Distinct ultraviolet-signaling pathways in bean leaves. DNA damage is associated with beta-1,3-glucanase gene induction, but not with flavonoid formation

Abstract

The enzyme beta-1,3-glucanase (betaGlu) was found to be strongly induced by ultraviolet (UV-B; 280-320 nm) radiation in primary leaves of French bean (Phaseolus vulgaris). This was demonstrated on the level of gene transcription, protein synthesis, and enzyme activity and was due to the expression of bean class I betaGlu (betaGlu I). In contrast to other proteins of the family of pathogenesis-related proteins, the induction of betaGlu I by UV correlated with the formation of photoreversible DNA damage, i.e. pyrimidine dimer formation. In conditions that allowed photorepair of this damage, betaGlu I induction was blocked. Therefore, UV-induced DNA damage seems to constitute a primary signal in the pathway leading to the induction of the betaGlu I gene(s). The induction was a local response because in partly irradiated leaves betaGlu I was selectively found in leaf parts exposed to UV. Although short wavelength UV (lambda < 295 nm) was most efficient in betaGlu I induction, longer wavelength UV (lambda > 295 nm) as present in natural radiation was still effective. In contrast to UV induction of betaGlu I, the induction of flavonoids in bean leaves was optimally triggered by much more moderate fluences from the UV wavelength range no longer effective in betaGlu I induction. UV induction of the flavonoid pathway shows no correlation with DNA damage and thus should be mediated via a different signal transduction pathway.

Figure 1.

A, Standard UV source used in studies for βGlu induction and UV/WL source used in experiments for flavonoid induction measured with a 250- to 800-nm double-monochromator spectroradiometer (model OL 754, Optronic, Orlando FL). A, Spectral irradiance of the standard UV source under transmission cut-off filters WG 360 (- - - -), WG 295 (· · · ·), WG 305 (—·—), and without filter (——). B, Spectral irradiance of the UV/WL source under transmission cut-off filters WG 360(—··—), WG 295 (· · · ·), WG 310 (- - - -), and under quartz (——). For better overview, the spectral irradiance in the UV range is given in linear scaling. Total spectral irradiance of the light sources (inset) is shown in logarithmic scale.

Figure 2.

Distinct effects of UV and wounding on βGlu I gene expression. Plants were irradiated for 15 min under the UV source; one of the primary leaves was covered with a WG 360 filter to exclude UV-B (control) while the opposite leaf was left uncovered. After UV irradiation, plants were kept in red light for the time indicated. For wounding response, one primary leaf was damaged using forcipes, while the opposite leaf was left untreated as a control. A, Time course of βGlu I protein accumulation. The position of βGlu I standard from tobacco is shown in the first lane. The apparent size in kilodaltons of the major immunoreactive band is indicated at the right. B, Time course of βGlu activity after UV treatment (•) or wounding (○) with controls (▾, UV cut-off filter WG 360; fl, untreated). C, Time course of βGlu I mRNA accumulation after UV treatment (•) with control kept under UV cut-off filter WG 360 (○). Inset, Northern blots (including loading control in first two lanes) 24 h after UV treatment. +UV, irradiation without filter; -UV, control irradiated under UV cut-off filter WG 360. Mean values and se from four independent experiments.

Figure 3.

βGlu and CPD induction in response to varied UV spectral ranges. Plants were irradiated for 15 min (black bars) or 30 min (gray bars) under the UV source, using transmission cut-off filters and then kept in red light for 24 h for measurement of βGlu induction. For analysis of CPDs, samples were taken from leaves in dim yellow light immediately after irradiation. A, Accumulation of βGlu I protein after 30 min of UV irradiation. The apparent size of the major 34-kD immunoreactive band is indicated at the right. B, Induction of CPDs. Representative experiment that was repeated twice with similar results. C, Induction of βGlu activity. Mean values and se of two independent experiments (n = 8).

Figure 4.

βGlu I protein accumulation under conditions allowing photoreactivation. Plants were irradiated for 15 min under UV; for control, one leaf was covered with UV transmission cut-off filter WG 360 to exclude UV-B. After irradiation plants were kept for 24 h either in red light (R) excluding the photoreactivating wavelength range or in WL including violet for photoreactivation. The apparent size in kilodaltons of 34-kD antigen is indicated.

Figure 5.

UV-induced flavonoid formation. Leaf discs were irradiated for 42 h under a combined UV/WL source in boxes covered with different UV transmission cut-off filters (WG 360, WG 310, WG 275) or quartz. Flavonoid content was quantified after PC separation by measuring the A₃₆₀ as described in “Materials and Methods.”Mean values and se from nine independent experiments.