6 mm) at room temperature using the mobile phase of 59 H2O : 40 MeOH : 1 HOAc and the flow rate of 1 ml min-1.

Elution of the Hx-glu was detected by absorbance measurement at 262 nm. The elution time for DIMBOA-glu was 5.78 min. The b-glucosidase activity was assayed using p-Nitrophenyl b-D-glucopyranoside (pNPG) as a substrate as described by Esen and Cokmus (Biochem. Genet. 28:319-336, 1989).

The data indicate that all four teosinte taxa contain the hydroxamic acid DIMBOA-glu (Figure 1), although its level varies from one taxon to the other. The highest concentration of DIMBOA-glu was found in Z. diploperennis and the lowest in Z. mays ssp. mexicana, which is almost the same as in the maize inbred K55 (Zea mays ssp. mays). These results show that the DIMBOA-glu concentration in Z. diploperennis is more than double its concentration in the maize inbred K55. As for the ?-glucosidase activity in teosinte leaves, the highest activity was measured in Zea mays ssp. mexicana, while Zea mays ssp. huehuetenangensis exhibited the lowest activity (Figure 1).

The presence of DIMBOA-glu and ?-glucosidase in teosintes suggests that the defense mechanism in the teosinte taxa is similar to that in maize and other cereals.

Figure 1. b-glucosidase activity and DIMBOA-glu concentration in the leaves of 15-day old light-grown maize and 4 taxa of teosinte. 1, Z. mays ssp. mays; 2, Z. mays ssp. parviglumis; 3, Z. mays ssp. mexicana; 4, Z. mays ssp. huehuetenangensis; and 5, Z. diploperennis.

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