Factors Affecting UV-B-Induced Changes in Arabidopsis thaliana L

Plant Cell Physiol. 39(7): 769-778 (1998)JSPP © 1998

Factors Affecting UV-B-Induced Changes in Arabidopsis thaliana L. GeneExpression: the Role of Development, Protective Pigments and theChloroplast Signal

Brian R. Jordan1, Patricia E. James2 and Soheila A.-H.-Mackerness2

1 New Zealand Institute for Crop & Food Research Limited, Palmerston North Research Centre, Private Bag 11030, Palmerston North,New Zealand

2 Department of Plant Genetics & Biotechnology, Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, U.K.

Gene expression is known to change in response toUV-B radiation. In this paper, we have investigated threefactors in Arabidopsis leaves that are likely to influencethese changes: development, protective pigments and the'chloroplast signal'. During late leaf development the ma-jor change in pigment composition, after exposure to UV-B radiation, is an increase in UV-absorbing pigments. Chiand Chi a/b ratio do not change substantially. SimilarlyChi fluorescence is not altered. In contrast, RNA tran-scripts for photosynthetic proteins are reduced more inolder leaves than in young leaves. To determine the role offlavonoids in UV-B protection, plants of Arabidopsis mu-tant tt-5, which have reduced flavonoids and sinapic esters,were exposed to UV-B and RNA transcript levels deter-mined. The tf-mutants were more sensitive to UV-B radia-tion than wild-type. To examine the role of the chloroplastsignal in regulating UV-B-induced changes in gene expres-sion, Arabidopsis gun mutants (genome uncoupled) havebeen used. The results show that UV-B-induced down-regu-lation still takes place in gun mutants and strongly suggeststhat the chloroplast signal is not required. Overall, thisstudy clearly demonstrates that UV-B-induced changes ingene expression are influenced by both developmental andcellular factors but not chloroplastic factors.

Key words: Arabidopsis thaliana — Chloroplast signal —Flavonoids — Gene expression — Leaf development —Ultraviolet-B radiation.

It has been established that depletion of the strato-spheric ozone layer is increasing the level of ultraviolet-Bradiation (UV-B: 280-320 nm) reaching the earth's surface(Blumthaler and Ambach 1990). A major effect of UV-Bradiation on plants is an inhibition of photosynthesis(reviewed in Bornman 1989, Jordan 1993, 1996). Extensivestudies have revealed a wide range of diverse functions that

Abbreviations: Chs, chalcone synthase; Lhcb, light harvest-ing complex binding proteins; psbA, Dl polypeptide of PSII;rbcL, large subunit of RUBISCO; RbcS, small subunit ofRUBISCO; S, Sandoz; UV-B, ultraviolet-B.

are affected, reflecting both the large numbers of moleculesabsorbing UV-B and the energetically unstable nature ofthe chloroplast (Bornman 1989, Strid et al. 1990). Thesechanges in photosynthetic function, however, take placeover different time periods ranging from minutes to days(Strid et al. 1994). One of the earliest effects of UV-B irradi-ation is an alteration in gene expression. For instance,mRNA levels for photosynthetic genes such as Lhcb andRbcS are substantially reduced within hours by supplemen-tary UV-B radiation (Jordan etal. 1991, 1992,1994, A.-H.-Mackerness et al. 1997, Strid et al. 1994). The rapid inhi-bition of chloroplast protein biosynthesis can account forthe slower UV-B-induced reduction in photosynthetic func-tion (Strid et al. 1994). The relative sensitivity of photosyn-thetic genes to UV-B radiation has been shown to be gene-specific, with a number of 'defense genes' up-regulatedwhile photosynthetic genes are down-regulated (Strid 1993,Jordan et al. 1994) and dependent on the developmentalstage of the tissue studied (Jordan et al. 1994, A.-H.Mackerness et al. 1997). For example, in pea, supplemen-tary UV-B reduced the relative levels of Lhcb transcripts inleaves and, to a lesser extent, in apical buds. In contrast,Lhcb levels were increased during greening of etiolatedbuds exposed to UV-B under the same conditions. The fac-tors involved in this regulation are, therefore, fundamentalto the reduction in photosynthetic efficiency.

Down-regulation of genes for photosynthetic proteins,especially those encoding the light-harvesting complex pro-teins (Lhcb), are frequently cited as evidence for a chloro-plast signal transduction pathway (Taylor 1989, Susek andChory 1992). This signal is associated with a lack of chloro-plast development or oxidative damage to the chloroplast.At present, it is unclear whether the signal is a positive(Hess et al. 1994, Taylor 1989) or a negative regulatorysignal (Susek et al. 1993). UV-B radiation is known to pro-duce oxygen radicals and peroxides and lead to oxidativestress (Arnotts and Murphy 1991, Murphy and Huerta1990, Beggs et al. 1986). Due to the high level of reactivemolecules within the chloroplast it is likely that one of theprimary sites of oxidative damage is within the chloroplastand consistent with this thylakoid membranes are perturb-ed very rapidly on exposure to UV-B radiation (Chow et al.1992). Thus, it is possible that UV-B-induced oxidative

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770 Factors affecting UV-B-induced change in gene expression

damage to the chloroplasts results in the repression of geneexpression for chloroplast proteins via the chloroplastsignal transduction pathway. To investigate the role of thissignal transduction pathway in UV-B regulation of photo-synthetic transcripts, we have used an Arabidopsis mutant,gun (genome uncoupled), in which gene expression for chlo-roplast proteins continues under conditions of oxidativedamage (Susek et al. 1993).

Plants can intercept a large percentage of solar radia-tion and assimilate it into organic material. This requiresthat visible radiation penetrates through the epidermallayers of leaves to the underlying palisade and mesophylltissues where the majority of photosynthesis takes place.A similar penetration of UV-B radiation into these cellswould, however, be harmful to the plant. In most plantsthat have been studied reflectance from the leaf surface isrelatively low (Day et al. 1993, Robinson et al. 1993) and at-tenuation by UV-B-absorbing pigments screens out thisharmful radiation (Caldwell et al. 1983, Tevini et al. 1991,Robberecht and Caldwell 1986). The major protective pig-ments are flavonoid compounds, a group of secondaryproducts which includes the fiavones, flavonols, isoflavo-noids and anthocyanins. Exposure to UV-B has beenshown to induce a rapid and coordinated increase in the ex-pression and activity of enzymes of the phenylpropanoidbiosynthetic pathway (Feinbaum and Ausubel 1992, Lois etal. 1989, Kubasek et al. 1992, Schmelzer et al. 1988) anda subsequent increase in the level of these flavonoid pig-ments (A.-H.-Mackerness et al. 1997, Jordan et al. 1994,Schnitzler et al. 1996). Other related phenolic compoundssuch as sinapate esters also accumulate in epidermal cellvacuoles after exposure to UV-B radiation and have alsobeen indicated as having a protective role against UV-Bradiation (Sheahan 1996, Li et al. 1993). Direct evidencefor the protective function of these phenolic pigments hascome from the use of Arabidopsis transparent testa (tt)mutants, tt-4 which has reduced flavonoids (Li et al.1993) and tt-5 which has both reduced flavonoids andsinapic esters (Ormrod et al. 1995, Li et al. 1993). Both ofthese rf-mutants were found to be more sensitive to UV-B ir-radiation than the wild-type plants. Thus both types ofphenolic compound are involved in UV-B protection. Inthe present study, we have used the tt-5 mutants to investi-gate the protective role of these compounds on gene expres-sion.

In this study we have investigated changes in gene ex-pression in Arabidopsis leaves and related them to photo-synthetic function and pigment composition. Three aspectswere investigated for their role in regulating UV-B-inducedchanges in gene expression: 1, Development; 2, The role ofprotective pigments; 3, The chloroplast signal.

Materials and Methods

Plant material and growth conditions—The wild type Arabi-dopsis thaliana Columbia and Landsberg ecotypes were usedroutinely in experiments as indicated in the text. In addition, wehave used the Arabidopsis mutants gun (genome uncoupled) andpoca (as transformed wild type controls containing the same Cab-3 promotor-driven transgene as gun mutants) in Columbia ecotype(Susek et al. 1993) and tt-5 mutant in Landsberg ecotype (Li et al.1993).

The Arabidopsis seeds were germinated in a mixture of LevinF2 : sand : vermiculite (6 : 1.5 : 1) and grown for either three orsix weeks in a controlled environment cabinet at 22°C. Irradianceof 40 W m"2 (200 fimo\ photons m~2 s"1: 400-700 nm) was provid-ed by 9 Philips TL 40 W cool white fluorescent tubes during a 12 hphotoperiod. The gun mutants were grown for 5 weeks under thesame conditions and then transferred to a glasshouse where theywere watered normally as control plants or with 5 mM Sandoz9789, a herbicide which leads to oxidative photo-destruction ofchlorophyll and bleaching of ieaf tissue (Frosch et al. 1979). After3 d the plants had bleached and were returned to the controlled en-vironment cabinets prior to experimental procedures. After thegrowth period, half of the plants were then transferred into anoth-er cabinet that supplied an equal amount of photosyntheticallyactive radiation but supplemented by a UV-B lamp (PhilipsTL 12, 40 W). Plants were watered or treated with Sandoz andsamples taken over a time period up to 7 d. The levels of UV-Bradiation were 14 and 35 mW m~2 nm"1 at 297 nm and 313 nm re-spectively, as determined by using an IL1700 Research Radiome-ter with calibrated photodetector/filters (International Light,Newburyport, U.S.A.). To eliminate UV radiation below 290 nmthe UV-B tube was wrapped in cellulose acetate which waschanged daily to prevent increased transmission of the shorterwavelengths due to degradation of the cellulose acetate. Thissource emits very low levels of UV-A and blue light, which wehave found are insufficient to affect the expression of the genesand levels of pigments studies. Comprehensive details of the UV-Btreatments are given in Jordan et al. (1994).

The inner 3 whorls of leaves and the outer leaves from therosettes were used in all experiments unless otherwise stated. Forthe purpose of this study, this division was considered to differ-entiate leaves at two different developmental stages.

All experiments were independently duplicated. Samples weretaken at random from different parts of the cabinets and from sep-arate plants selected for their uniformity.

Measurement ofPSII Chi fluorescence—Chi fluorescence wasdetermined by using a Hansetech plant efficiency analyzer (Hanse-tech, Kings Lynn, U.K.). The leaves were dark adapted for 5 minprior to measurement for 30 s.

Pigment analysis—Leaf tissue (0.5 g) was ground to a finepowder in liquid nitrogen. The powder was then extracted in 80%acetone buffered with 50 mM Tris-HCl pH7.5 (final volume 10ml). The mixture was clarified by centrifugation at 5,000 xg for10 min in a Sorvall RC5 centrifuge. Chi was determined in the su-pernatants by the method of Porra et al. (1989).

For UV-absorbing pigment determination, the frozen powderwas extracted in methanol (5 ml per 0.5 g of fresh weight), centri-fuged and the absorbance scanned between 200 nm and 450 nm asdescribed in Markham (1982). Anthocyanins were determined byextraction of the frozen powder in \% HC1 (v/v) in methanol (5ml per 0.5 g), centrifuged and the absorbance scanned between480 nm and 700 nm (maximum anthocyanin absorbance being at530 nm) as described by Hirose et al. (1990).

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Factors affecting UV-B-induced change in gene expression 771

The experiment was repeated twice with similar results anddata from one set of experiments is presented.

Purification of total RNA, Northern blotting and hybrid-isation—RNA was extracted as described by Jordan et al. (1992)and stored at — 70°C. The RNA was separated on a 1.5% agarosegel containing 6% (v/v) formaldehyde in 2 x MOPS (Maniatis etal. 1982). Each gel was examined under UV light both to checkRNA integrity and to ensure that equal amounts of RNA wereloaded in each lane (data not shown). The RNA blotting, prehy-bridisation, hybridisation and washing were carried out as de-scribed in Jordan et al. (1992). Autoradiography of the filters wasat — 70°C using Fuji RX film with a single intensifying screen.Blots presented are representative of blots obtained from two inde-pendent experiments.

Protein extraction and Western blotting—Total protein wasextracted and analysed by Western blotting essentially as de-scribed in A.-H.-Mackerness et al. (1997). The LHCB antibodyused was a monoclonal antibody specific for the light harvestingcomplex II (LHCII) and is described in Darr et al. (1986).

DNA sequences—The Lhcb cDNA sequence (pAB 96) is a1.8 kbp cDNA clone from pea and is described in Corruzzi et al.(1983). The RbcS sequence is a genomic fragment from Arabi-dopsis and is described in Krebbers et al. (1988). The psbA se-quence is an 850 bp Hindlll fragment containing the 3' 60% of thegene from spinach (Jordan et al. 1989). The rbcL sequence is a657 bp Pstl fragment from Maize (Jordan et al. 1989). The Cns se-quence is a 3.8 kbp Hinilll genomic clone from Arabidopsis(Feinbaum and Ausubel 1992).

Results

Developmental variation in response to UV-B irradiationPigment composition and morphology—The effect of

UV-B on the morphology and pigment composition of 3week old seedlings and the inner (younger) and outer

(older) leaves of 6-week-old Arabidopsis var Columbiaplants was determined.

Young seedlings were allowed to establish (3 weeksfrom germination) and then half were transferred into asupplementary UV-B cabinet. The seedlings were visuallyinspected daily and showed very little physical differencescompared to control plants after a period of 3 weeksexposure to supplementary UV-B radiation. Significantchanges in Chi content per fresh weight (Control 1.41 ±0.181 mgg"1; UV treated 1.39±0.02mgg~') or a/b ratio(3 : 1) were not detected. However, UV-B exposure didresult in an increase in the levels of UV-absorbing pigments(Fig. la) and anthocyanins (Fig. lb).

In contrast to seedlings, 6 week old rosettes of Arabi-dopsis inner and outer leaves showed visible damage to theleaf surface after only a single day of exposure to UV-Bradiation. This took the form of a silvery 'glaze', predomi-nantly on the older outer leaf whorls. Chi content was sig-nificantly increased in only the inner leaves in response toUV-B exposure, with no significant changes detectable inthe outer leaves (Fig. 2a). In addition, no significant changewas observed in the Chi a/b ratio at either developmentalstage (Fig. 2b). Methanol extracts from inner and outerleaves treated with and without UV-B were scanned in anabsorption spectrophotometer between 200 and 500 nm.There was a rapid increase in absorption within the UVregion in the outer (Fig. 3b) and, to a greater extent, innerleaves (Fig. 3a) on exposure to UV-B. This increase was de-tectable after 8 h and continued over the experimental peri-od. The absorption above 380 nm was slightly decreased inouter leaves and increased in inner leaves compared to con-

g

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Fig. 1 The (a) flavonoid and (b) anthocyanin profiles obtained from 3 week old Arabidopsis seedlings treated with (—) or without(—) supplementary UV-B for 3 weeks.

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Fig. 2 Pigment content in inner and outer leaves of 6 week old Arabidopsis plants exposed to supplementary UV-B for a period of 7 d.(a) Total chlorophyll, (b) Chlorophyll a:b ratio. Each bar represents the mean value obtained from three independent experiments and er-ror bars indicate S.E.

trols and paralleled the duration of exposure to UV-B. Thisis mainly due to changes in Chi content which were deter-mined more accurately using acetone extracts and de-

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Fig. 3 The flavonoid profile obtained from (a) inner and (b)outer leaves of 6 week old Arabidopsis plants treated without(-UV-B) or with (+UV-B) supplementary UV-B for (—) 8h( - ) 3 d, (•-) 7 d.

scribed above. In contrast, no change in anthocyanin levelswas detected in response to the UV-B treatment in the 6week old plants (data not shown).

From these experiments, the effect of UV-B on olderrosettes was shown to be more pronounced than in youngerseedlings and, therefore, more extensive studies were car-ried out using 6 week old Arabidopsis plants. The effect ofUV-B was determined in both the inner and outer leaves inorder to further access variation in response between leavesat different developmental stages, to supplementary UV-B.

Chi fluorescence—The effect of UV-B on the Chi fluo-rescence of both the inner younger leaves and the olderouter leaves of 6 week old Columbia rosettes were deter-mined for 3 d (data not shown). The maximum quantumyeild of PSII photochemistry, as monitored by FV/FM

ratio, was not affected by UV-B treatment in the innerleaves while a small decrease was detected in the outerleaves. However, the maximum reduction in ratio was onlyca. 10% after 3 d.

Transcript levels—The mRNA transcript levels for anumber of genes (representing nuclear-encoded, chloro-plast-encoded and defense genes) were determined inyoung and older leaves of 6 week old Arabidopsis plants(Fig. 4). The response to UV-B irradiation of Lhcb mRNAtranscripts differed between the outer (old) and inner(young) leaves (Fig. 4a). In inner leaves, a reduction inLhcb mRNA levels was detectable after 3 d of UV-B ex-posure at which time transcripts had declined by approxi-mately 50%. This reduced level was maintained over the 7d period. The reduction in Lhcb mRNA was considerablyfaster in the outer leaves than in the inner leaves (Fig 4a).Thus, after only 1 d the Lhcb mRNA transcripts had de-clined by over 50% and the level continued to fall until thelast sampling day (7 d). To determine if the decline in LhcbmRNA levels is reflected in LHCB protein content, protein

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Factors affecting UV-B-induced change in gene expression

Id 3d 7d

773

(a) Lhcb

Q L 7dr

(b) RbcS

(c) rbcL

(d) psbA

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Fig. 4 Northern blots showing levels of (a) Lhcb (b) RbcS (c)rbcL (d) /K&<4 and (e) Chs transcripts in inner (i) and outer (o)leaves of 6 week old Arabidopsis plants treated with supplemen-tary UV-B for a period of 7 d.

was isolated from inner and outer leaf tissue after variouslengths of UV-B exposure. The proteins were separated bySDS-PAGE and Western blotting (Fig. 5). Using LHCBantibody, no change in LHCB protein levels could be de-tected in the inner or outer leaves in response to supplemen-tary UV-B, although there was an overall decline in thelevels with time, particularly in the outer leaves (Fig. 5).The effect of UV-B on transcript levels of another nuclear-encoded chloroplast protein, the small subunit ofRUBISCO, encoded by RbcS, was similar to that on Lhcb,with levels in both inner but, to a greater extent, outerleaves falling after 3 d (Fig. 4b).

The effect of UV-B on two chloroplast-encodedgenes, rbcL and psbA, encoding the large subunit ofRUBISCO and the Dl polypeptide of PSII, respectively,was also studied (Fig. 4c, d). The effect of UV-B on rbcLtranscripts was similar to that of the Lhcb transcriptsbut not as rapid (Fig. 4c). Thus, the mRNA level in theinner leaves was maintained for 3 d, before declining.Again in the older leaves, the rbcL mRNA level decreasedmore rapidly, within 1 d, and remained at a lower levelthroughout the period of exposure. In contrast, psbAmRNA transcript levels, after an initial decline (1 d) weremaintained at a high level throughout the subsequent daysof UV-B exposure in both inner and outer leaves (Fig. 4d).

control

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I O I

Fig. 5 Western blot analysis LHCB protein levels from inner (I)and outer (O) leaves of 6 week old Arabidopsis plants treated with(UV-B) or without (control) supplementary UV-B for a period of7d.

To test the response of an important defense gene, themRNA levels of chalcone synthase (Chs) was studied(Fig. 4e). In control tissue the Chs mRNA levels were unde-tectable or present only in trace amounts. UV-B exposureresulted in a dramatic increase in Chs mRNA transcriptlevels in both the inner and outer leaves. After only Id ofUV-B exposure the levels have increased in inner leaves by10-fold and in the outer leaves by more than 5-fold. By 3 d,however, the levels in the leaves started to decline and con-tinued to fall up to the last sampling day (7 d). The max-imum Chs mRNA transcript levels in the older outer leaveswas 70% of the level in the younger inner leaves.

The effect of UV-B radiation on Arabidopsis tt-5mutants—Arabidopsis tt-5 mutants and wild type Land-sberg were grown for 6 weeks and then exposed to UV-Bradiation for 3d. Inner and outer leaves were then ana-lyzed for Lhcb, psbA, rbcL and Chs gene expression(Fig. 6). The responses of Landsberg and Columbia ecotypeswere very similar (Compare Fig. 4, 6). The reduction inLhcb, rbcL and psbA mRNA was more extreme in re-sponse to UV-B in both the outer and inner leaves of the tt-mutants than in the wild type plants (Fig. 6a, b, c). Theeffect of UV-B on the tt-5 mutant, similar to the observa-tion made for wild type plants, was greater on the nuclear-encoded than chloroplast-encoded genes and the effect ontranscripts in outer leaves was more pronounced than thatin the inner leaves. To extend these data, Chs mRNA levelswere also determined in tt-5 mutants as well as in wild typeplants (Fig. 6d). The wild-type Arabidopsis Landsbergecotype responded like Columbia with a rapid increase inChs mRNA. In contrast, the tt-5 did not show a similar dra-matic increase, although the levels were higher in UV-treat-ed plants than in control plants. In addition, similar to thewild type, the increase was to a higher level in the innerleaves of tt-5 than the outer leaves (Fig. 6d). The level offlavonoids and anthocyanins were also measured in the in-ner and outer leaves of the //-mutants in response to UV-B.

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a LhcbmRNAtranscripts

UV-B exposure

8h 3d

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c psb - A mRNAtranscripts

d chs mRNAtranscripts

#**# • * • • « • • •

% • • Hwt tt5wt tt5 wt tt5wt tt5wt tt5 wt tt5

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Fig. 6 Northern blots showing levels of (a) Lhcb (b) rbcL (c) psbA and (d) Chs transcripts in inner (I) and outer (O) leaves of 6 weekold Arabidopsis wild-type (wt) and tt-5 mutant (tt5) treated with supplementary UV-B for a period of 3 d.

The level of flavonoids and anthocyanins were ca. 10 x and1,000 x lower, respectively, in the tt-5 mutants than in thewild type at both developmental stages. They were similarat both developmental stages and no increase in either com-pound could be detected in tt-5 on UV-B exposure (datanot shown).

The role of the chloroplast signal in UV-B responses—The herbicide Sandoz 9789 inhibits phytoene desaturaseand blocks the synthesis of yS-carotene. This leads to oxida-tive photo-destruction of Chi and bleaching of leaf tissue(Frosch et al. 1979). Under the conditions of the controlledenvironment cabinets application of herbicide had no effecton the plants due to the relatively low irradiance. Conse-quently, Sandoz was supplied to 5 week old plants whichwere then transferred to a glasshouse under natural irra-diance. Within 3 d the inner younger leaves (within theinner 3 whorls) were completely photobleached and contain-ed no Chi. The outer older leaves remained green and show-ed little evidence of photobleaching. The plants were thentransferred back to growth cabinets for UV-B treatments.The herbicide-treated plants lasted up to 3 d in a suitablecondition for experimentation. To determine the role ofthe chloroplast signal in UV-B-induced changes in gene ex-pression, we determined the effect of UV-B on the level ofphotosynthetic transcripts in the inner leaves of the Arabi-dopsis gun mutant treated with Sandoz or water (Fig. 7)

and Arabidopsis gun and poca (transgenic controls)plants treated with Sandoz (Fig. 8). Although gun mu-tants photobleached with Sandoz had reduced levels ofthe nuclear-encoded Lhcb and RbcS mRNAs compared tothe water treated controls (Fig. 7a, b), the expression levelswere still substantial. After 3 d of UV-B irradiation the con-trol water-treated gun mutants showed a small reduction inLhcb and RbcS mRNA levels. In contrast, Sandoz-treatedgun mutants exposed to UV-B showed severe reduction inLhcb and RbcS mRNA levels (Fig. 7a, b). The mRNA tran-script levels for the two chloroplast-encoded genes, psbAand rbcL, were also determined in Sandoz and water treat-ed plants exposed to supplementary UV-B (Fig. 7c, d). Inwater-treated controls, rbcL and psbA expression was de-creased slightly after 3 d exposure to UV-B radiation.Again, in Sandoz treated tissue the levels of these tran-scripts were reduced in control plants and declined furtheron exposure to UV-B radiation (Fig. 7c, d). In addition, us-ing poca, which expresses the same Cab-3 promotor-driventransgene as gun mutants, as the control, the effect of sup-plementary UV-B was determined in poca and gun mutantstreated with Sandoz (Fig. 8). The level of all photosynthetictranscripts mpoca and gun mutants prior to any treatment,was comparable (data not shown) as previously reported(Susek et al. 1993). However, relative to poca, Lhcb andRbcS transcript levels remained relatively high in gun

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Factors affecting UV-B-induced change in gene expression

(a) Lhcbplants (Fig. 8c, d). The same response was found withshort-term exposure to UV-B (8 h), but to a lesser degree(data not shown).

(b) RbcS

(c) rbcL

(d) psbA.

c sc u su

Fig. 7 Northern blots showing levels of (a) Lhcb (b) RbcS (c)rbcL and (d) psbA transcripts in inner leaves of 6 week old Arabi-dopsis gun mutants sprayed with Sandoz (S) or water and treatedwith (U) or without (C) supplementary UV-B for a period of 3 d.

mutants after Sandoz treatment (Fig. 8a, b). UV-B ex-posure (3 d) resulted in the down-regulation of both thesetranscripts in gun and poca plants. Similarly, rbcL andpsbA transcripts were higher in gun mutants than in pocaplants after Sandoz treatments and again UV-B exposure(3 d) resulted in a decline in levels in both gun and poca

Gun Poca

J2S-SB1

(a) Lhcb

(b) RbcS

(c) rbcL

(d) psbA

SC SU SC SU

Fig. 8 Northern blots showing levels of (a) Lhcb (b) RbcS (c)rbcL and (d) psbA transcripts in inner leaves of 6 week old Arabi-dopsis gun and poca plants sprayed with Sandoz (S) and treatedwith (U) or without (C) supplementary UV-B for a period of 3 d.

Discussion

The sensitivity to UV-B was found to be heavily de-pendent on the developmental stage of the leaves studied.The effect of UV-B was monitored primarily in the inner(young) and outer (old) leaves of 6 week old rosettes ofArabidopsis var Columbia plants. The parameters used todetermine sensitivity were changes in physical appearance,Chi fluorescence and transcript levels encoding three keyphotosynthetic proteins (Fig. 4). Considering all parame-ters, the outer leaves were found to be more sensitive toUV-B than the inner leaves. Physically the outer leavesshowed symptoms earlier and Fv/Fm was affected by UV-Bonly in these leaves (data not shown). Lois (1994) alsofound a gradient of sensitivity to UV-B in Arabidopsis withthe older leaves becoming damaged by UV-B faster andmore extensively than the younger leaves. The effect of sup-plementary UV-B on transcripts encoding photosyntheticproteins has been previously reported at different develop-mental stages, to our knowledge, only in pea seedlings(A.-H.-Mackerness et al. 1997, Jordan et al. 1994). Similarto studies in pea, supplementary UV-B resulted in thedown-regulation of photosynthetic genes, with nuclear-en-coded transcripts (Lhcb and RbcS) (Fig. 4a, b) reduced at agreater rate than those encoded by the chloroplast (psbA)(Fig.4d). However, compared to psbA transcripts, thedecline in the chloroplast-encoded rbcL transcripts wasmore rapid in response to UV-B and possibly, as in pea,reflects the co-ordinated control of rbcL with RbcS tran-scripts (Fig. 4c). The difference in response of genes encod-ed by the nucleus and the chloroplast appears to be a reflec-tion of the different ways in which UV-B affects transcriptabundance: nuclear-encoded photosynthetic transcripts areregulated primarily at transcription while those encoded bythe chloroplast are primarily regulated post-transcriptional-ly (A.-H.-Mackerness et al. 1997 for more detailed discus-sion). UV-B resulted in down-regulation of photosynthetictranscripts faster in the outer older leaves than the inneryounger leaves (Fig. 4) as has been previously reported forpea (A.-H.-Mackerness et al. 1997, Jordan et al. 1994).Therefore, as in pea, although supplementary UV-B re-sulted in the down-regulation of photosynthetic transcriptsat both stages of leaf development, the inhibition andhence sensitivity of the older outer leaves was greater thanthe younger inner leaves.

Exposure to supplementary UV-B resulted in an in-crease in the levels of UV-absorbing pigments in both inner(Fig. 3a) and outer (Fig. 3b) leaves of six week old Colum-bia plants. However, the increase was faster and to a higherlevel in the inner leaves which is consistent with previous

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data (Lois 1994, Jordan et al. 1994). To our knowledge,spectral analysis of UV-absorbing pigments in inner andouter leaves of Arabidopsis, as denned in this paper, in re-sponse to UV-B, have not been determined. However, thespectra obtained in this study from outer leaves, under con-trol and UV conditions, was similar to the profiles obtainedfrom whole rosettes (Lois and Buchanan 1994). The innerleaves comprise a smaller proportion of whole rosettes andthus it is not surprising that whole plant spectra resemblethose obtained from the outer leaves.

Increases in UV-absorbing pigments, particularly fla-vonoids, in response to UV-B have been shown to be dueto dramatic changes in the rate of transcription for en-zymes of the phenylpropanoid pathway (Feinbaum andAusubel 1992, Chappell and Hahlbrock 1984). In Arabi-dopsis, UV and blue light appear to be primarily involvedin regulating Chs expression in older seedlings (Fuglevandet al. 1996, Kubasek et al. 1992, Feinbaum et al. 1991,Kaiser et al. 1995). Our results are thus in agreement withprevious results showing a UV-B inducible, rapid buttransient increase in Chs mRNA (Fig. 4e) and subsequentcomparable increases in flavonoid levels (Chappell andHahlbrock 1984, Jordan et al. 1994, A.-H.-Mackerness etal. 1997).

A tight coupling between the flavonoid content andUV-resistance in developing Arabidopsis seedlings hasbeen reported and suggests a role for flavonoids in UV-Bprotection (Lois 1994). As discussed above, a similar corre-lation was found between level of UV-absorbing com-pounds and sensitivity of inner and outer leaves to UV-B ex-posure. In order to determine the importance of thesephenolic pigments in the UV-B mediated effects on photo-synthetic transcript levels at different developmental stageswe have used tt-5 mutants which have lower levels offlavonoid and sinapic esters (Li et al. 1993). Our results in-dicate that the level of these protective pigments does affectthe accumulation of the photosynthetic transcripts in re-sponse to UV-B as the level of transcripts declined faster inthe tt-5 mutants than in the wild-type plants (Fig. 6). How-ever, the absence of these pigments did not affect therelative levels of the photosynthetic transcripts in the innerand outer leaves indicating that the differences in photosyn-thetic transcript accumulation, at different developmentalstages, is unlikely to be correlated to the levels of phenolicpigments present in the tissue. One of the proposed roles offlavonoids in response to UV-B exposure has been shieldingDNA from UV-induced damage (Harborne 1988, Stafford1990, Stapleton and Walbot 1994). It is likely, therefore,that the greater reduction in photosynthetic transcripts anddiminished increase in Chs expression in tt-5 mutants, com-pared to wild-type plants, in response to UV-B (Fig. 6), wasdue to an increase in UV-induced damage to DNA in themutants. However, as the developmental variation in tran-script accumulation was still apparent in tt-5 mutants, al-

though there was no difference in the levels of flavonoidsand anthocyanins between inner and outer leaves in thesemutants (data not shown), it is unlikely that DNA damagecould be the primary mechanism by which transcripts areup- or down-regulated in response to UV-B in wild-typeplants.

A key component of plastid development is the coordi-nation of gene expression between two different genomeswith the nucleus playing a major role in determining the de-velopmental fate of the chloroplast (Taylor 1989, Kirk andTilney-Basser 1978). However, the developmental stage ofthe chloroplast itself also appears to regulate the expressionof nuclear genes coding for chloroplast proteins. Notably,under conditions of photooxidative damage or in plantswere chloroplast development is arrested, the expression ofnuclear-encoded chloroplast RNAs and cytosolic mRNAsencoding chloroplast proteins is blocked (Mayfield andTaylor 1987, Burgess and Taylor 1988). It has been hypoth-esised that lack of chloroplast development and photooxi-dative damage to the chloroplast leads either to the destruc-tion (Hess et al. 1994, Taylor 1989) or generation (Susek etal. 1993) of a factor or signal of chloroplast origin that reg-ulates transcription of nuclear genes encoding chloroplastproteins, for example, Lhcb and RbcS. The nature of thissignal is at present unknown but direct evidence for its ex-istence has come from the isolation of Arabidopsis gunmutants that allow the partial expression of nuclear-encod-ed chloroplast proteins in the absence of chloroplast devel-opment (Susek et al. 1993). In plants, UV-B exposure isknown to lead to generation of active oxygen species andeventually result in oxidative stress (Arnotts and Murphy1991, Murphy and Huerta 1990, Beggs et al. 1996). Chloro-plast membranes, rich in polyunsaturated fatty acids, areparticularly vulnerable to oxidative damage and one of theconsequences of UV-B exposure is in fact perturbation ofthe chloroplast membrane (Chow et al. 1992). It is possi-ble, therefore, that the chloroplast signal could be involvedin the down-regulation of nuclear genes encoding photosyn-thetic proteins. The treatment of the gun and poca mutantswith Sandoz 9789 illustrated the absence of this signal ingun mutants. The levels of nuclear-encoded transcripts forchloroplast proteins were maintained at higher levels in thegun mutants than in poca under the conditions of photo-bleaching and hence photooxidative stress (Fig. 8a, b) asreported previously by Susek et al. (1993). The experimentswith gun mutants clearly show that UV-B-induced down-regulation of Lhcb and RbcS occurs even in the absence ofthe chloroplast signal (Fig. 7a, b, 8a, b). Reduction of chlo-roplast-encoded transcripts in response to oxidative stressis not thought to be mediated by the chloroplast signal butis a result of the loss of chloroplast function and hence tran-scription. As reported previously (Susek et al. 1993), lowlevels of chloroplast-encoded transcripts (rbcL and psbA)were detected in the photobleached gun mutants. The levels

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Factors affecting UV-B-induced change in gene expression 777

of these transcripts were further reduced on exposure toUV-B indicating that the down-regulation of chloroplast-encoded photosynthetic proteins, as would be expected, isnot affected in these mutants (Fig. 7c, d, 8c, d). Thus it isunlikely that the chloroplast signal is involved in the down-regulation of Lhcb and RbcS in response to supplementaryUV-B.

In conclusion, we have shown that there is a clear de-velopmental sensitivity to UV-B with respect to effects onphotosynthetic transcripts and that this is not a result ofdifferences in protective pigments present at the different de-velopmental stages. Protective pigments do, however, playan important role in reducing UV-B-induced down-regula-tion of gene expression, as illustrated by the experimentswith ^-mutants. In addition, the results presented in thispaper clearly indicate that the down-regulation of nuclear-encoded photosynthetic transcripts, in response to UV-B ex-posure, is not dependent on the chloroplast signal.

This research was supported by the Biotechnology and Biolog-ical Sciences Research Council, U.K. We would like to thank Dr.Feinbaum for supplying the Chs cDNA, Dr. Corruzzi for theLhcb cDNA, Dr. Graham King, HRI, Wellesbourne, U.K. for theRbcS cDNA, Dr. Joanne Chory and Dr. Ronald Susek, Salk In-stitute for Biological Sciences, San Diego, U.S.A. for supplyingthe gun mutants and to Dr. P. Thornber at the University ofCalifornia, Los Angeles, C.A. for supplying the LHCB antibody.We are grateful to Dr. Richard Napier and Dr. Brian Thomas forcritical review of the paper.

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(Received October 23, 1997; Accepted May 6, 1998)

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Factors Affecting UV-B-Induced Changes in Arabidopsis thaliana L

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