UV radiation-sensitive norin 1 rice contains defective cyclobutane pyrimidine dimer photolyase

Abstract

Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase-dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase.

Figure 1.

UV Radiation Damage and Photorepair in UV Radiation–Sensitive and –Resistant Rice Cultivars. (A) UVB radiation induces growth stunting and browning of UV radiation–sensitive Norin 1. (B) Slow dimer photorepair (open triangles) in Norin 1 seedlings; dimer removal in the dark (filled triangle) was not observed. (C) UVB radiation has little effect on UV radiation–resistant Sasanishiki seedlings. (D) Rapid dimer photorepair (open circles) in Sasanishiki seedlings; dimer removal in the dark (filled circle) was not observed. (A) and (C) Plants were grown for 35 days in a phytotron under visible light without (−) or with (+) supplemental UVB radiation. (B) and (D) Seedlings exposed to UV radiation at 302 nm were harvested immediately and kept in a dark box (filled triangle and filled circle) or exposed to blue fluorescent lamps filtered through UF4 plexiglass (open triangles and open circles). Seedlings were harvested at increasing times, their DNA isolated, and CPD frequencies were measured in at least duplicate samples. Lines for photorepair are least-squares fits. Lines fit to the excision data are superimposed on the x axis; the lines shown were displaced slightly upward for clarity. Symbols show averages with error bars; individual determinations are shown without error bars. Error bars indicate population standard deviations.

Figure 2.

Schematic Representation of Photoflash Analysis. DNA in plants (or in vitro) is irradiated with UV to produce high CPD frequencies. Samples are incubated in the dark to allow formation of photolyase–CPD (enzyme–substrate) complexes. A high-intensity (sufficient for photolysis of all existing enzyme–substrate complexes), sub-millisecond (time short enough that no photolyase can bind to a second CPD) photoflash is administered to the complexes. Because each photolyase–CPD complex repairs one dimer, measurement of the level of CPD photolyzed directly reflects the concentration of active photolyase–CPD complexes at the instant of the flash.

Figure 3.

Determination of CPD Levels and Flash Intensities Required for Photoflash Analysis in Rice Seedlings. Sasanishiki seedlings were irradiated with increasing UVB doses, kept in the dark for 15 min to allow formation of photolyase–CPD complexes, and then exposed to a single, full-intensity photoflash. The level of photorepaired CPDs was measured in at least replicate determinations, and the no-UV radiation (no dimer) values were subtracted from each initial dimer value. The vertical error bars show standard deviations of photorepair obtained in the flash; the horizontal error bars show the measured range of initial net CPD concentrations. The inset shows the amount of photorepaired CPD (initial value, 80 CPD Mb⁻¹) versus the intensity of the photoflash. Lines were fit by least squares (insert) or provided to guide the eye (main graph). Small filled symbols indicate individual data points; large open symbols indicate averages.

Figure 4.

Anticipated and Experimental Time Courses of Association of Photolyases with CPD in Rice Seedlings. (A) Anticipated kinetics of photolyase–substrate complex formation if Norin 1 contains a regulatory mutation that affects the cellular photolyase content. The final amount of complexes differs between Norin 1 and Sasanishiki. (B) Anticipated association kinetics if Norin 1 contains a structural mutation in photolyase protein. The rate of association is slower in Norin 1, but the final amount of complexes is similar to that in Sasanishiki. (C) Experimental photoflash data for photolyase–CPD complex formation in Norin 1 (filled triangles) and Sasanishiki (filled circles). The third leaves of seedlings were exposed to 6 kJ m⁻² of unfiltered UVB radiation, inducing ∼80 CPD Mb⁻¹; then they were kept in darkness until, at various times (shown in minutes) after UVB irradiation, they were exposed to a single full flash. DNAs were isolated, and CPD frequencies were measured in at least replicate determinations. The points indicate individual measurements; lines are provided to guide the eye. The experimental data fulfill the prediction of (B) for a structural mutation.

Figure 5.

Photoflash Analysis of Thermal Stability of Photolyase–CPD Complexes in Vitro. (A) Scheme for photoflash analysis of photolyase–CPD complexes. Complexes are incubated at different temperatures and after various times are subjected to a single flash. The level of photorepaired CPDs indicates the level of complexes persisting after incubation at that temperature for the times tested. (B) Photorepair in rice seedlings in extracts in vitro and in plants in vivo. Extracts of Sasanishiki or Norin 1 were mixed with UV-irradiated λ DNA, incubated for 15 min at 28°C for photolyase–CPD complex formation, and then exposed to continuous illumination; CPD repair was measured as a function of time. Values represent the mean ±sd of three samples for Norin 1 (open triangles) and Sasanishiki (filled triangles). The inset shows CPD photorepair in vivo of both cultivars (data from Figure 1B).

Figure 6.

Photoflash Analysis of Thermostability of Complexes Containing CPD and Photolyases from Norin 1 or Sasanishiki. Extracts of the two cultivars were mixed with λ DNA containing CPD, incubated for 15 min at 28°C for formation of photolyase–CPD complexes, and then underwent different treatments. (A) Incubation of extracts was shifted to 0°C. (B) Incubated extracts continued to be incubated at 28°C. (C) Incubation of extracts was shifted to 45°C. (D) Incubation of extracts was shifted to 60°C. At various times, samples were exposed to a single photoflash, and CPD frequencies were measured. The photorepair activity of UV radiation–resistant Sasanishiki (circles) and Norin 1 (triangles) was measured as a function of time after the shift in temperature. Lines in (A) to (C) are least-squares fits; lines in (D) guide the eye. Error bars indicate sd.