Survival of Plants During Short-Term BOA-OH Exposure: ROS Related Gene Expression and Detoxification Reactions Are Accompanied With Fast Membrane Lipid Repair in Root Tips

Abstract

For the characterization of BOA-OH insensitive plants, we studied the time-dependent effects of the benzoxazolinone-4/5/6/7-OH isomers on maize roots. Exposure of Zea mays seedlings to 0.5 mM BOA-OH elicits root zone-specific reactions by the formation of dark rings and spots in the zone of lateral roots, high catalase activity on root hairs, and no visible defense reaction at the root tip. We studied BOA-6-OH- short-term effects on membrane lipids and fatty acids in maize root tips in comparison to the benzoxazinone-free species Abutilon theophrasti Medik. Decreased contents of phosphatidylinositol in A. theophrasti and phosphatidylcholine in maize were found after 10-30 min. In the youngest tissue, α-linoleic acid (18:2), decreased considerably in both species and recovered within one hr. Disturbances in membrane phospholipid contents were balanced in both species within 30-60 min. Triacylglycerols (TAGs) were also affected, but levels of maize diacylglycerols (DAGs) were almost unchanged, suggesting a release of fatty acids for membrane lipid regeneration from TAGs while resulting DAGs are buildings blocks for phospholipid reconstitution, concomitant with BOA-6-OH glucosylation. Expression of superoxide dismutase (SOD2) and of ER-bound oleoyl desaturase (FAD2-2) genes were contemporaneously up regulated in contrast to the catalase CAT1, while CAT3 was arguably involved at a later stage of the detoxification process. Immuno-responses were not elicited in short-terms, since the expression of NPR1, POX12 were barely affected, PR4 after 6 h with BOA-4/7-OH and PR1 after 24 h with BOA-5/6-OH. The rapid membrane recovery, reactive oxygen species, and allelochemical detoxification may be characteristic for BOA-OH insensitive plants.

Keywords: BOA-OH isomers; Detoxification; FAD2-2; Lipids; Membrane repair; ROS; SOD2.

Fig. 1

Nitro precursors 1–4 synthesized as precursors for the hydroxy BOAs 5–8

Fig. 2

General Synthesis of the four isomeric hydroxy BOAs 5–8

Fig. 3

Maize root hairs (A) were cut and extracted. The extracts and washing solutions obtained from the root hair zone including the mucilage drop (B) were analyzed for benzoxazinoid contents. The extracts of cut root hair contain DIMBOA-glc, DIMBOA and MBOA (n = 12), the wash solution only DIMBOA and MBOA (n = 7) as major compounds

Fig. 4

A, D: Maize root surface peroxidase assay (ABTS) exposes youngest root zones, the tips covered by the root cap and the bases of emerging roots as sites of peroxidase activity. B: Left to right: control, and after 24 h incubation with BOA-4-OH, BOA-5-OH, BOA-6-OH, BOA-7-OH. Dark polymers derived from BOA-OHs precipitate at lateral root emerging sites but not at the surface of root hairs or the tip, C: After BOA-OH incubations, ring-like structures developed preferentially at root zones prior to lateral root emergency. D: Root tips and root caps are dark after assaying peroxidase with ABTS. E: Dark spots at the emerging sites of lateral roots after BOA-OH incubations

Fig. 5

Reactions of maize roots. All BOA-OH isomers elicit weak to strong (shown here) bubble formation in the root hair zones with 0.5 mM BOA-OHs. 1A: Control, tap water + 500 µl MeOH; 1B: BOA-4-OH; 1C: BOA-5-OH; 1D: BOA-6-OH; 1E: BOA-7-OH. Arrow heads point to root hairs with bubbles at their tips and to root hairs of the control seedlings (1A). 2A: 500 µM of BOA-6-OH elicited no catalase activity developed during the first hours, arrow points to a white root tip (see gene expression study). 2B: Low catalase activity after 6 h; 2C: Whole plant to A2; 2D: Control plant

Fig. 6

Total fatty acids in maize roots. Left side: unsaturated fatty acids; Right side saturated fatty acids. A: RTa (young root tissue), B: RTb (older root tissue). RTa und RTb samples were taken without (t0) and after exposure to 0.5 mM BOA-6-OH for 10, 20, 30-, 40-, 50- and 60-min. The figure shows mean values and standard deviations (n = 3). Significance with reference to t0 (t-test): *p < 0.05; **p < 0.005; ***p < 0.0005)

Fig. 7

Total fatty acids in A. theophrasti root tips. Left side: unsaturated fatty acids; Right side: saturated fatty acids. A: RTa (young root tissue), B: RTb (older root tissue). RTa und RTb samples were taken without (t0) and after exposure to 0.5 mM BOA-6-OH for 10, 20, 30-, 40-, 50- and 60 min. The figure shows mean values and standard deviations (n = 3). Significance with reference to t0 (t-test): *p < 0.05; **p < 0.005; ***p < 0.0005)

Fig. 8

Maize (a:ZmRTa/b:ZmRTb) and A. theophrasti (a: AbRTa/b:AbRTb). Major structural phospholipids: PC phosphatidylcholine, PE phosphatidylethanolamine, PG phosphatidylglycerol, PI phosphatidylinositol. Glycolipids: MGDG, DGDG. Grouped columns present control and incubation times (left to right: 0 = control, 10, 20, 30, 40, 50, 60 min) of the different glyco/phospholipids. Molecular species are given in Fig. S1. Significance with reference to t0 (t-test): *p < 0.05; **p < 0.005; ***p < 0.0005)

Fig. 9

Maize TAGs: Series ZmRTa: A decrease in many molecular species is found after 20 min, while other species are not affected (52.5; 54.3) or increase (54.7). In ZmRTb, the major tendency was an increase of many species, although contents varied strongly. A. theophrasti TAGs: Series AbRTa: A decrease of most of the molecular species is found after 10 min. In AbRTb, the 52. 5–2 molecular species are less affected than the 54.7–3 ones. TAG 54.6 and 54.5 decreased during the first 20 min. Grouped columns present control and incubation times (left to right: 0 = control, 10, 20, 30, 40, 50, 60 min). Significance with reference to t0 (t-test): *p < 0.05; **p < 0.005; ***p < 0.0005)

Fig. 10

Defined DAGs are significantly affected in A. theophrasti (Ab) but not in maize (Zm) sample series RTa and RTb. Grouped columns present control and incubation times (left to right: 0 = control, 10, 20, 30, 40, 50, 60 min). Molecular species with low abundance (for instance 18:1/18:3 in Abutilon and 16:0/18:1 and 18:1/18:2 in maize are not affected. Significance with reference to t0 (t-test): *p < 0.05; **p < 0.005; ***p < 0.0005)

Fig. 11

Maize genes affected by BOA-OH isomers. Relative transcript abundance of genes encoding enzymes involved ROS detoxification (SOD2; CAT1, CAT3), fatty acid desaturation (FAD2.1; FAD2.2) and genes related to pathogenesis and resistance (PR1, PR2, NPR1, POX12), mean values ± STD, n = 3, shown as fold-changes (log₂^{− ΔΔCt}). Responses to BOA-OH isomer exposure are shown for 30 min, 1 h and 6 h incubation times

Fig. 12

A: Contents of detoxification products in maize roots exposed to BOA-OHs for 24 h. HPLC analysis of methanolic extracts from maize roots reveals the accumulation of BOA-6-O-glucoside and BOA-5-O-glucoside, but almost no products of BOA-4-OH and BOA-7-OH. B: BOA-6-O-glc accumulation in root tips during the first h of incubation