Effects of High Light Stress by lzi10112

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									Plant Physiol. (1990) 94, 1663-1670                                                                         Received for publication April 23, 1990
0032-0889/90/94/1 663/08/$01 .00/0                                                                                     Accepted August 21, 1990


          Effects of High Light Stress on Carotenoid-Deficient
                     Chloroplasts in Pisum sativum'
                                        Anurag D. Sagar2 and Winslow R. Briggs*
               Carnegie Institution of Washington, Department of Plant Biology, Stanford, California 94305

                            ABSTRACT                                           vum) grown in the presence of the herbicide Norflurazon3
                                                                               (Sandoz 9789) in the context of its effect on a variety of
   The effects of high light stress on chloroplast ultrastructure              nuclear-encoded genes for photosynthetic proteins (24). The
and protein and mRNA composition were investigated in carote-                  primary site of action of Sandoz 9789 and a closely related
noid-deficient peas (Pisum sativum, L.). In low light, the thylakoid
membrane polypeptide pattem was altered, with several promi-                   derivative (Sandoz 6706) appears to be inhibition of the
nent chlorophyll-binding proteins present in diminished amounts.               dehydrogenation step of carotenoid synthesis (4, 6) such that
This change was found to be reflected in the ultrastructural                   carotenoid precursors phytoene and phytofluene accumulate.
organization of intemal chloroplast membranes. In contrast to the              If plants grown under low fluence-rate light are exposed to
normal grana stacking found in the controls, carotenoid-deficient              higher intensity light conditions in the absence of carotenoids,
plastids contained long, unstacked lamellae. Exposure to pho-                  damage to lipids as well as proteins and nucleic acids can
tooxidative light that resulted in destruction of >70% of chloro-              occur, presumably as a consequence of the intracellular for-
phyll did not lead to changes in total RNA and total cellular protein          mation of reactive free radical species (16). Despite this dam-
pattems. This treatment did lead to gross alterations in the                   age, however, plants grown in the presence of Norflurazon
chloroplast structure. Within 24 hours the plastid was seen as a               show normal photomorphogenetic responses (e.g. refs. 14,15).
swollen vesicle with only a few membrane remnants still present.                  Norflurazon-treated pea seedlings develop relatively nor-
Accumulation of five plastid-encoded mRNAs encoding a diverse                  mally under nonphotooxidative light conditions. Although
array of photosynthetic proteins was found to be affected in                   carotenoid biosynthesis is inhibited, some Chl accumulation
different ways. While psaA mRNA was rapidly reduced by more                    occurs. Furthermore, nucleus-transcribed mRNA species cod-
than 75%, levels of psbF/E and atpB/E were reduced by 50%.
psbA and petA mRNAs, on the other hand, appeared to be more
                                                                               ing for several chloroplast polypeptides are expressed at levels
resistant to photobleaching and remained relatively unchanged                  near those in control plants (24). When these seedlings are
during 24 hours of high fluence-rate light treatment.                          transferred to photooxidative light conditions, there is a dra-
                                                                               matic change both in the pigments and in the pattern of
                                                                               mRNA expression. Chl levels decrease almost threefold within
                                                                               8 to 10 h of the transfer, and accumulation of the nuclear
                                                                               mRNAs encoding chloroplast components is inhibited while
                                                                               cytoplasmic rRNA and levels of at least one cytoplasmic
                                                                               mRNA do not change. Similar phenomena have been ob-
   Long-term exposure of a green plant to strong light can                     served in maize, barley, and mustard (26).
result in injury to the photosynthetic apparatus, the damage                      We have investigated further the events occurring during
being caused in large part as a consequence of light- and                      chloroplast photooxidation in herbicide-treated peas. Previous
oxygen-dependent destruction of the photosynthetic pig-                        studies (24) indicated that although mRNA levels of Cab of
ments. This bleaching is an extemal symptom of the intracel-                   the light-harvesting complex of PSII in Norflurazon-treated
lular photooxidative damage occurring within the chloroplast.                  peas grown in dim red light were like those in the control
In higher plants damage caused as a consequence of Chl                         seedlings, the amount of the protein itself was reduced dra-
photooxidation has been investigated in several plant species                  matically. It was, therefore, of interest to do electron micro-
(3, 5, 7, 11-13, 18, 19) mainly in relation to the study of the                scopic investigations of the structure of the pea chloroplasts
effects of chlorosis-inducing herbicides. More recently, exper-                in 6-d-old pea seedlings grown in dim red light both prior to
iments with herbicides inhibiting carotenoid biosynthesis have                 and following exposure to strong white light for 24 h, with or
provided a useful system to study this process with respect                    without Norflurazon treatment. The biochemical basis of the
both to plastid damage and to its consequences for nuclear                     ultrastructural differences between control and photooxidized
gene expression (26).                                                          plastids was investigated by analysis of changes in soluble and
   We have previously characterized some of the damaging                       membrane polypeptides. Together with these biochemical
effects of photooxidative light on pea seedlings (Pisum sati-                  studies, the effects of photooxidation on the accumulation
   'This is Carnegie Institution of Washington Department of Plant                'Abbreviations: Norflurazon, 4-chloro-5-(methylamino)-2-(a,a,a-
Biology Publication No. 1043.                                                  trifluoro-m-tolyl)-3(2H)-pyridazinone; Cab, Chl a/b-binding protein;
   2Address: Plant Science Institute, Department of Biology, Univer-           RbcS and rbcL, small and large subunits of ribulose 1,5-bisphosphate
sity of Pennsylvania, Philadelphia, PA 19104.                                  carboxylase.
                                                                        1663
1664                                                    SAGAR AND BRIGGS                                      Plant Physiol. Vol. 94, 1990


pattern of plastid-encoded photosynthetic protein mRNAs            zon samples were heated. Electrophoresis was on 10 to 20%
are   reported.                                                    SDS polyacrylamide gradient gels at 4°C. Proteins were
                                                                   stained with Coomassie following electrophoresis.
                  MATERIAL AND METHODS                               Protein quantitation was carried out by a simplification of
                                                                   the Lowry assay (22) or the Bradford assay (8).
Plant Materials and Light Treatments
   Pisum sativum L. cv Alaska (W. Atlee Burpee Co., War-           Electron Microscopy
minster, PA) seedlings were treated with Norflurazon and              Sections (1 mm) of apical buds of peas grown in appropriate
grown as described previously (24). Peas were grown for 6 d        light conditions were fixed for 4 h in 2% glutaraldehyde and
in continuous, low-fluence-rate red light (0.7 ,umol m-2 s-')      1% paraformaldehyde and post-fixed in 1% phosphate-buff-
followed by transfer to continuous high-fluence-rate white         ered osmium tetroxide. En bloc staining was with 1 % aqueous
light conditions (100 ,mol m-2 s-', cool-white fluorescent).       uranyl acetate for 1 h, and followed by dehydration in a
Apical buds were harvested at various time points in the white     graded series of ethanol concentrations and infiltration in
light and either weighed and placed in dimethylformamide           Spurr's standard formula for firm resin overnight on a rotator.
for Chl determination or frozen in liquid N2 to be used for        Polymerization was carried out at 60 to 70°C for 24 h. Silver-
pigment, RNA, and protein analysis. The designation 0 h            gray sections 75 to 80 nm in thickness were cut on a Reichert
refers to buds harvested just prior to the transfer.               Ultracut E microtome and examined through a Phillips 410
                                                                   TEM after staining with Reynold's lead citrate.
Chi Determinations
  Chl quantitation was carried out as described (24), following                               RESULTS
the technique of Moran (21). Carotenoids and Chl were                 Pea seedlings grown in continuous red light (0.7 ,umol m-2
assayed by HPLC as described by Thayer and Bjorkman (27).          s-') were transferred to white light (100 ,umol m-2 s-') for 24
                                                                   h on d 6, and terminal buds were harvested at intervals
RNA Extraction and Analyses                                        thereafter for analysis of changes in fresh weight, Chl, RNA
  Cytoplasmic RNA extraction was carried out as described          and protein. These data calculated on a per bud basis are
in Sagar et al. (25) except that the extraction buffer contained   shown in Table I. Bud fresh weight of both control and
0.25 M sucrose and the filtrate was centrifuged for 10 min at      herbicide-treated seedlings is seen to increase in parallel, with
7,000 rpm. Preparation and treatment of slot blots was ac-         the carotenoid-deficient seedlings showing a small but con-
cording to published protocols (24). Each experimental slot        sistently higher value. As the table illustrates, while Chl/bud
was loaded with 2.5 ,ug of RNA and the results were quanti-
                                                                   increases 3-fold in control and decreases about 2.6-fold in
tated as described elsewhere (24). The chloroplast plasmid         herbicide-treated plants, a concomitant 1.5-fold increase oc-
                                                                   curs in RNA in both plants (although calculated on a unit
clones used for plastid RNA analysis were kindly provided by
Dr. Neal Woodbury. Isolation and properties of the chloro-         fresh weight basis one would see no change). Total protein
plast plasmid clones used have been described (29).                levels remain constant throughout the fresh weight increase
                                                                   in both herbicide-treated and control plants. The herbicide-
                                                                   treated peas synthesize RNA and protein and increase in fresh
Protein Analyses                                                   weight in a normal fashion. A detailed HPLC analysis of the
   Extraction and characterization of membrane proteins and        photosynthetic pigments (Table II) shows the complete ab-
electroblot analyses were as described previously (24). Crude      sence of all xanthophylls except lutein under low light con-
membrane preparations enriched for chloroplasts were ob-           ditions and reduced lutein and 13-carotene (3% and 13% of
tained by filtering homogenates through Miracloth and then
pelleting them by centrifugation at 15,000g for 10 min. The
remaining proteins, the chloroplast-depleted fraction obtained     Table I. Characteristics of Red Light-Grown Norflurazon-Treated
following filtration and pelleting of the chloroplast-enriched     and Untreated Pea Buds Transferred to High Fluence Rate White
fraction, were precipitated with 10% trichloroacetic acid. Fol-    Lighta
lowing centrifugation at 15,000g for 10 min the protein pellet          Time in                   Fresh wt.      RNA     Protein     Chl
obtained was washed in 90% ice-cold acetone and sonicated
in 0.1 M each DTT and Na2CO3, boiled in 5% SDS for 1 min,                 h                         mg            Ag           mg
and kept frozen at -70°C until electrophoresis. Antibodies to              0       Control          22        66    1.21             8.6
RbcS and rbcL were kindly provided by Dr. Arthur                                   Norflurazon      27        92    1.38             5.3
Grossman.                                                                  8       Control          29        83                    17.6
   Thylakoid membrane proteins were extracted according to                         Norflurazon      34       102                     3.0
the protocol of Metz and Miles (20). Extracted samples were                        Control          31        84    1.10            27.1
                                                                          16       Norflurazon      34        93    1.90             1.0
stored at -70°C. Prior to electrophoresis, samples were thawed                     Control          34       105    1.14            32.2
and denatured with lithium dodecylsulfate and DTT to final                24
                                                                                   Norflurazon      43       139    1.60              1.4
concentrations of 2% and 30 mm, respectively. Samples were
then either heated to 70°C prior to loading or loaded directly         Data are expressed on a per bud basis (average of 20         buds per
onto gels. In one experiment, neither control nor Norfluora-       experiment).
                                              PHOTOOXIDATION AND PLASTID STRUCTURE                                                           1 665


Table II. HPLC Analysis of Pigment Levels in Control and Norflurazon-Treated Peas Before and After Transfer to High Fluence Light
                       Hours in                                                 Pigment Levels
                      White Light     Chl a       Chl b     13-Carotene     Neoxana      Violaxan     Antheraxan     Zeaxan         Lutein
                                                                                  nmoles/bud
     Control               0          9.36        2.40         0.84           0.34         0.99           0.00        0.00           2.40
     Norflurazon           0          5.05        0.85         0.11           0.00         0.00           0.00        0.00           0.08
     Control              24         43.37      15.04          1.89           2.24         4.01           0.13        0.00          10.27
     Norflurazon          24           0.14       0.01         0.01           0.00         0.00           0.00        0.00           0.00
  a Neoxan, violaxan, antheroxan, zeaxan: neoxanthin, violaxanthin, antheroxanthin, zeaxanthin, respectively.



control levels) in Norflurazon-treated seedlings grown under              Biochemical Analysis
dim red light. Chl a and b were also somewhat reduced (to
about 54 and 34%, respectively). (Tables I and II present                    It was of interest to investigate the possible biochemical
results of single experiments, but each was repeated 2-3 times            basis of the ultrastructural differences between control and
with similar results.) The 24 h white light treatment virtually           herbicide-treated chloroplasts depicted in Figures 1 and 2. Gel
eliminated all photosynthetic pigments save for a small                   electrophoretic analyses of the chloroplast-enriched and the
amount of Chl a.                                                          remaining protein fractions were therefore carried out and are
                                                                          illustrated in Figures 3 and 4. Figure 3, panel A, shows the
Plastid Ultrastructure
                                                                          pattern of chloroplast-depleted proteins from control and
                                                                          Norflurazon-treated seedlings, prior to and at various times
   Figures 1 and 2 are electron micrographs ofsections through            following transfer to high light. Although the overall protein
terminal buds of peas grown in the absence (panels A and C)               profiles in both cases are remarkably similar, there are some
or presence (panels B and D) of Norflurazon. Panels A and B               polypeptides, for example a 14 kD protein near the bottom
show sections of 6-d-old seedlings grown under low fluence-               of the gel, which appear to be diminished. Antibodies specific
rate red light. Panels C and D are from seedlings exposed to              to two major soluble proteins, the small (RbcS; -14 kD) and
an additional 24 h of high fluence-rate white light.                      large (rbcL; -54 kD) subunits of ribulose 1,5-bisphosphate
   In control seedlings grown in dim red light, plastids are              carboxylase were used in a Western blot analysis. The results
oval to round in shape with an internal membrane organiza-                shown in Figure 3, panel B, indicate a reduction in the
tion that appears to be fairly typical of plastid development             abundance of both these polypeptides in the seedlings
in low light. Prolamellar bodies with thylakoid membranes                 undergoing photooxidation. Both proteins are almost unde-
radiating from them can be seen, together with numerous                   tectable by 24 h. In the case of RbcS, levels are reduced after
osmiophilic globules (which in many instances are seen to be              8 h of light treatment. In the control seedlings both protein
clustered near these crystalline arrays). Grana stacks and the            subunits show a gradual increase in light.
interconnecting lamellae are clearly visible. Plastids from                  Differences in the pattern of polypeptides from the chloro-
seedlings grown under dim red light in the presence of Nor-               plast-enriched fraction between control and Norflurazon-
flurazon, on the other hand, appear to be more irregular in               treated seedlings are seen in Figure 4, panel A. In this case a
structure (panel B). Although there is an extensive array of              dramatic reduction in the accumulation of several polypep-
internal membranes, these membranes are largely unstacked                 tides is observed. Some of these polypeptides are diminished
(overlaps of 2 thylakoids can be seen at several positions).              in the membranes of the treated seedlings even prior to
Prolamellar bodies are absent although there are numerous                 exposure to the photooxidative light conditions. To charac-
peripheral vesicles (which also may be involved in forming                terize more specifically the changes seen in the membrane
lamellae; see Bachmann et al. [2]). These prolamellar bodies              profiles for the chloroplast-enriched fraction, thylakoid mem-
are seen more clearly in a micrograph of the whole cell shown             branes were purified and subjected to electrophoresis on
in Figure 2, panel B.                                                     polyacrylamide gels. The pattern of the stained proteins is
   Panels C and D in Figures 1 (single chloroplast) and 2                 shown in panel B. The thylakoid membrane polypeptide
(entire cell) show the structure of plastids in control (panels           profile again demonstrates that several proteins fail to accu-
C) or Norflurazon-treated (panels D) seedlings that were                  mulate normally in Norflurazon-treated plants even under
placed in photooxidative light for 24 h. In the control plants            the nonphotooxidative light conditions. A 66 kD protein
one sees an oval chloroplast, again with well stacked thylakoid           the Chl-a-apoprotein of photosystem I-is reduced at 0 h.
membranes. Prolamellar bodies are no longer present, and a                Also, quite conspicuous is the decrease in the 27-29 kD
few starch grains are visible. Plastids from the carotenoid-              polypeptides, which likely represent the light-harvesting-com-
deficient seedlings are very dissimilar to those seen in panel            plex proteins of PSII.
B. Chl photooxidation (and presumably lipid peroxidation)
have brought about massive damage to the internal mem-                    Effects on Chloroplast mRNAs
brane structure. Although a few disorganized membranes are
still evident, the plastid appears otherwise like a round, swol-            Previously we characterized the effects of photooxidation
len vesicle.                                                              on nuclear-encoded chloroplast protein mRNAs in carote-
1 666                                                        SAGAR AND BRIGGS                                         Plant Physiol. Vol. 94, 1990

noid-deficient peas (24). Therefore, we also investigated the               show differing response patterns in high light. The psaA
changes in plastid-encoded mRNAs. Figure 5 shows the                        mRNA level declines -75%, but petA mRNA shows almost
changes, over a 24 h time period in high fluence-rate white                 no change. The psbA mRNA decreases slightly (-25-30%).
light, in mRNAs coding for different components of the                      This slow, small decrease is in contrast to the rapid and
photosynthetic apparatus. Results are shown for control and                 dramatic increase seen in the control seedlings. The mRNAs
Norflurazon-treated seedlings. psaA represents the Chl apo-                 corresponding to atpB/E and psbF/E decrease by 50%.
protein of the P700 complex of PSI; psbA codes for the Dl
protein of the reaction center of PSII; psbF/E code for the                                             DISCUSSION
small and large subunits of cytochrome b-559; atpB/E repre-
sents the f3 and e subunits of the coupling factor ATP synthase;               We have investigated the changes that occur in pea seedlings
petA codes for the Cyt fapoprotein. Although mRNA levels                    under light conditions that lead to Chl photooxidation. Al-
increase in the control plants, and there is a decrease in the              though the physiological effects of photooxidative stress have
mRNA in the Norflurazon-treated seedlings, the extent of the                been the subject of investigation for a number of years, the
response varies in each case. The psbA mRNA shows the                       effects at the molecular level are not well understood. We are
most dramatic induction, increasing almost 4-fold in 24 h of                interested in exploring the molecular basis of this process.
white light, while psaA and psbF/E mRNAs increase about                     Since plants lacking photoprotective carotenoids are particu-
1.5-fold, and petA and atpB/E show an intermediate 2-fold                   larly susceptible to photobleaching, the herbicide Norflurazon
increase. Similarly, the mRNAs in the photooxidative plastids               provides a convenient laboratory tool to study the biochemi-




                                                                              .I -




C




Figure 1. Electron micrographs illustrating effects of photooxidative light on plastid ultrastructure; 6-d-old seedlings grown in low fluence-rate
red light in the absence (A) or presence (B) of Norflurazon. Crystalline lattices of the prolamellar bodies and grana stacks were present in the
control plastids (A), but absent from the carotenoid-deficient plastids (B) which contained long, unstacked lamellae. High fluence-rate white light
treatment on control (C) and Norflurazon-treated (D) seedlings for 24 h drastically alters the structure of the chloroplasts of the latter. A few
internal membrane remnants and lipid droplets were visible. No change in cell anatomy (except for specific changes in plastid ultrastructure) was
apparent, nor were organelles visibly affected. Magnification x25,000.
                                             PHOTOOXIDATION AND PLASTID STRUCTURE                                                                1667




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Figure 2. Micrographs of sections through terminal buds of peas. Experimental conditions were the same as in Figure 1. Magnification x7,500.
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1 668                                                          SAGAR AND BRIGGS                                                                            Plant Physiol. Vol. 94, 1990


                            A^   ,\            ;'@"-   ,'ite
                                                                         grown in continuous red light-a condition in which Chl
                                                                         accumulation can occur.
                                                                            Figures 1 and 2 show that, whereas the control seedlings
                                                                         have a normal plastid structure with thylakoid membranes

                                      a.l I .                            radiating from prolamellar bodies and grana stacks as ex-
                                                                         pected, the herbicide-treated peas have a rather different
                                                                         plastid structure, with virtually no grana stacking seen. Similar
                                                                         observations were made in wheat (3). Frosch et al. (13) did
                                                                         not discern any differences in plastid membrane structure
                                                                         between control and herbicide-treated plants grown in far red
             B.. i.                                                      light. The most likely cause of lack of membrane stacking
                                                                         seen in our micrographs is the reduction of light-harvesting
                        4             |ll J.
                                         ~.
                                                                         complexes in these plants. Because carotenoids together with
                 ii                        _v..,         .v              Chl are known to be a component of these membrane-
                                                                         associated pigment-protein complexes, these results in turn
                                                                         indicate that normal assembly of light-harvesting complexes
                                                                         is probably dependent on the presence both of Chl and of
                                                                         carotenoids.
                                                                            Other thylakoid membrane-associated proteins including
                                                                         Cytfand the Dl protein (the Q3 herbicide-binding protein of
                                                                         PSII), are also found to be reduced under these conditions
                                                                         (not shown), presumably because of aberrations in membrane
                                                                         integration, although a more direct effect of photooxidation
                                                                         itself cannot be ruled out. Relatively fast turnover of the Dl
                                                                         protein has been demonstrated in excessively high light con-
                                                                         ditions (>3,000 uE) in Chlamydomonas (17). Not surpris-
                                                                         ingly, the herbicide-treated seedlings with a perturbed internal
                                                                         membrane structure are rather incompetent photosyntheti-
Figure 3. A, SDS-polyacrylamide gel electrophoretic analysis of pro-     cally as deduced from fluorescence measurements (our un-
teins from chloroplast-depleted fractions from peas. Seedlings grown     published observations).
for 6 d in continuous, low fluence-rate red light were transferred to       Following the 24 h of photooxidative light treatment which
high fluence-rate white light and terminal buds harvested at the times
indicated for protein analysis. B, Protein blot analysis investigating   is sufficient to bleach >80% of the Chl, the chloroplast
effects of high light on accumulation of the small and large subunits    structure is drastically altered and the plastid appears as a
of ribulose 1,5-bisphosphate carboxylase. The proteins were trans-       swollen vesicle with a few internal membrane remnants still
ferred to nitrocellulose membranes following electrophoresis. Levels
of the two subunits were assayed by visualization of antigen-antibody
complexes as described in "Materials and Methods."
                                                                          A                    ll]B

cal and molecular aspects of photooxidative stress. Under low
light conditions plants grown in the presence of the herbicide
can develop in a morphologically normal fashion. When                     ...
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transferred to high light, Chl levels are reduced, although fresh                                                                                   $: g                **.*|w.
weight as well as total RNA and protein continue to show an                                                        -i i::S         X*-+      t+j.   "
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increase (Table I), indicating normal cellular and metabolic              .:EiM:-

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   Although the seedlings accumulate Chl as well as mRNAs                                                                                           .*e
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coding for chloroplast proteins, HPLC analysis of carotenoid              wi a E[E 4,
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levels indicated an almost complete lack of xanthophylls and                          y
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very much reduced levels of A-carotene. It was therefore of               ___s_
                                                                                                                                                           <            * * P #} Z e
interest to analyse the plastid ultrastructure in the herbicide-
treated seedlings as well as the untreated controls. Although
previous studies have looked at the effects of nuclear muta-
tions (1-3, 5, 7, 23, 28) or inhibitors ofcarotenoid biosynthesis        Figure 4. Comparison of changes in the protein profiles from chlo-
                                                                         roplast-enriched fractions from carotenoid deficient (+Norflurazon)
(3, 13) on plastid structure, there have not been any detailed           and control seedlings. Denaturing polyacrylamide gel electrophoretic
studies correlating accumulation of mRNA and proteins in-                pattern of (A) total membrane proteins prior to (O h) and following
volved in photosynthesis with the structural development of              transfer to high fluence-rate white light, and (B) thylakoid membrane
the plastid. Moreover, this is the first study to analyse plastid        proteins. In the latter case, photooxidative exposure was for 8 or 96
structure in pea seedlings (both treated as well as untreated)           h as indicated.
                                                                       PHOTOOXIDATION AND PLASTID STRUCTURE                                                                                                        1 669


      200    -psa A                          0                           200        - petA                                                             0
                          0                                                                                                            .00
      150                                                                150        .   0
              -       0            0.                         .0                            *0               *

      100'                                                                                                        0
                                                                         100                ..   ..P..0.o                                         0
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       50
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        0       4     8           12    16           20           24       D                4            8        12        16             20         24
                                                                                                                                                                      Figure 5. Time course of photooxidation-in-
                                                                                                                                                                      duced changes in mRNA abundance for five
                                                                         200[ atpB/E                                                   S                              plastid-encoded genes. Buds were harvested
                                                                                                                       0                          0
                                                                                                                                                                      every 2 h following transfer to high fluence-rate
                                                                         150 -                                                             0                          white light. Each time point represents the av-
      400
              psb A
                                                                         100p           *~~~~~
                                                                                             0m                                                   0
                                                                                                                                                                      erage of three independent experiments. The 0
                                                                                                                                                                      h points were designated as 100% in both con-
                                                          0                                          * t.r
                                                                                                     '°"
                                                                                                                  0~~~~~
                                                                                                                                       g o
                                                                                                                                                                      trol and Norflurazon-treated plants; all other data
                                                                          50                                                                          0
 I
                                                 0
                                                                                                                                                                      points were normalized to these values. In each
 a)
      300                                                                       I                                                                                     panel the closed symbols represent the control,
 c
 co                                                                        0                4            8        12        16             20         24              and the open symbols represent mRNA levels in
                                                                                                                                                                      Norflurazon-treated seedlings. RNA levels in
                      00      0                                                                                                                                       control and Norflurazon-treated seedlings at 0 h
                                                                                                     1                                                      1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
                                                                                                                                                                      were similar (as was the case for nuclear RNAs
 z
      200                 0                                              200p[          psbF/E
                                                                                                                                                                    [24]).
 E                                                                       150-                            *             0
                          0                                                                      0                                                0
      I00                                                                                                                                  0
                          0"'°          0                     0          100
                                             ..
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        0       4     8           12    16       20               24       0                4            8        12        16             20         24

                                   Time in high light (hours)                                                     >



present. Interestingly, this damage to the plastid, although it                                                              have detected changes in several plastidic membrane and
does not affect overall RNA and protein accumulation, is                                                                     soluble polypeptides in carotenoid deficient maize leaves.
sufficient to cause changes in nuclear gene expression resulting                                                                Finally, to complete our analysis of the effects of high light
in the decrease in abundance of several nuclear encoded                                                                      stress on the chloroplast, we analysed the changes in the
mRNAs. This effect of photobleaching has been seen in                                                                        accumulation of plastid-encoded mRNAs. Earlier studies on
several plant species (26). In peas, the Chl a/b-binding protein                                                             the nuclear-encoded mRNAs demonstrated a diverse array of
mRNA was found to be most severely affected, while accu-                                                                     effects on the different mRNA species, with the Cab mRNA
mulation of the mRNAs coding for other chloroplast com-                                                                      being affected most strongly, while others, e.g. ferredoxin
ponents, e.g. ferredoxin and RbcS were less susceptible (24).                                                                mRNA, were rather more resistant to photooxidation (24).
   The fact that several cytosolic mRNAs coding for chloro-                                                                  Similarly, the plastid genes showed a variety of responses with
plast components are relatively unaffected is also reflected in                                                              the psaA (P700 apoprotein) mRNA being reduced by >75%
the overall protein patterns of total soluble proteins. Surpris-                                                             after 24 h of high light, while the petA (CytJ) and psbA (Dl
ingly, in the face of the dramatic alteration of plastid structure                                                           protein) mRNAs showed a relatively small decline. Levels of
these protein profiles appear essentially unchanged, except for                                                              psbF/E and atpB/E were reduced 50%. In the case of the
some specific proteins, e.g. RbcS and rbcL, which were found                                                                 nuclear genes the drop in mRNA levels could, at least in some
to be less abundant. Investigations of membrane polypeptides                                                                 cases, be ascribed to a drop in transcriptional activity. For
indicated that proteins in the 27 to 29 kD range, as well as a                                                               plastid genes, it has been postulated that mRNA turnover,
66 kD protein, were diminished. These membrane polypep-                                                                      and not transcription rates, is primarily responsible for mod-
tides are more clearly visualized in the thylakoid membrane                                                                  ulating genetic activity (9, 10). These results would then
profile (shown in Fig. 4, panel B) as the light-harvesting                                                                   indicate a short half-life for the psaA gene mRNA and a much
complex proteins PSII and the Chl-a apoprotein of PSI,                                                                       longer turnover rate for the psbA or petA mRNAs.
respectively. These proteins were found to be reduced even                                                                      In future experiments it will be of value to examine the
under the growth conditions with low fluence-rate red light                                                                  effects of photooxidative stress in plants acclimated to growth
conditions, probably a consequence of instability in the ab-                                                                 in high light in contrast to shade adapted plants. These studies
sence of carotenoids (see above). Similarly Mayfield et al. (18)                                                             would not only help to provide greater insight into effects of
1670                                                          SAGAR AND BRIGGS                                    Plant Physiol. Vol. 94, 1990

high light stress on sun versus shade chloroplasts, but also                  1017-1022
lead to understanding the adaptive strategies used by plants              12. Feierabend J, Winkelhusener T (1982) Nature of photooxidative
to protect their photosynthetic apparatus.                                      events in leaves treated with chlorosis-inducing herbicides.
                                                                                Plant Physiol 70: 1277-1282
                                                                          13. Frosch S, Jabben M, Bergfeld R, Kleinig H, Mohr H (1979)
                       ACKNOWLEDGMENTS                                          Inhibition of carotenoid biosynthesis and the herbicide San
                                                                                9789 and its consequence for the action of phytochrome on
   We would like to thank Fran Thomas for her generous help and                 plastogenesis. Planta 145: 497-505
guidance with the electron microscopy, Susan Thayer for the HPLC          14. Gorton HL, Briggs WR (1980) Phytochrome responses to end-
analysis, Olle Bjorkmen for helpful discussions, Timothy W. Short               of-day irradiations in light-grown corn grown in the presence
for his careful review of the manuscript, and Loretta Tayabas for help          and absence of Sandoz 9789. Plant Physiol 66: 1024-1026
                                                                          15. Jabben M, Deitzer GF (1979) Effects of the herbicide Sandoz
with the manuscript.                                                            9789 on photomorphogenic responses. Plant Physiol 63: 481-
                                                                                485
                                                                          16. Krinsky NI (1979) Carotenoid protection against oxidation. Pure
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