R. E. GROSS~ AND W. M. DUGGER, Department
Peroxyacetyl nitrate inhibits autotrophic and heterotrophic growth of Chlumydomonas. Experimental results show both photosynthesis and respiration are adversely affected by PAN. Photosynthesis is more severely damaged than is respiration, when measured as percentage of reduction and as the ability to recover after treatment. The presence of white or blue light during treatment enhances the effects of PAN on photosynthesis. Measurable sulfhydryl levels of cells show linear reductions with increasing amounts of PAN applied. Pigment analysis after treatment indicates a differential destruction of the chlorophylls, with chlorophyll b being more stable than chlorophyll a. Carotenoids are also destroyed, commencing with PAN treatment. The pattern of pigment destruction by PAN in whole cells is repeated in vitro, when the pigments in hexane-acetone are treated with PAN. The implications of PAN damage to Chlumydomonas are discussed in view of similar effects on higher plants.
Pcroxyacetyl nitrate (PAN) a strong oxidant formed photochemically in polluted air is a major component of smog. The phytotoxicity of the oxidant (Darley et al., 1963) has been related to PAN damage on the ultrastructure of chloroplasts (Thomson et al., 1965) and further examined in tissues, isolated cell fragments, and enzyme systems. (Mudd, 1965; &din and Altman, 1965; Dugger et (II., 1966). Differences in the effect of PAN on the photoreductivc system of bean plants due to age and physiological conditions were reported by Dugger and Ting (1968). The degree of light-dependent damage to the photosynthetic system was correlated by these workers, with the sulfhydryl contents of the green tissues. The present study utilizes a unicellular, sexual, green alga, Chlamydomonas rei~~hardtii, as the test organisms for further studies on the mechanism of PAN damage. Ease of culturing and handling of large homogeneous cell populations, which are metabolically closely related to higher plants, make it possible to examine, simultaneously, several cc,11functions under the influence of PAN. This study is also designed to observe some of the immediate changes taking placr after short exposures to PAN, with special emphasis on the pigment system. Furthermore, an attempt will be made to correlate the degree of PL4N damage on cell populations to structural differences between the wild type and two uvinduced mutants. MATERIALS
Chlamydomonus reinhardtii Dangeard (-) strain obtained from the Indiana Culture Collection ( #90) served as the wild-type test organism. From a series of ’ The work reported in this paper has been supported in part by a research grant ( AP-40) from the Division of Air Pollution, Bureau of State Services, Public Health Service. * Environmental Sciences Post Doctoral Trainee, USPHS, National Institutes of Health. 256
RESPONSE TO PAN
uv-induced pigment mutants, two were designated U-l and U-2, respectively, and also used as test organisms. All cultures were kept bacteria-free and unialgal through repetitive streaking on agar plates. The basal inorganic growth medium previously described (Gross, 1968) was modified by changing the N source to 1 g/liter of NH,NO, and the buffer system to I g/liter of K,HPO, and 0.5 g/liter of KH,PO, with the starting pH adjusted to 7.2. For heterotrophic growth 2.0 g/liter of sodium acetate, 0.5 g/liter of SOdium citrate, and 0.1 g/liter of CaCl, were added to the basal medium. In addition the buffer system was changed to 2 g/liter of KH,PO, and 1 g/liter of K,HPO, with the starting pH at 6.4. For growth on solid media 1.7%of agar was added to the liquid media. Cultures in %--mm diameter tc>sttubes were gassed with 1%CO,-in-air through glass tubing held in place bv a cotton plug. The tubes were suspended in a Plexi,glas water bath maintained at 25 + .l”C. Two banks of Ken-Rad cool white fluorescent lamps provided 1.2 x lOa ergs/set-cm’ of irradiance on both sides of the bath. Shake cultures \vere grown in a New Rrunswick reciprocal water bath shaker at 25°C. A bank of overhead fluorescent lights provided 1.8 ,y 10’ ergs/ set-cm’ of irradiancc at the lcvcl of the 125-ml cotton-plugged Ehrlenmeyer flasks used, Inocula wcrc in the log-phase of growth. The initial optical densities, determined in the culture tubes. were approximately 0.04 OD units as measured on a Coltmnn Junior spectrophotometer, Model 6A, at 550 m/l. Oxygen measurements were made with a model 53 Yellow Springs Instrument Co. oxygen monitor attached to a model SR Sargent recorder. The cells were contained in a 2.4~ml Plexiglas chnmbcr which could be illuminated from one side. 4 small magnetic stirrer with a stirring bar inside the chamber kept the cells in suspensionand the dissolved oxygen homogeneous. Chlorophylls and carotenoids were extracted and analyzed in a Cary 15 spectrophotometer accordin g to the method of Weybrew (1957). Carotenoids were extracted and partially separated in columns of weakcmcd aluminum oxide as described by Krinsky and Levine ( 1964). Xanthophylls were further purified by the cellulose thin-layer method of Schneider ( 1966). Sulfhydryl content was measured by use of the modified amperometric assay of Dugger and Ting (1968) adapted for algae. Cells were harvested by centrifugation and directly resuspended in 5 ml aliquots of 0.5 hl sucrose and 0.04 M K,HPO, solutions which for some tests was also made 0.01 hl with sodium ascorbate. The cells were then broken in a Riosonik sonicator with a 45second treatment and the entire sample added to 15 ml of sulfhydryl mix for assay. Peroxyacetyl nitrate was prepared, diluted with nitrogen, and assayed as described by Stephens et (11.( 1961). The algae were exposed to PAN in liquid supsensionthrough which metered amounts of the gaseousmixture were bubbled. Samples treated with N, scn.cd as controls. After treatment with PAN the samples were bubbled with air for 1 minute to drive off residual PAN and reestablish a normal gaseous equilibrium. For most tests the average PAN concentration was 125 ppm in N, and the treatments usually lasted several minutes. Exact concentrations figures will be given for each test. White light during treatments and for measuring photosynthesis was provided
by a G. E. 1493 filament lamp which was focused at the samples. Monochromatic light was obtained with a Bausch and Lomb high intensity grating monochrometer. RESULTS
When the gassing of an autotrophic culture of C. reirzlzardtii, wild type was changed for several minutes from 1%CO,-in-air to 100 ppm PAN-in-N, a complete cessation of growth was observed. Figure 1 shows the ensuing lag period during
(Growth of Chhnydonmm reinhardtii wild type bubbled with 1% CO?-in-air in FIG. 1. the light, or air in darkness, as a function of PAN treatment in darkness and subsequent incubation in the light and in darkness. Closed circles, untreated control. Open circles, 20 mpmoles P.4N/l >( 10’ cells. Solid lines, incubation in the light. Dashed lines, incubation in darkness.
which the cells that were kept in the light lost most of their green color resulting in a drop of OD, at 550 nm where chlorophyll absorption was at a minimum. If the treated cells were placed into darkness and bubbled with air, the cultures remained green; but this procedure did not speed up the resurgence of growth over a culture that was returned to the light immediately after the PAN treatment (Fig. 1) . When the treated cells were left in the light and bubbled continuously with N, they kept an intermediate green color. Addition of 0.0% S amino levulinic acid to the treated cultures did not affect the bleaching or regreening cycle of the cultures discussed in Fig. 1. However, the acid had some preventive effect. with respect to chlorophyll loss, in less severely damaged cultures. Complete protection from the PAN damage described in Fig. 1 could bc achieved by the addition of 0.08%reduced glutathione before, but not after, PAN treatment. A regenerated culture was grown for several doublings and then subjected to PAN again, under identic,21 conditions. The pattern of inactivation and resurgenct of growth repeated itself, as has been shown in Fig. I; in fact, this was repeated 8 times with the progeny of the original culture without the development of an)
obvious resistance. A similar sequence with hctcrotrophically grown cells ga\~ the same results. The effects of P.4N on the respiration of Ch.la221ydo222onrr.~ were examined h!using a culture of U-l, a yellow-green mutant, which responded strongly to the presence of acetate with increased rates of 0, uptake. Short PAN treatments subsided quickly to nea caused a rise in O1 uptake (c.g., Table I, 3) which rndogcmous levels. Longer treatments (e.g.. Table I, 2. 3) caused inhibition which took hours to dissipate. The reduction of substrate-induced 0, uptake was much greater (Table I, 6) than the reduction of endogenous respiration (Table 1, 3) when equal amounts of PAN were applied. The reduced levels of 0, uptake of the two treatments were similar.
I !) x 1 .s 2 0 4.0 1.1 ::.!I
The initial rise of 0, uptake upon short treatments ( Table I. 4) may he partiall>~ dues to a substrate effect of the PAN breakdown prodrlcts, specifically the acetate moiety of the molecule. The substrate effect was clearly shown (Table I, 7) \vhen ~~~11swcw introduced to a buffer solution that was previousI\; bubbled \\-ith PAN. .,\ comparison of photosynthesis and rrspiration of \\:hoI~ wild type ~11s after a P.4N treatment showed a marked difference in immediate damage as well as rcanwy potential. The percentage of reduction of 0, evolution in the light for tluw treatment levels was much greater than the percentage of reduction of 0, uptake, in darkness (Fi,q. 2). It is also apparent from the graph that recover!’ in darkness proceeded at il greater rate in the less srvrwly damaged process of endogenous wspiration. Immediate titrations for sulfhydryls after increasing PAN treatments showed a correlated decrease (Fig. 3) of SH content. Treatments alld titrations vwro esecutccl in room light, since conditions of light or darkness during trcatmcmts showc~l no marked diflercnces. A decrease in SH content equivalent to ;I treatnrcwt with 0.6 ~rmolcs PAS was also aehicwed by placin ‘,’ cells in buffer into darkness and brlhbling with air for 16 hours. Conditions of light and darkness during the treatment with P,4N had a marked effect on the severity of photosvnthesis inhibition. Figure -1 shorn-s the enhance-
Recovery of net photosynthesis and endogenous respiration in cells of wild type in buffer and darkness as a function of PAN treatments in darkness at 0 time. Continuous lines, net photosynthesis. Broken lines, endogenous tion. (a) 50 mpmoIes PAN/IO’ celIs; (m ) 100 mpmoles PAN/lo’ cells; (A) 200 PAN/lo’ cells. domonus
ment of this photosynthesis inhibition in the presence of 1.8 x 10” ergs/set-cmL’ white light. Blue light in general was more effective than red light for increasing damage. Figure 5 shows the difference in O2 evolution between a dark treatment and three PAN treatments under monochromatic light of 440, 450, and 485 nm all adjusted to 6.3 X lo3 ergs/set-cm2, as well as white light of 1.8 x 10’ ergs/set-cm2. White light irradiance of 6.3 X lo3 ergs/set-cm3 during PAN treatment was equivalent to darkness. Pigment extractions of cell samples treated with increasing amounts of PAI% showed a continuous loss of chlorophyll a and cartenoids while the amount of chlorophyll b did not change for the first 20 minutes after commencing the PAN
FIG. 3. The reduction in total measurable sulfhydryls in cells of Chlumydomonas wild type as a function of increasing concentrations of PAN, studied in room light.
HOURS FIG. 4. The effect of PAN treatments evolution in the light (net photosynthesis) Cells in buffer and light after treatment.
RESPONSE TO PAN
in the in cells
light and in darkness on subsequent of Chlumydomonas reinhardtii wild
treatment in darkness (Fig. 6). In terms of percentage reduction the loss of carotenoids is much greater than that of chlorophyll a, but the loss of both commenced immediately after the beginning of PAN treatment. A strong white light 13 X lo5 ergs/see-cm? irradiance) directed at the cells during treatment increased
FIG. 5. PAN type as a function evolution between after treatment.
damage to net photosynthesis in cells of Chlamydomonas reirlhardtii wild of light quality during treatment. Damage expressed as the difference in 01 cells treated in darkness and treatments in the light. Cells in buffer and light
FIG. 6. A. Reduction of pigments extracted from type as a function of PAN concentrations. B. Loss 1.6 ,rmoles of PAN as a function of light or darkness in darkness. Broken lines, cells shaken in the light.
cells of Chlam~~domonas of pigments from the after treatment. Solid
reinhardtii wild cells treated with lines, cells shaken
the rate of loss of carotenoids and chlorophyll a, and after 10 minutes of bubbling a reduction of chlorophyll b was also apparent. When the pigments of untreated cells were extracted, dried under N-, and redissolved in hexane-acetone it was possible to bubble the solution with PAN. Figure 7 shows the in r;ifro differential destruction in light and darkness of the PAN-treated pigments. The figures are calculated from N, bubbled controls which actually increased shghtly due to solvent loss. Aluminum oxide column and cellulose thin-layer chromatography of carotenoid pigments showed that the fastest disappearing pigment of this fraction upon treatment with PAN was ,&carotene. This pigment was identified by R,. and A,,,:,, values in petroleum ether (451 nm) separate and mixed with an authentic sample. The oxygenated carotenoids were tentatively identified by R, as violaxanthin, lutein, neoxanthin, and trollein. All were adversely affected by the PAN treatment of the whole cells, but since the degree of destruction was differential and some breakdown products interfered with a clear-cut separation, an analysis seemed premature. Table II lists the amounts of chlorophyll, total carotenoids. and SH contents of the wild type and two mutants, U-l and U-2, on a dry weight basis as well as chlorophyll a/b ratios. Also given are data from an average of three tests each. which show that after treatment with PAN in darkness the rate of recovery of net photosynthesis was different for the three cultures. U-l, the culture with the lowest SH count and the highest a/b ratio, recovered faster than U-2 which had
MINUTES FIG:. 7. Percentagr of in vitro reduction :ls a function of darkness (A) and light (B) 2. Chlorophyll a, 3. Total carotenoids.
of chloraplast pigments during continuous PAN
in hesane-acetone (7 : 3 ) gassing. 1. Chlorophyll h,
the highest SH level and the lowest a/b ratio. The wild type showed intermediate values for both SH and a/h, and its recovery potential was intermediate. also. DISCUSSION
Preliminary experiments (unpublished data) with the colorless alga Prototlzeca elcarly showed PAX damage on endogenous and substrate-induced respiration independent of conditions of light or darkness during treatment. Working, then, under the assumption that pigments are not required for inductions of PAN injury.
111lt re:tt~rtl cells nnalyri.5 (.%I1 per mg dry weight !
1 :; 1 I
the green alga Chk~wlytlomonus was used to study the effect of PAN on photosynthesis of whole cells against the background of damage to respiration. In order to assure realistic P.4N concentrations, all trcutments were so designed that the majority of a cell population was still viable at the end of an experiment. PAN damage to subsequent photosynthesis of whole wild type cells of cl&znl!Jrlo~~lonn.~ is clearly accentuated if white light is applied during treatment (Fig. 4). The differential rate of recovery of ccl1 samples gassed separately in light or darkness (Fig. 5) is another measure of difference. Increasing the amount of PAN applied durin g a dark treatment to make the initial inhibition equivalent to a light treatment with less PAN did not produce the same rccovcry kinetics for the two samples. This would be expected in ;I complex system \vherc lightindependent proccsses nre also damaged. Taylor et nl. ( 1961) found PAN injury to primary leaves of pinto beans is absolutely light-dependent, Unless light was applied before. during, and after PAN treatment no damage could lx observed in terms of bronzing of the lower leaf surfaces. However, it was later reported by Dugger ef al. (1963) that photosynthetic “CO, fixation by similarv treated bean leaves was equivalent to an untreated control for at least 1 hour after treatment or even until initial visible damage symptoms were apparent 3 hours latclr. It swnis that n close coupling bctwccn n photoreceptor and a later damaged process exists. It is not surprising for “CO, incxporation to come to a halt, once damage lwcomes apparent. Clearly, there is n continuous destructive process after PAN treatment of pinto leaves which acts initially on a level different from CO, fixation. In view of our findings that PAN d~tmagc~ immediately after treatment is greater for photosynthesis than for respiration (Fig. 2). ns well as the fact that PAS rc,duces the pigment contents of Ch1amy~~omonas (Fig. 6), it is tempting to associate the post-treatment damage in pinto leaves with a continuous loss or alteration in essential ,pigni&ts or chloroplast structures. In Chlunz!/do172of1(r,y \w observed a continuous breakdown of pignwnts cwn when the cells were gassed with PAN in darkness (Fig. 6). It is significant, ho\vever, in connection with the light-dependent damage in pinto leaves that after a strong P,4N treatment of Clz(nmytlo,r~oruls light induced the complete destruction of all pigments while darkness had a protective effect. The accelerated lxcnkdown ir2 c&o of pigments in hesanc, during treatment with PAN in the light (Fi& 7i also supports the al)ovc olxervations. Dllggcr et art. ( 1965) reported 011experiments in \vhich bean plants were esposccl to PAN and chloroplasts prepared 3 hours after fumigation, before visible damage developed, Significantly, the percentage of reduction in the Hill reaction of these chloroplasts ~1s the snmc whether the plants were left in the light OI placed into darkness for the 3 hours. In C711amyt707,lo12(1s./~707?~01~~~.~~ too, we olxervccl a cessation of Hill activity in chloroplast fragments from cells which were c~rposcd to 2AN and subsequently shnkcn in light or darkness, before preparation of the chloroplasts ( unpublished data). Recently Koukol et ~1. ( 1967) found cyclic photophosphorylation of chloroplasts isolated from Black Valentine lwau leaves was inhibited 1,~ PAN irregarclless of light or darkness during trratmcnt. This result is in good agrecmcwt with our observations on the effect of light or darknesc
during treatments of Chlnm~domonas, although we did not check this particular cell function. The effects of PAN on the respiration of Chlamgdomonns were quite varied, depending on the length of the treatment. Endogenous respiration of U-l was accelerated by short treatments, while longer treatments caused increasing inhibition (Table I, 2, 3, 4). Not shown on the table is the effect of 60 or 90 mpmolrs of PAN on similar cell samples. The rise in respiration still occurs. but is preceded by a short period of inhibition. This phenomenon of increased respiration due to oxidant treatment is somewhat analogous to the results of Todd ( 1958) who found a rise in respiration in ozone-treated pinto bean lcaves. Tl’r do not know if our results wcrc due to an uncoupling effect. Substrate-induced respiration was reduced to a similar low level as endogenous respiration (Table I, 6) by an equivalent PAN treatment’; but the percentage of reduction in this case was much greater. Thch rise of respiration when celk were introduced into buffer which had previously been b~lhhled with PAN (Tahlc I, 7) is, in our estimate, a substrate effect due to the acetate moiety from broken C~OWJ> PAN and is inclistinguishahl(1 from an acetntc-indllccd rise in respiration (Table 1. 5). Mudd ( 1965) has shonTnthat PAN inactivated enzymes. but some’w(‘re S~O~VJJ to bc prottctcd by substratc~sand cofactors. Hc conc~luclrd the inactivation VX> due to oxidation of the enzyme sulfhydryl groups. In a recent paper 1111ggerand Ting (1968) correlated susceptibility of Gan plants to PAN \vith the levels ot sulfhydryl compounds in the tc>stplants. Age of the plants and li$t-dark chan:~cs were shown to be the most prominent factors influclncing SH contents. Plants with h$ier levels of sulfhydryls were markedly more [email protected]
to PAX under thcb conditions of the experiments. Also after PAN trctattnt&, when rmdt~rsurfxc glazing of the lraves b~~camc~ apparent, thev found a significant rrclriction in Sll c,ontents of their test plants. Tn ~:hI~2l22!/~70222o~2ns. too. \\-e consistently fouxl that PAN rrduccs th the highest a/b ratio, alth:jugh \\.c arc‘ aw:irc of tlic fact that total chlorophyll of this mutant is low. On the other hand. tli(s c~tlls slojvest to r(‘cover. U-2. have the lowest a/b ratio (Table II). Dugger CI (11.( 1963) were nhlc to abtain ;m action spectrum of light-medi:ttc%d PAN damage to pinto beans. On the basis of increased damage when blue light between 300 and 500 nm was applied they [email protected] a possible photo-reaction between carotcnoicl pigments and PAN. Tl ic> 111uc light r+fect. espxklly at 440
and 485 nm (Fig. 5), is also apparent in Chlamydornontr.~ enoids were shown to be severely affected by PAN.
and, of course, tarot-
REFERENCES DARLEY, E. F. cf al. (1963). Plant damage hy pollution derived from automobiles, iirch. Environ. He&h 6, 761-770. Effect of peroxyDUGGER, W. hl., JR., KOUKOL, J.. REISD, W. D., AXU PALMER, R. I,. ( 196:3). acetyl nitrate on C”O1 fixation by spinach chloroplasts and pinto bran plants. Plant Ph~siol. 38, 468472. DUGGER, W. M., Ju., Tarpon, 0. C., KLEIN, W. H., AND SHROPSHIRE, FL-., JH. ( 1963). Action spectrum of peroxyacetyl nitrate damage to bean plants. Nature 198, 7576. DUGGEB, W. M., JR., MUDII, J. B., ASD KOUKOL, J. (1965). Effect of PAN on certain photosynthetic reactions. Arch. Environ. Health 10, 195-200. DUGGER, W. AI., JR., KOUKOL, J., AND PALMER, R. L. ( 1966). Physiological and biochemical effects of atmospheric oxidants on plants. J. Air Pollution Control ilssoc. 16, 467-471. DVT(:C.ELI, W. hf., JR.. .4x1) TTSC:, I. P. ( 1968). The effect of perosyacetyl nitrate on plants: Photoreductive reactions and susceptibility of bean plants to PAN. Phytopathology. In press. CROSS, R. E. ( 1968 ). Xlechanism of toxicity and resistance to D-mannose and certain derivatives in species of the genus Chlorellu Beij. J. Ph!/col. 4, 140-151. ~OUSOL, J., DUCCER, W. \I., JR.. AiXD P.chim, R. L. (1967). Inhibitory effect of peroxyacetyl nitrate on cyclic photophosphorylation by chloroplasts from Black Valentine bean leaves. Plant Physiol. 42, 1419-1422. KRINSKY, N. I., AND LEVINE, R. P. (1964). Carotenoids of wild type and mutant strains of the green alga, Chlamydomonas reinhardi. Plant Physiol. 39, 680-687. MUDD, J. B. (1965). Responses of enzyme systems to air pollutants. Arch. Enoiron. Health 10, 201-206. ORDIN, L., AND ALTMAX, A. ( 1965). Inhibition of phosphoglucomrrtase activity in oat coleoptiles by air pollutants. Ph~ysiol. Phntarrun 18, 790-797. E’ me einfache Methode zur diin:rschichtc1~ron~atograpl~ischen SCHNEIDEH, H. A. W. (1966). Trennung von Plastidenpigmenten. J. Chromutog. 21, 448453. STEPHEM, E. R., DARLEY, E. F., TAYLOR, 0. C., AND SCOTT, W. E. ( 1961). Photochemical reaction products in air pollution. Intern. J. Air Water Pollution 4, 79-100. TAYLOR, 0. C., DUGGER, \f'. hl.. JR., CAKDIFF, E. A., AND DARLEY, E. F. (1961). Interaction of light and atmospheric photochemical products (‘smog’) within plants. Nature 192, 814816. ( 1965). Elfects of peroxyacetyl THOMSON, W. W., DUGGER, W. M.. JR., ANII PALMER, R. L. nitrate on ultrastructure of chloroplasts. Botan. Ga-. 126, 66-72. TODD, G. W. (1958). Effect of ozone and ozonated I-hexene on respiration and photosynthesis of leaves. Pkunt Physiol. 33, 416420. WEYBREW, J. A. (1957). Estimation of the plastid pigments of tobacco. Tobacco 144, 18-22.