Plant Responses to Changing Environmental Stress: Cyclobutyl Pyrimidine Dimer Repair in Soybean Leaves

Photochemistry and Photobiology, 1996, 64(3): 464-468

Symposium-in-Print

Plant Responses to Changing Environmental Stress: Cyclobutyl Pyrimidine Dimer Repair in Soybean Leaves

Betsy M. Sutherland,*l Shinnosuke Takayanagi: Joe H. Sullivan3 and John C. Sutherland’ ‘Biology Department, Brookhaven National Laboratory, Upton, NY, USA; 2Department of Biology, School of Medicine, Toho University, Tokyo, Japan and 3Department of Horticulture and Landscape Architecture, University of Maryland, College Park, MD, USA

Received 15 January 1996; accepted 14 June 1996

ABSTRACT

We have determined the capacity of soybean seedlings to repair DNA damage by UV doses that do not produce apparent injury in the plants. They remove cyclobutane pyrimidine dimers by both excision and photoreactivation. The rates and relative contributions of these repair processes were determined as a function of initial level of cyclobutyl pyrimidine dimers. Photoreactivation was detected in seedlings at all initial dimer levels. Although excision was not observed at the lowest dimer frequencies, at higher initial dimer levels it was quite effective in dimer removal. The rates of repair in soybean were sub- stantially higher than in alfalfa seedlings at the same DNA damage levels.

INTRODUCTION

Plants meet extraordinary environmental challenges and have evolved a multiplicity of strategies for meeting them. First, the overwhelming majority of plants are sessile. More- over, their obligate energy source, the sun, presents delete- rious UV radiation along with light effective for photosynthesis. Not only does the absolute UV flux vary naturally (with time of day, season, cloud cover and other factors), but human activities can also alter the quantity and quality (wavelength range) of UV reaching the biosphere.

Damage to DNA is well understood to be detrimental to simple, single-cell organisms, whose replication requires integrity of their genome. However, plant reproductive structures and many growing tissues are internal, shielded by cellular structures. It may thus be less obvious why plants should need to protect the integrity of DNA in vegetative structures, which can undergo expansion with little or no cell division. However, it is clear that plants can suffer UV damage: UVB-sensitive cultivars of soybean may manifest UVB-

*To whom correspondence should be addressed at: Biology De- partment, Building 463, Brookhaven National Laboratory, Upton, NY 11973-5o00, USA. Fax: 516 344-3407; e-mail: Sutherl3 @ bnl.gov.

0 1996 American Society for Photobiology 003 1-8655/96 $5.00+0.00

induced damage as reduced photosynthesis ( I ,2) , biomass (3.4) or seed yield (5). A possible key factor may be the devastating effect that DNA lesions can have on transcription, partially or completely halting mRNA production. Con- tinued production of mRNA is essential for vegetative me- tabolism, especially if critical proteins are themselves UV sensitive and thus must be replaced more frequently under UV stress conditions.

A clear indication that UV poses a major threat to plant survival is that they have evolved a panoply of protective mechanisms to prevent UV-induced damage to DNA, and an extensive system of repair responses that allow them to reverse or remove UV damage from cellular DNA. The range and integration of these responses are only now being determined. Furthermore, the strategies employed by various plant species differ.

Although it is desirable to determine repair capacity of plants under field conditions, the many uncontrollable vari- ables encountered in natural conditions make this a daunting task for analytical studies. We have therefore undertaken a study of UV responses of plants grown under controlled conditions of day length, temperature and illumination. Further- more, in the field, plants carry out repair simultaneously with formation of damage by impinging sunlight, making difficult the deconvolution of initial damage levels from the net level of damage that is induced minus damage that is repaired. We thus treated plants with UV in temporally brief exposures to minimize simultaneous repair and provide accurate measures of initial damage levels and the rates and extents of repair by photoreactivation and by excision. These studies should provide basic information on the repair capabilities of different plant species and cultivars to provide insight for designing and interpreting field repair studies. Determining the roles of different DNA damages, their repair and especially the coordination of these responses is essential to understanding the ability of different plant cultivars, species and integrated plant communities to cope with today’s and tomorrow’s environments.

MATERIALS AND METHODS Planr marerial. Soybean (Glycine mux (L.) Merr., cultivar Forrest) seeds were placed on Whatman # I filter paper in small petri dishes

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with 5 mL sterile double-distilled water and kept in the dark at room temperature for 3 days until the roots were 2-3 cm long, then trans- planted into pots with a 5:l:OS mixture of Custom Blend 90-1 (Grace Sierra Co.. Millipilas, CA): sand : vermiculite (Schundler Co., Metuchen, NJ) dampened with diluted Sierra slow release fertilizer solution (19:7:10, 15 cc/gallon water). The pots were placed in a shallow dish containing sterile double-distilled water to provide a constant water supply and grown in an environmental chamber il- luminated with cool white fluorescent bulbs (SylvanidGTE, Dan- vers, MA) filtered by UF4 Plexiglas (Rohm and Haas, Philadelphia, PA) that excludes most wavelengths shorter than -400 nm. The seedlings were grown (20°C; 16 h photoperiod) for 10 days and the fully expanded first true leaves were used for irradiation experiments.

UV irradiation. Seedlings were irradiated with Westinghouse FS20 lamps, which emit UVB, UVA and UVC radiation to produce initial cyclobutyl pyrimidine dimer (CPD)? frequencies between -10 and -60 siteslmegabases (Mb, megabase, 1 million bases); UV exposure times were held constant at 5 min, and the incident intensity varied (up to 400 FW/cmZ) to give the desired initial dimer frequency. Radiation was monitored using a Jagger meter ( 6 ) cali- brated vs a YSI radiometer (Yellow Springs Instruments, Yellow Springs, OH). All subsequent manipulations were carried out in dim yellow light (General Electric, 25 W; A > -500 nm) to minimize uncontrolled photoreactivation of pyrimidine dimers.

Photoreactivation. Samples to be photoreactivated were exposed to UV as described above, then to light from two Philips 15T8/B blue fluorescent lamps (North American Philips Lighting, Somerset, NJ), filtered through a UF4 Plexiglas tilter, resulting in a spectrum with a maximum at about 450 nm. The combination of blue lamps and the UF4 filter provided visible light for photorepair while ex- cluding almost all long-wavelength UV radiation (7).

DNA extractinn. DNA was obtained from soybean seedlings by a modification of the method of Quaite et al. (8.9). The first true leaves were sliced in buffer A (0.5 M ethylenediaminetetraacetate [EDTA; Mallinckrodt AR, Paris, KY], 10 mM Tris-HCI [United States Biochemical, Cleveland, OH, enzyme grade], pH 8.0, I % sar- cosy1 [Sigma, St. Louis, MO], 1 rng/mL proteinase K [Boehringer Mannheim, Indianapolis, IN]), vacuum-infused for 3 W O s and incubated at 45°C for 30 min. The slurry was mixed with an equal volume of 1.4% low melting point agarose (Seaplaque; FMC, Rock- land, ME) and agarose plugs were prepared. The plugs were digested (buffer A, 50°C. 3 days), rinsed with TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA), followed by TE containing 2.5 mM phenylme- thylsulfonyl fluoride, TE and UV endonuclease buffer (30 mM Tris HCI, pH 8.0, 40 mM NaCI, 1 mM EDTA).

Treatment of soybean DNA with UV endonuclease from Micro- coccus luteus. The UV endonuclease was partially purified from M. luteus by streptomycin and ammonium sulfate precipitations and its specificity and activity toward CPD and lack of nonspecific cleavage were determined using supercoiled DNA containing and lacking dimers (10) and by analysis of cleavage using DNA sequencing gels ( 1 I ) . Plugs containing soybean seedling DNA were digested with sufficient UV endonuclease to yield complcte cleavage at all dimer sites, and a duplicate sample was incubated in the buffer (without endonuclease) under identical conditions. Reactions were stopped and the DNA denatured by adding alkaline stop mix (0.5 M NaOH, 50% vol/vol glyerol and 0.25% wt/vol bromocresol green) and in- cubating for 30 min at 37°C.

Alkaline unidirectional pulsed jield electrophoresis (UPFE). DNAs were dispersed according to their single-strand molecular lengths by alkaline agarose UPFE ( I 2). Agarose (SeaKem LE; FMC) gels (0.4% wt/vol) were prepared in 1 mM EDTA, 50 mM NaCI, equilibrated with alkaline electrophoresis solution (2 mM EDTA, 30 mM NaOH) for 30 min. Sample DNAs were electrophoresed along with DNAs of known lengths that served as molecular length mark- ers (DNA from bacteriophages C [750 kb], T4 [I70 kb], A [48.5 kb], T7 139.9 kb], a BglI digest of T7 [22.5, 13.5 and 4 kb] and a Hind111 digest of A (23, 9.4, 6.6, 4.4, 2.3. 2.0. 0.56 and 0.13 kb]).

tAbbreviations: CPD, cyclobutyl pyrimidine dimer; EDTA, ethylenediaminetetraacetate; Mb, megabase; TE, Tris-EDTA buffer, pH 8; UPFE, unidirectional pulsed field electrophoresis.

Electrophoresis was for 16 h (15 V/cm; 0.3 s pulse, 10 s interpulse period; 10°C with buffer recirculation); the plugs were then removed from the wells and electrophoresis resumed using the same conditions for 8 h. Gels were neutralized (2 X 30 min in 0. I M Tris-HCI. pH 8). stained with ethidium bromide ( I FglmL) for 30 min and destained in H 2 0 overnight at 4°C.

Electronic imaging of gels and pyrimidine dimer analysis. The DNA in the destained gels was quantitated by imaging the distribution of the fluorescence of DNA-bound ethidium using an im- proved version of the electronic imaging system described previously (13). The CPD frequencies were calculated as previously described using the method of moments (8.9.14). DNA profiles of sample and length-standard lanes were obtained from the quantitative image data; a dispersion function (molecular length vs migration position) was constructed from the molecular length standards. Us- ing this curve and the quantity of DNA at each molecular length position from the quantitative image data. the number average molecular length, L,, of each DNA distribution was calculated from the equation

where L(x) is the length of the DNA molecules in Mb that migrated to position x, and p(x) is the intensity of ethidium fluorescence from DNA molecules at that position. From the number average lengths of the DNA populations, the frequencies of lesions were obtained from the equation

(2) where C$ is the frequency of dimers (CPDhlb), L,( +e) is the number average length (in Mb) of the population of molecules treated with the UV endonuclease, and L,(-e) is the number average length (Mb) of the population of molecules not treated with the enzyme.

C$ = L;'(+e) - L;'(-e)

RESULTS Photoreactivation is well known to play a major role in repair of CPD in plants, but excision has been generally thought to be absent or inconsequential (15-20). We thus tested the ability of soybean plants to carry out excision as well as photorepair. To characterize CPD repair, we exposed the seedlings to UV radiation to induce higher levels of dimers (-50 CPD/Mb). (The soybean plants grown in the UV- free growth chamber, then exposed to these UV levels, showed no detectable injury, but did appear greener within a few days after UV treatment.) They were then harvested immediately or incubated for increasing times in the dark or in the presence of blue photorepair light. The CPD frequency was then determined from alkaline agarose gel electrophoresis, electronic imaging, computation of number average molecular lengths (see Eq. I ) and from them, the frequency of CPD (see Eq. 2) . Panels C and F of Fig. I show that soybean plants can carry out photorepair (Fig. IC) and excision (Fig. IF) of pyrimidine dimers.

In alfalfa seedlings, the rates of excision and photorepair-as well as their relative contributions to the total repair of d imersdepend on the initial CPD frequency. To determine whether the rates of repair in soybean might also de- pend on the initial damage level, we subjected seedlings to UV exposures inducing increasing levels of CPD and determined time courses of repair for the different CPD levels. Figure 1 shows the results of such experiments. The left column of Fig. IA, B and C shows the time courses of photoreactivation by seedlings challenged with initial dimer frequencies ranging from about 10 to almost 60 CPD/Mb. Al-

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Figure 1. Photorepair and excision repair in soybean seedlings. Seed- lings were exposed to UV to produce CPD levels from -12 to -60 CPDl Mb, then either exposed to photoreactivating light (A, B and C) or incubated in the dark (D, E and F). Samples were harvested at increasing times and their DNA isolated; dimer frequencies were determined by UV endonuclease digestion, agarose gel electrophoresis. ethidium staining, quantitation by electronic imaging, computation of number average molecular lengths and from them, the frequency of dimers. Initial CPD frequencies (in CPDIMb) for the panels were A, 12.6; B, 19.4; C, 57.2; D, 16.5; E, 22; and F, 46 (solid line) and 40.4 (dotted line). The data points represent the mean of at least two replicate determinations. The initial rates of repair were determined by a linear least-squares fit; the least- squares lines are shown as solid or dotted lines through the points used in the calculation; the dashed lines were fit by eye to the other data points.

Photorepair Time (mid Incubation Time (mid

though the photorepair rate is clearly highest at the higher initial dimer frequencies (cf: -0.9 CPD/Mb/min at 11 dimerslMb to 5 CPD/Mb/min at -50 dimersNb), it is clearly observable even at the lowest dimer level examined.

Panels E and F of Fig. 1 show that soybean plants carry out excision repair as well as photorepair. However, at the lowest initial dimer level, excision could not be detected (Fig. ID). Furthermore, Fig. IE and F show that, in this plant, excision rates at intermediate damage levels (-20 CPDIMb) are similar to those observed at about double that level (0.97 CPD/Mb/min at 20 CPD/Mb vs 1.06 CPDNblmin at initial levels of either 40 or 46 CPD/Mb).

Figure 2 (panel A) shows the dependence on the initial CPD level of photorepair and excision rates in soybean plants. Excision is not detectable at the lowest initial damage levels but is clearly demonstrable at the higher CPD frequencies. Photorepair also shows a significant dependence on initial CPD level but is quite effective even at the lowest dimer level studied. Comparison of these results with those for alfalfa obtained by Quaite et al. (21) (Fig 2B) shows both important similarities and striking differences. Figure 2 shows a comparison of dependence of photorepair and excision rates on initial dimer levels for soybean and for alfalfa. Both species show significant increases in rates of both

repair processes with increasing damage levels. However, the rates of excision and of photorepair in soybean are much higher than in alfalfa: the maximum excision rate we observed in soybean plants was - 1 CPD/Mb/min, whereas in alfalfa seedlings it was -0.25 CPD/Mb/min. Similarly, soybean plants carry out photorepair more rapidly than do alfalfa seedlings: although soybean plants can photorepair as rapidly as -5 CPD/Mb/min, the most rapid rate observed in alfalfa seedlings was one-tenth that value. Inspection of Fig. 2A and B also shows that, even with its high excision pro- ficiency, the rate of photorepair in soybean is five times higher than in alfalfa. Thus this highly excision-proficient plant still relies principally on photoreactivation to repair pyrimidine dimers in its DNA.

DISCUSSION

Conflicting data on the presence of excision repair in plants, and its contribution to dimer repair, have been obtained by different investigators. Although dimer excision could not be observed in Ginkgo ( 1 3 , Nicotiana ( 16) or Chlarnydorn- onas (17). Howland (18) and Eastwood and McLennan (19) observed low levels of CPD excision in carrot protoplasts. Small and Greimann (22) demonstrated both photorepair and

Photochemistry and Photobiology, 1996, 64(3) 467

Figure 2. Dependence of photorepair and excision rates in soybean (A) and alfalfa seedlings (B) on initial CPD level. (A) Soybean: the initial rates of the repair processes from Fig. 1 are plotted vs the initial dimer frequencies measured in each panel of Fig. I . Both photorepair and excision rates increase with increasing levels of CPD. (B) Alfalfa: initial rates of removal of CPD by excision and photorepair were measured in seedlings exposed to UV levels inducing increasing levels of CPD (data from Quaite et al. (21)). The slopes of the lines in both panels were determined by linear least-squares analysis.

ioybean A 0.60

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excision in chloroplasts, and Degani et al. (23) showed sub- stantial levels of excision but even more rapid photorepair in the aquatic plants Wolffia microscopicu and Spirodela po- lyrhizu. In wild-type Arubidopsis, Pang and Hays (24) could detect excision, but found that photorepair predominated; in the strain transparent testa, defective in UV-absorbing flavonoid production, Britt et al. (20) showed efficient photoreactivation but found little contribution of excision to CPD removal. One possible explanation for conflicting results was provided by the work of Quaite et al. (21), who showed that photorepair could be detected in alfalfa seedlings at all initial dimer levels examined, but excision was measurable only at higher damage levels. Our results on soybean show that this is the case in this plant as well.

Plants are exposed to varying levels of UV radiation daily, with higher levels during the midday hours; furthermore, not only does the absolute quantity of UV vary, the ratio of UV to visible radiation changes both daily and seasonally. The response of a plant to UV radiation depends both on its genotype and on its environment. For example, soybean cultivars differ in UVB sensitivity, with about one-half of the 60 cultivars tested showing UV sensitivity (3,4,25). Envi- ronmental conditions may also alter responses to UV. For example, concurrent exposure to drought (1.2) or nutrient deficiency (26) may mask UVB sensitivity, while exposure to UVB radiation under low light conditions may increase apparent UVB sensitivity (26). Even within a population, individuals may experience different microenvironments- shading from neighbors of the same or different species, wind stress and water conditions-that may affect both the damage levels and repair capacities.

As far as preservation of genetic integrity is concerned, it would be effective for a plant to employ all its repair capacity for even the lowest damage levels. Generally speak- ing, however, repair is not “free.” For all enzymatic processes, production of the repair proteins requires transcription from the DNA and translation into protein, all of which require expenditure of cellular energy. Making the simpli- fying assumption that all repair enzymes will have approx- imately the same half times in the cell and ignoring the re- quirements of some proteins for phosphorylation or for pro-

duction of a cofactor, we see that a multienzyme pathway will require the expenditure of more cellular resources than a repair pathway with few or a single enzyme. Thus excision repair, merely because of the participation of multiple enzymes in the incision, excision, resynthesis and ligation steps, is more expensive for an organism than is photorepair, a single-enzyme repair path.

Furthermore, photoreactivation is efficient for the cell in its use of light as an energy source for catalysis. Because plants are exposed to visible and near UV light that can be used for photorepair simultaneously with damaging UV radiation, they can, defacto, carry out repair virtually simultaneously with infliction of damage. Because photorepair re- verses the two pyrimidines covalently bonded in a CPD to two parental monomers, it also comprises an error-free repair path. Its specificity for CPD and light requirement do convey two limitations: it has no or little activity against non-CPD lesions and its activity ceases with the close of daylight.

Excision provides a complementary repair mode: as discussed above, it requires multiple enzymes, each one of which poses a debt against the energy budget of the cell. Further, the execution of nucleotide excision repair requires the expenditure of ATPs and incorporation of nucleoside tri- phosphates. Thus, in toto, excision is an expensive repair process for the cell. In balance, however, it offers a path for removal of bulky lesions other than CPD, as well as a mech- anism of removing CPD and other lesions in the absence of light.

Because CPD are induced at high frequencies by UV and interfere with transcription and with replication, organisms have evolved both repair paths for removing them and strategies for coordinating such paths. Soybean clearly utilizes primarily photorepair to advantage, both at low and at high damage levels. At extremely low CPD initial frequencies, this plant apparently does not use excision in dimer repair, but at higher damage levels, soybean clearly utilizes excision to repair CPD.

Comparison with the strategy used by alfalfa reveals striking differences: first, excision in soybean leaves is approxi- mately four times higher than the highest rate found in alfalfa (21). It might be hypothesized, then, with such effective

468 Betsy M. Sutherland ef a/.

nucleotide excision repair, that photorepair would be less important in the overall scheme for dimer removal. Surpris- ingly, however, photorepair is extremely rapid in soybean, about five times higher than the maximum rate of excision. The maximum photorepair rate in soybean is also some 10-fold higher than the highest rate we found in alfalfa.

Examination of the repair strategies in these plants reveals two major common themes: the predominance of photorepair and the increase in rates of excision and of photorepair with increasing initial CPD level. However, repair in soybean is distinctly different from that in alfalfa: repair overall is much more rapid, with both photorepair and excision rates many multiples of those observed in alfalfa. Further, as discussed above, even with highly effective excision repair, photorepair is used even more in soybean than in alfalfa for removal of CPD. These results indicate that delineation of repair in a variety of plant species under different conditions is nec- essary to understand repair strategies in communities com- posed of many individual species, as well as their ability to cope with environmental stresses under today’s conditions and under naturally occurring variation and those changes induced as a result of human activities.

Acknowledgements-This research was supported by the Office of Health and Environmental Research of the U S . Department of En- ergy and grants 92-02277 and 89-37280-4798 from the U.S. De- partment of Agriculture.

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Plant Responses to Changing Environmental Stress: Cyclobutyl Pyrimidine Dimer Repair in Soybean Leaves