the effect of supplementary ultraviolet-b radiation on mrna

Journal of Experimental Botany, Vol. 48, No. 308, pp. 729-738, March 1997Journal ofExperimentalBotany

The effect of supplementary ultraviolet-B radiation onmRNA transcripts, translation and stability of chloroplastproteins and pigment formation in Pisum sativum L.

Soheila A-H. Mackerness1'3, Brian Thomas1 and Brian R. Jordan2

1 Horticulture Research International, Wellesbourne, Warwick CV359EF, UK2 New Zealand Institute for Crop and Food Research Limited, Levin Research Centre, Kimberley Road,Private Bag 4005, Levin, New Zealand

Received 23 November 1995; Accepted 23 October 1996

Abstract

The effect of supplemental UV-B radiation on geneexpression for three photosynthetic proteins (chloro-phyll a/b-binding protein, D1 polypeptide of PS II andRUBISCO) and on flavonoid composition has beeninvestigated in expanded leaves and in apical buds ofpea seedlings. In the expanded third leaves, UV-Bcaused a decrease in nuclear-encoded Lhcb and RbcStranscript levels within 2 d of UV-B treatment and alater decline in the SSU and LHCB polypeptides. Theeffect of UV-B on the chloroplast-encoded rbcL andpsbA genes was more complex. Levels of LSU and D1initially appeared to be regulated at the translationaland/or post-translational level and only later in theUV-B treatment, at the level of mRNA abundance. UV-Balso accelerated the degradation of the D1 polypep-tide. For all genes studied the UV-B-induced inhibitionof transcripts was greater in leaves than in the apicalbuds, indicating that the bud is less sensitive to UV-Bdamage. The mRNA transcript levels for chalcone syn-thase [chs) were also measured. In leaves, chs RNAtranscripts were initially present in low amounts andexposure to supplemental UV-B resulted in a transientincrease in the level of these transcripts. In contrast,chs transcripts in the apical buds were initially presentat high levels, with UV-B exposure resulting in a slowprogressive decline in transcript levels. These resultsindicate that the response to UV-B is complex and isdependent on the organ studied.

Key words: Flavonoids, pea, protein turnover, RNAtranscripts, UV-B stress.

Introduction

A decrease in the stratospheric ozone concentrationhas led to an increase in the ultraviolet-B (UV-B;280-320 nm) radiation reaching the earth's surface(Blumthaler and Ambach, 1990). This increase is likelyto continue into the foreseeable future, inevitably havinga negative impact on biological organisms. Plants cannotavoid sunlight and are, therefore, particularly at risk. Theimpact of an increase in UV-B on the physiologicalparameters and morphological features of plants has beenextensively studied (Bornman and Teramura, 1993).However, the knowledge of the effect of UV-B at thebiochemical and molecular levels is limited (reviewed inJordan, 1996).

It is well established that a major site of damage byUV-B is the chloroplast, leading to impairment of photo-synthetic function (Bornman, 1989). Recently, a fewstudies have focused on the molecular mechanism under-lying UV-B sensitivity of photosynthesis. SupplementalUV-B was found to cause a decline in total RNA, enzymeactivity and protein levels of several key photosyntheticproteins including RUBISCO (Jordan et al., 1992), Dlpolypeptide of photosystem II, chlorophyll a/6-bindingprotein (LHCB; Jordan et al., 1991) and the ATPasecomplex (Zhang et al., 1994). Nuclear-encoded genes aremore sensitive than genes encoded in the chloroplast(Jordan, 1996). The relative sensitivity of transcripts toUV-B radiation is also dependent on the developmentalstage of the tissue studied (Jordan et al., 1994).

In contrast to the down-regulation of genes encodingphotosynthetic proteins, UV-B results in the up-regulationof some defence genes such as chalcone synthase (Jordan

3 To whom correspondence should be addressed. Fax: +44 1789 470 552. E-mail:[email protected]

© Oxford University Press 1997

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et al., 1994) and glutathione reductase (Strid, 1993).Glutathione reductase is thought to be an important partof a system designed to scavenge active oxygen producedin response to oxidative damage caused by exposure ofplants to a multitude of stresses (Smith et al., 1990).Chalcone synthase is a key enzyme in the synthesis offlavonoids. Flavonoids are produced under a variety ofconditions including UV-B exposure and are thought toact as UV-B screening pigments (Caldwell, 1981;Wellman, 1983). Increases in the level of mRNA for thesegenes indicate that repression of gene expression forchloroplast proteins is a specific response to UV-B treat-ment and not a result of non-specific damage to theDNA. Similarly, Lhcb genes are up-regulated in etiolatedbuds while down-regulated in green leaves under thesame UV-B treatment (Jordan et al., 1994). In addition,absence of photolyase involvement in protection of photo-synthetic transcripts against UV-B damage, under highPAR conditions (A-H Mackerness et al., 1996), alsoindicates that UV-B effects on gene expression are specific.

Studies of the effects of UV-B on mRNA levels indicatethat UV-B-mediated inhibition of photosynthetic per-formance can, to some extent, be related to decreases inthe levels of photosynthetic proteins. They do notshow, however, whether the effect of UV-B arisesthrough changes in transcription, translation or post-transcriptional events. The accumulation of flavonoidcompounds in response to UV-B light has been shown tobe due to an increase in the rate of transcription of thechalcone synthase (chs) gene (Chappell and Hahlbrock,1984). However, the level at which UV-B down-regulatesthe genes coding for chloroplast proteins has not beenstudied.

The aim of this study was 2-fold: firstly, to investigatefurther the effects of UV-B on gene expression and proteinlevels of chloroplast proteins in fully expanded leaves,and to determine at what level UV-B is affecting theirsynthesis and degradation. In addition, to compare theresponse of two different organs to UV-B exposure interms of changes in RNA levels and flavonoids.

Materials and methods

Plant material and experimental conditions

Pea (Pisum sativum L. cv. Feltham first) seedlings were grownin a controlled environment cabinet, partitioned in two halvesusing a piece of hard wood covered on both sides with adhesive-backed aluminized Melinex (Ritrama Ltd, Manchester, UK)with 12h light (22°C), 12 h dark (16°C) cycles, at 70%humidity for 17 d. Incident radiation was provided by Philipswarm white fluorescent tubes giving an irradiance of 150fimolm~2 s"1 PAR. Half of the plants were then given supplementaryUV-B radiation from two UV Lamps (Philips TL 40) duringthe 12 h photoperiod. To exclude UV radiation below 290 nm,the UV-B lamps were wrapped in cellulose acetate sheets whichwere changed daily. The other half of the plants (controls) were

subjected to the same treatment but in the absence of activatedUV-B lamps. For the feeding experiments, illumination wascontinuous with or without UV-B supplementation. The spectralirradiance was determined between 260 and 320 nm, using anOptronics 740A spectroradiometer (Optronics Laboratories,inc, Orlando, Fl, USA). The incident dose rate was estimatedto be equivalent to 182 mW m"2 (UV-BBE based on generalizedplant action spectrum of Caldwell (1971), normalized to unityat 300 nm). This level of UV-B represents 8.5% at 297 nm and7.5% at 313 nm of UV-B radiation used in a comparable study(Jordan et al., 1991, 1992). The third pair of leaflets or theapical bud were routinely used for experiments as indicated inthe text and samples taken 6 h into the photoperiod unlessotherwise stated.

All experiments were independently duplicated or triplicatedas indicated in the text. Samples were taken at random fromdifferent parts of the cabinets and from separate plants selectedfor their uniformity.

Purification of total RNA

Leaves (2g) and the apical buds (1 g) were removed fromplants, frozen in liquid nitrogen and stored at — 70 °C. TotalRNA was extracted as described in Jordan et al. (1991). TheRNA was stored at - 7 0 ° C .

RNA blotting

RNA (20 fig) was separated on a 1.5% agarose gel containing6% (v/v) formaldehyde in 2 x MOPS (Maniatis et al., 1982).Each gel was visualized under UV light both to check RNAintegrity and to ensure that equal amounts of RNA were loadedin each lane (data not shown). The RNA was transferred toHybond-N (Amersham International, UK) by capillary blotting(Maniatis et al., 1982). Northern blots were hybridized withDNA probes labelled with a-[32P] deoxycytosine 5'-triphosphate(a-[32P]dCTP, 3000 Ci mmor1, Amersham International, UK)by using a BRL Random prime DNA labelling system (LifeTechnologies, UK) as per manufacturers instructions. The blotswere prehybridized for 12 h at 65 °C in 5 x SSC, 50 mMphosphate buffer pH 7, lOOmgml"1 denatured calf thymusDNA, 0.1% (w/v) SDS and 1 x Denhardt's solution (0.1% Ficoll(molecular biology grade, Sigma), 0.1% bovine serum albumin(BSA), 0.1% polyvinylpyrrolidone (molecular biology grade,Sigma). The denatured probe was then added and the blotincubated for a further 12-14 h at 65 °C. The blots were washedtwice in 0.3 x SSC; 0.1% (w/v) SDS, for 15 min at 25 °C, andonce for 15 min at 65 °C. Autoradiography of the filters was at— 70 °C using Fuji RX film with two intensifying screens.Relative amounts of hybridization to specific bands werequantified by excising the appropriate area of the filter andquantification of the bound radioactive probe by using liquidscintillation spectrometry (Beckman LS 6000 TA) in Omnisafescintillation fluid (Life Technologies, UK).

Quantitative data from Northern blots is presented aspercentage transcripts from UV-B treated plants as comparedto transcripts from control plants. Values are quoted as resultsfrom three independent experiments.

Preparation of proteins

Total protein was isolated from leaf discs (1 cm2) cut from thethird pair of leaflets or from the leaflets by extraction of c. 0.5 gof tissue with 8 M urea, 6.25 mM TRIS-HC1 pH 6.8, 2% (w/v)SDS and 5% j3-mercaptoethanol (0.5 cm3 per 0.5 g). Thesamples were spun at 2700 g for 10 min and precipitated withacetone (80% (v/v) final concentration). The protein pellet wasresuspended in 0.2 M NaOH and the concentration of the


samples determined using a Pierce BCA protein assay (LifeTechnologies, UK).

SDS-PAGE, Western blotting and immunostaining

Protein samples were prepared for SDS-PAGE by addition ofan equal volume of 20% glycerol, 0.02% Bromophenol Blue,2% SDS, 6.5 mM TRIS-HC1, pH 6.8 and 10% (v/v) 0-mercaptoethanol and boiling for 10 min. Gels were loaded onan equal protein basis (30 ^g) on to a discontinuous SDS-polyacrylamide gel (Laemmli, 1977) consisting of a 12% (w/v)acrylamide resolving gel and a 4% (w/v) stacking gel, using aBiorad mini gel system, and electrophoresed for 3 h at 100 V.After electrophoresis the gels were either stained with Coomassieblue or blotted on to nitrocellulose using a Bio-Rad mini blottersystem and immunostained with the appropriate antibody as inBesford et al. (1993). An image of the blots was obtained byscanning using a Umax PowerLook Scanner enabling quanti-fication of the proteins using Image 1.47 programme fromApple Macintosh.

Quantitative data from Western blots is presented as proteinlevels in UV-B treated plants as a percentage of protein levelsin control plants. Values are quoted as results from threeindependent experiments.

DNA sequences and antibodies

The sequence for the rbcL is a 657 bp Pst I fragment frommaize (Jordan et al., 1989) and the rbcS sequence is a 250 bpHind III cDNA subclone from pea, pSSUl (Bedbrook et al.,1980). The psbA sequence is an 850 bp Hind III fragmentcontaining the 3' 60% of the gene from spinach (Jordan et al.,1989). The Lhcb cDNA sequence (pAB 96) is a 1.2 kbp Pst IcDNA clone from pea and is described in Corruzzi et al.(1983). The chalcone synthase 1 (chs) sequence is a 1.4 kbpEcoRI/Hind III cDNA clone from pea (Harker et al., 1990).

The antibody against RUBISCO was raised against tomatoRUBISCO and is described in Besford (1990). The Dl antibodywas raised against the N-terminal portion of the Dl polypeptideand is described in Besford et al. (1993). The Lhcb antibodywas a monoclonal antibody specific for the light harvestingcomplex II (LHCII) and is described in Darr et al. (1986; ClassVI). This antibody cross-reacted most effectively with the28 kDa LHCB protein and therefore, the effect of UV-B onthis LHCB protein is described in this paper.

In vivo turn-over of proteins

Rates of in vivo protein synthesis and degradation weredetermined by radioactive labelling with35S methionine asdescribed by Greenberg et al. (1989). Ten leaf discs (1 cm2)were floated adaxial side up in 20 ml of 0.1% Tween containing3fiCiml~" of 35S methionine (1000 Cimmol"1; AmershamInternational, UK). The discs were then incubated in thepresence or absence (control) of supplemental UV-B asdescribed earlier. To observe degradation of the proteins, theleaf discs were radiolabelled with 35S methionine for 3 h undercontrol conditions and chased in 1 mM methionine for 4, 8 or24 h under control or UV-B conditions. To observe synthesis,the leaf discs were radiolabelled with 3!S methionine for 1 hunder control or UV-B conditions. For both synthesis anddegradation experiments, after the incubation period, the discswere washed three times in distilled water and then frozenimmediately in liquid nitrogen. The samples were prepared forelectrophoresis as described above and equal amounts of protein(40 fig) analysed by SDS-PAGE. The gels were treated withIntensify (NEN Research Products) before autoradiographywith Hyperfilm 0-max (Amersham International, UK).

UV-B, chloroplast proteins and flavonoids 731

Quantification of the radiolabelled proteins was made by usinga scanning densitometer (Biorad model 620). Peak heightsfrom the autoradiographs are quoted relative to a standard(the 46 kDa protein), when I fA of 14C labelled molecularweight markers were loaded (MW 14.3-220 kDa, 5fiC\m\~\Amersham International, UK) which was normalized to unity.The results are quoted relative to this value, as relative peakheights (rph) and are results from two independent experiments.The position of the proteins was determined by immunoblottingwith the appropriate antibody as described above.

The effect of the protein synthesis inhibitors cycloheximide(Clix) and streptomycin (St) on the protein levels, under controland UV-B conditions, were also determined by using leaf discs.The leaf discs were floated as above in either 50 mg ml"1 Chxor St or on dH2O. After appropriate incubation periods thediscs were washed and stored as above. The samples wereprepared for electrophoresis, separated on SDS-PAGE andspecific proteins detected by western blotting and immuno-staining. The experiment was repeated three times with similarresults and data from one set of experiments is presented.

Flavonoid analysis

For flavonoid determination, c. 20 mg samples of leaves orbuds were ground into a fine powder under liquid nitrogen.The frozen powder was extracted in methanol (5 cm3 per 0.5g), microfuged at 13 000 rpm and the absorbance scannedbetween 200 and 450 nm, as described in Markham (1982),using a Perkin-Elmer 557 dual wavelength, double beamspectrophotometer. The experiment was repeated three timeswith similar results and data from one set of experiments ispresented.

Statistical analysis

An analysis of variance (ANOVA) was performed for eachexperiment unless otherwise stated. Standard errors of differ-ences (SED) were determined from the ANOVAs.

Results

Effect of UV-B radiation on mRNA levels from leaves andbuds

Data from quantitative Northern blot analysis of tran-scripts for Lhcb, RbcS, rbcL, and psbA, from UV-Btreated plants as compared to transcripts from controlplants, on equivalent days, are plotted in Fig. 1.

In leaves, the nuclear-encoded Lhcb and RbcS (Fig. la,b) transcripts were reduced dramatically after 2 d ofUV-B treatment, and had fallen to c. 19.5% and 5.4% ofcontrol values after 4 d UV-B treatment. The declinecontinued up to 8 d where the transcripts were onlypresent in trace amounts. The effect of UV-B on thechloroplast-encoded genes was not as dramatic.Transcripts from rbcL were not significantly reduced after2d of UV-B supplementation (Fig. lc). The levels beganto decline by the third day of treatment and were at 45%of control values after 4 d treatment. The psbA was theleast affected by the UV-B supplementation over the first4 d of treatment, by which time transcript levels were stillat 88% of control values (Fig. Id).

The mRNA levels for all four photosynthetic geneswere reduced less in the buds than in leaves (Fig. 1).



0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8

UV-B exposure (d)

Fig. 1. Changes in the amounts of leaf (O) and bud ( • ) mRNA duringexposure to UV-B, relative to control values. Northern blots, probedwith cDNA probes, were quantified by excision of the appropriate areaof the filter and determining the amount of bound radioactive probeusing liquid scintillation spectrometry. The relative hybridization isdefined as the percentage c.p.m. obtained from samples from UV-treatedplants with respect to c.p.m. from samples from control plants, whichwere treated under the same conditions, but in the absence of UV-B,on equivalent days of treatment. Each point represents the mean valueobtained from three independent experiments. Error bars represent thestandard errors of differences of the means. Probes used were (a) Lhcb,(b) RbcS, (c) rbcL, (d) psbA.

Lhcb and RbcS transcript levels were only significantlyreduced after 5 d and 4 d of UV-B treatment, respectively.Lhcb transcripts were reduced to c. 76% and RbcS tran-scripts to 58% after 5 d and 4 d of UV-B treatment,respectively (Fig. la, b). For the chloroplast-encodedgene, rbcL, the decline in transcript levels was delayed inbuds as compared to in leaves, dropping after 3 d ofUV-B treatment and falling at a slower rate than tran-scripts in leaves (Fig. lc). In contrast, psbA transcriptlevels were not significantly reduced by exposure to UV-Buntil the 5th day of UV-B treatment (Fig. Id). Furtherstudies on the four photosynthetic genes were carried outusing leaves which were more responsive to UV-B thanthe young buds.

In both organs studied, the level of transcripts for allfour photosynthetic genes studied remained relativelyconstant, throughout the experiment, under controlconditions.

Protein levels in leaves

Total proteins were separated by SDS-PAGE, transferredto nitrocellulose and examined by Western blottingand immunostaining. Quantitative data obtained fromWestern blot analysis, comparing protein levels fromUV-B treated plants with levels in control plants, onequivalent days, are shown in Fig. 2. Relative to controls,the levels of the nuclear-encoded LHCB protein and theSSU of RUBISCO, isolated from leaves, dropped signi-

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Fig. 2. Changes in the amounts of photosynthetic proteins, in leaves,during exposure to UV-B, relative to control values. Western blots,immunoblotted with the appropriate antibody, were quantified usingthe Image 1.47 package for Apple Macintosh. The protein levels aredefined as the percentage density of the protein bands obtained fromUV-treated plants with respect to densities from control plants, whichwere treated under the same conditions but in the absence ofsupplemental UV-B, on equivalent days. Each point represents themean value obtained from three independent experiments. Error barsindicate the standard errors of differences of the means. Antibodiesused were against (a) RUBISCO SSU ( • ) , LHCB protein (O), and(b) RUBISCO LSU (A), Dl (A).

ficantly over the 8 d of treatment (Fig. 2a). There wassome delay in the decline in protein levels compared toRNA levels. The levels of LHCB protein and SSU poly-peptide did not significantly change for the first 2 d and1 d of UV-B exposure, respectively, after which levels ofboth proteins began to decline. In contrast, the decreasein the levels of the chloroplast encoded Dl and LSU ofRUBISCO preceded a measurable decline in their corres-ponding mRNA levels. The levels of Dl polypeptidebegan to drop within 2d of UV-B treatment (Fig. 2b),but psbA mRNA levels were not significantly affected forup to 4 d of treatment (Fig. Id). For rbcL, the mRNAlevels remained unaffected for the first 2 d of UV-Bsupplementation (Fig. lc), while in contrast, the LSUpolypeptide levels began to decline by the second day ofUV-B treatment and after 3 d, continued to decline alongwith rbcL mRNA levels (Fig. 2b).

The level of all four photosynthetic proteins studiedremained relatively constant, throughout the experiment,under control conditions.

Protein biosynthesis in leaves

It is possible that the apparent discrepancy betweenprotein levels and mRNA levels for the chloroplast-encoded proteins was due to enhanced degradation of Dl


and LSU polypeptides under UV-B conditions. To testthis, the rates of in vivo protein synthesis and degradationwere determined. The data from two independent experi-ments is illustrated in Fig. 3. The LHCB protein wasrelatively stable over the 24 h chase period and was notaffected by UV-B exposure (Fig 3b). A loss of label fromLSU and SSU of RUBISCO was seen within 24 h butonly under UV-B conditions. UV-B treatment resulted ina fall in the level of labelled LSU and SSU to 85% (±3.1)and 88% (±2.2) of original values, respectively, as com-pared to 96% (±3.2) and 97% (±2.8) under controlconditions (Fig. 3a, b). In contrast, the Dl polypeptidewas rapidly lost throughout the chase period. The rate ofloss was greater under UV-B conditions. 54% (±5.6) ofDl remained after 8 h supplemental UV-B as comparedto 69% (±2.8) under control conditions (Fig. 3a). Inaddition, synthesis of Dl was also increased under UV-Bconditions by 9.5% (±2.1) (data not shown). The rate ofincorporation of 35S-methionine into the LSU and SSUof RUBISCO and LHCB protein was too low to allowreliable measurements of instantaneous synthesis rates bythis method.

To determine the effects of UV-B on degradation overa longer period, streptomycin (St) and cycloheximide

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0 4 8 12 16 20 24 28

Chase time (h)

Fig. 3. Densitometric analysis of autoradiographs of in vivo translationproducts obtained from 33S pulse chase experiment, analysed bySDS-PAGE. The values represent the mean relative peak heights (rph),from two independent experiments The rph is defined as the peakheights obtained relative to a standard described in material andmethods. The error bar refers to the standard error of the difference(SED) which was derived from analysis of variance using theexperiment x treatment interaction as error term. Open symbols repres-ent values from control treatments and closed symbols for UV-Btreatments, (a) LSU (O) and Dl (O) and (b) LHCB protein (0) andSSU (O).


(Chx), inhibitors of chloroplast and nuclear protein syn-thesis, respectively, were fed to leaf discs under controland UV-B conditions for 3d. Total proteins wereextracted, separated by SDS-PAGE and analysed byWestern blotting. A representative blot is illustrated inFig. 4. The level of LHCB protein was not visibly reducedby Chx treatment even under UV-B conditions (Fig. 4a).The enhanced loss of Dl polypeptide in response toUV-B, observed in the 35S methionine feeding experimentswas confirmed in the St treatment. The level of Dl wasdecreased under control conditions in the presence of St,however, the decline under UV-B conditions was consider-ably more pronounced (Fig. 4b).

In the presence of Chx and St, respectively, the levelsof SSU and LSU of RUBISCO were not visibly reducedunder normal illumination. In contrast, there was asubstantial decrease in the levels of both the subunitsafter exposure to UV-B in the presence of antibiotics(Fig. 4c, d). A 3d UV-B treatment resulted in a declinein LSU, SSU and Dl levels in the controls withoutantibiotics. However, the effects were small as comparedto antibiotic treatments (Fig. 4).

Changes in flavonoid composition and gene expression ofchs in leaves and buds

Representative absorbance profiles of flavonoid extractsof leaves and buds over the period of 8 d with supple-mental UV-B radiation are shown in Fig. 5. The spectralprofiles obtained form the buds and leaves are quitedifferent. The UV-absorbance peak (A^J was c. 320 nmin leaf samples after 1 d of UV-B exposure. Subsequentexposure resulted in a shift in the A,,^ towards lowerwavelengths, with a slight decrease in absorbance, up to4 d treatment (Fig. 5a). The increased absorbance at 8 dis probably not a true increase in flavonoid content ofthis organ, but more likely due to the over-estimation of

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Fig. 4. Western blot analysis of total protein isolated from leaf discs.The protein was isolated from control discs (C) or those exposed tosupplemental UV-B (U), treated without antibiotic (dH2O), or withstreptomycin (St) or cycloheximide (Chx) and then immunoblottedwith the appropriate antibody. Blots shown are representative resultsobtained from one of three experiments. Antibodies used were against(a) LHCB protein, (b) Dl, (c) RUBISCO SSU, (d) RUBISCO SSU.



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Fig. 5. The flavonoid profiles obtained from (a) leaves and (b) budsafter exposure to UV-B for (—) 0 d, (—) 1 d, ( • ) 4 d, and ( )8 d. The profile shown are representative results obtained from one ofthree experiment.

the flavonoid contents as a result of a reduction in freshweight of the tissue resulting from UV-B exposure. Incontrast, in buds, there was a progressive, slow decline inflavonoid content on exposure to UV-B. The shift in theAmax observed in leaves was also not observed in the buds(Fig. 5b). Spectral absorbance profiles obtained formleaves and buds did not vary significantly throughout the8 d period under control conditions (data not shown).

The chs RNA levels were quantified using Northernblots probed with the chs cDNA. Data from quantitativeNorthern blot analysis comparing transcripts from UV-Btreated plants as compared to transcripts from controlplants, on equivalent days, is illustrated in Fig. 6. Therewas a relationship between the chs mRNA levels and thelevels of flavonoids present in the two organs studied.The initial mRNA level in the leaves was very low. UV-Bexposure lead to an increase of c. 6-fold in the c/istranscripts in 2 d, after which the levels progressivelydeclined over subsequent light-dark cycles (Fig. 6). Incontrast, in buds, the expression was initially high, fallingafter only 1 d UV-B supplementation to 69% of controlvalues, and continued to decline slowly for the remainderof the experiment (Fig. 6).

0 1 2 3 4 5 6 7 8

UV-B exposure (d)

Fig. 6. Changes in the amount of leaf (O) and bud ( • ) mRNA duringexposure to UV-B, relative to control values. Northern blots, probedwith chs cDNA probes, were quantified by excision of the appropnatearea of the filter and determining the amount of bound radioactiveprobe using liquid scintillation spectrometry. The relative hybridizationis denned as the percentage counts obtained from samples fromUV-treated plants with respect to counts from samples from controlplants, which were treated under the same conditions, but in theabsence of UV-B, on equivalent days of treatment. Each pointrepresents the mean value obtained from three independent experimentsError bars represent the standard errors of differences of the means.

Discussion

UV-B-induced changes in nuclear-encoded genes

Treatment of pea leaves with supplemental UV-B resultedin a dramatic decrease in the level of mRNA for thenuclear genes studied. Lhcb and RbcS RNA levels werereduced substantially after only 2 d UV-B treatment andfell to trace amounts after a further 6d (Fig. la, b).There was a comparable decrease in SSU polypeptidelevels over this period (Fig. 2a). The turnover ofRUBISCO is normally quite slow and the protein ismaintained at a relatively stable level in mature leaf tissue(Iwanij et al., 1975; Nikolau and Klessig, 1987).Biosynthesis and degradation of this protein can, how-ever, be regulated independently by many physiologicaland environmental factors (Jordan et al., 1986; Thomasand Stoddart, 1980). UV-B exposure resulted in anincrease in the rate of degradation of RUBISCO (Figs 3,4). This enhanced degradation rate could occur eitherthrough UV-B-induced damage of RUBISCO followedby selective degradation of the damaged protein, oralternatively through an effect of UV-B on cellular proteo-lytic mechanisms. The present study does not allow theseand perhaps other possibilities to be distinguished. Thecombination of enhanced SSU degradation and thedecline in rbcS transcript levels in response to UV-Bexposure, resulted in SSU pools being depleted at agreater rate than normally observed under natural condi-tions. In contrast, UV-B did not enhance the degradationof the LHCB protein (Fig. 3). Hence, although LhcbRNA levels were dramatically reduced by UV-B exposure,the effect on protein levels was delayed and protein levelsfell at a slower rate than RNA levels (Fig. la, 2a).

For both LHCB and SSU, UV-B exposure caused adecrease in the levels of mRNA transcripts. This could


be due either to a decrease in transcription rates orenhanced degradation of the transcripts. Nuclear-encodedgenes for chloroplast proteins are frequently regulated atthe level of transcription (Kuhlemeier et ai, 1987) andboth Lhcb and RbcS have been previously shown to becontrolled at the level of transcription in pea (Gallagherand Ellis, 1982). On the other hand, regulation of RNAdegradation has been shown to be involved in establishingthe steady-state levels of SSU in soybean (Shirley andMeagher, 1990). Nuclear runoff experiments will berequired to discriminate these two possibilities. Thus whileit is clear that a decrease in mRNA abundance is aprimary cause of the reduced levels of the nuclear-encodedchloroplast proteins (Fig. 1), stability of proteins is alsoan important factor in determining the levels of theseproteins in response to UV-B irradiation (Fig. 2).

UV-B-induced changes in chloroplast-encoded genes

RbcL transcripts were not affected by UV-B radiation upto 2 d, after which levels began to fall sharply (Fig. lc).In leaves, the levels of LSU, however, had begun todecline on the second day of UV-B exposure (Fig. 2b)and, therefore, prior to any loss in the level of rbcLtranscripts (Fig. lc). UV-B irradiation was found toenhance the degradation of LSU (Figs 3, 4). Manyprevious studies have shown regulation of chloroplastgene expression by translational control (Gruissem, 1989;Mullet, 1988). Increases in the level of LSU polypeptidein pea in response to light have been shown to beprimarily at the level of translation (Inamine et ai, 1985).In addition, inhibition of transcription of the RbcS geneby a-amanitin (Sasaki, 1986) or reduction of mRNAtranscripts by antisense technology (Rodermel et ai,1988) did not result in the down-regulation of rbcLtranscription. However, the levels of LSU polypeptidewere co-ordinated with the loss in SSU synthesis via post-translational control (Rodermel etai, 1988). It is, there-fore, possible that the loss of LSU by the second day ofUV-B supplementation, before any decrease in rbcLmRNA levels was detected, was not only as a result ofenhanced degradation of LSU, but also linked to a declinein SSU synthesis.

On the third day of UV-B exposure, there was a sharpdecrease in the levels of rbcL mRNA (Fig. lc). This dropwas not reflected by a sharp decrease in the levels of LSUpolypeptide, which continued to decline along with theSSU polypeptide levels (Fig. 2b). The reduction in rbcLgene expression was not due to a build-up of unboundLSU as no excess free LSU was detected in any of thesamples after analysis on native gels nor was any changedetected in the stoichiometry of RUBISCO (data notshown). Sasaki (1986) also noted that the pool of freeLSU was not increased in the absence of the synthesis ofSSU. Degradation of free LSU is a key control in the


co-ordination of LSU levels with SSU levels (Kirk andKirk, 1985; Berry et ai, 1986). The decrease in the levelof rbcL mRNA on the third day is either due to UV-Binduced damage to DNA and transcriptional apparatusor a specific response to UV-B at the level of RNAabundance. The effect of UV-B on gene expression isspecific, and although some genes are rapidly down-regulated, others are up-regulated, for example, chs levelsin this study (Fig. 6) and in Jordan et ai (1994). It is,therefore, unlikely that the plant's transcriptional appar-atus was significantly damaged within 3 d. It appears,therefore, that in response to UV-B exposure, LSU poly-peptide levels are initially affected at the translational/post-translational level. Longer term exposure, however,results in control also being exerted at the level of RNAabundance, either through the down-regulation of rbcLtranscription or through an increase in RNA turnover.

The Dl polypeptide of PS II has been studied in detailand has been observed to have a high rate of light-induced turnover (Mattoo et ai, 1984). In the presentstudy the psbA mRNA levels were not measurably reduceduntil 4 d UV-B exposure when plants were showing severesigns of stress (Fig. Id). The level of Dl was, however,affected dramatically by exposure to UV-B. There was aprogressive loss of Dl with increasing exposure tosupplemental UV-B (Fig. 2b). Dl synthesis is strictlylight-dependent (Prasil et ai, 1992) and under limitedtranscriptional regulation. The increases in the amountof this protein are effectively controlled at the post-transcriptional levels (Deng and Gruissem, 1987; Kleinand Mullet, 1986). It appears that UV-B also affects Dlat this level. UV-B irradiation did not significantly affectpsb A transcript levels until the 5th day of the treatment.However, in vivo translation assays (Fig. 3a) and inhibitorstudies (Fig. 4b) did indicate that the Dl protein isconsiderably more unstable under UV-B conditions thanunder white light. Spectral evidence has indicated thatthe major UV-B photosensitizer for Dl degradation isplastoquinone (Greenberg et ai, 1989) although proteincomponents such as tyrosine residues may also be respons-ible for photo-trapping of UV-B energy (Friso et ai,1994). In the present study, after 8 h of supplementalUV-B, 54% of the Dl protein remained as compared to69% under white light (Fig. 3a). These results are inagreement with those observed by Greenberg et ai (1989).Higher rates of Dl degradation were noted in Spirodelapolyolorrhiza illuminated with UV-B irradiation than withvisible or far red light. Greenberg et ai (1989) also notedthat the rate of synthesis of Dl was also elevated inresponse to UV-B, and the results are consistent withthis. During high irradiance, the concentration of PS IIincreases relative to other thylakoid membrane complexes(Callahan et ai, 1990; Kettunen et ai, 1991; Melis, 1992).Exposure of pea leaves to UV-B results in an increase in


736 A-H Mackemess et al.

the degradation of Dl either directly or indirectly. Thisis partly offset by increased synthesis, but the rate of lossexceeds the capacity of the cells to compensate and henceleads to the observed decrease in the level of the Dlpolypeptide.

Different organs have differing sensitivities to UV-Birradiation and levels of protective pigments

In the apical buds, the levels of transcripts for the fourchloroplast proteins studied were not reduced as dramat-ically as in leaves. Therefore, although, in both organsmRNA transcripts were reduced by UV-B exposure, theinhibition and hence sensitivity of leaves to UV-B isgreater than that of buds. These results are in agreementwith a comparable study by A-H Mackemess et al. (A-HMackerness, Liu, Thomas, Jordan, Thompson, andWhite, unpublished data) in pea and by Lois (1994) andJordan et al. (Jordan, James and Anthony, unpublisheddata) in Arabidopsis.

The flavonoid profiles obtained from the leaves andbuds showed qualitative differences between the organs.In addition, the pattern of expression of the chs geneswas different between pea leaves and buds. However,there was a correlation between the level of gene expres-sion and the concentration of flavonoids in the twoorgans. In leaves, the levels of expression of the chs geneand flavonoid were very low prior to UV-B exposure(Figs 5a, 6). The levels of transcripts and flavonoids rosesharply to a peak after 2 d falling slowly during progress-ive samples. The increased absorbance at 8 d is probablynot a true increase in flavonoid content of the leaves, butmore likely due to the over-estimation of the flavonoidcontents as a result of a reduction in fresh weight of theleaves resulting from UV-B exposure (Fig. 5a). Similarrises have been previously observed in the expression ofchs genes (Strid, 1993; Takeda et al., 1993; Brans et al.,1986) and in levels of flavonoids (Jordan et al., 1994;Strid and Porra, 1992). The increase in chs expression inresponse to UV-B radiation has been shown to be due toincreases in the transcription rates of the gene (Chappelland Hahlbrock, 1984). In the buds, however, level ofexpression and flavonoids were highest in the absence ofUV-B; supplemental UV-B causing a progressive decreasein the level of chs mRNA and flavonoids (Figs 5b, 6).The mechanism and reason for the decrease or inhibitionin the expression of this gene, and hence the decrease inflavonoid levels, cannot be determined from the resultsobtained in this study.

It can be concluded that the effects of UV-B radiationon gene expression in pea are complex and the level atwhich different photosynthetic proteins are affected isdependent on the location within the plant and on theorgan being studied. In addition, these UV-B-inducedchanges are specific being dependent upon the particular

gene studied. The primary effect of UV-B being on mRNAabundance for nuclear-encoded genes, with some genes(e.g. chs) being up-regulated while others (e.g. Lhcb andRbcS) down-regulated. For chloroplast-encoded genes,however, the primary effects are post-translational withchanges in RNA abundance, where they occur, being lateand, therefore, probably secondary events.

Acknowledgements

This research was supported by the Biotechnology andBiological Sciences Research Council, UK. We would like tothank Dr P Thornber at the University of California, LosAngeles, CA, USA for supplying the LHCB antibody and DrR T Besford at the Horticulture Research International,Wellesbourne, UK for the RUBISCO and Dl antibodies. Weare grateful to Ron Pierce for running the Weiss cabinets, toDr A Thompson and Dr M White for critical review of thepaper, to Dr John Fenlon for the statistical analyses and finallyto the two anonymous reviewers for their helpful comments.

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the effect of supplementary ultraviolet-b radiation on mrna

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