Light Control of hliA Transcription and Transcript Stability in the Cyanobacterium Synechococcus elongatus Strain PCC 7942

The high-light-inducible proteins (HLIPs) of cyanobacteria arepolypeptides involved in protecting the cells from high-intensitylight (HL). The hliA gene encoding the HLIP from Synechococcuselongatus strain PCC 7942 is expressed in response to HL orlow-intensity blue or UV-A light. In this study, we explorevia Northern analysis details of the transcriptional regulationand transcript stability of the hliA gene under various lightconditions. Transcript levels of the hliA gene increased dramaticallyupon a shift to HL or UV-A light to similar levels, followedby a rapid decrease in UV-A light, but not in HL, consistentwith blue/UV-A light involvement in early stages of HL-mediatedexpression. A 3-min pulse of low-intensity UV-A light was enoughto trigger hliA mRNA accumulation, indicating that a blue/UV-Aphotoreceptor is involved in upregulation of the gene. Low-intensityred light was found to cause a slight, transient increase intranscript levels (raising the possibility of red-light photoreceptorinvolvement), while light of other qualities had no apparenteffect. No evidence was found for wavelength-specific attenuationof hliA transcript levels induced by HL or UV-A light. Transcriptdecay was slowed somewhat in darkness, and when photosyntheticelectron transport was inhibited by darkness or treatment withDCMU, there appeared a smaller mRNA species that may representa decay intermediate that accumulates when mRNA decay is slowed.Evidence suggests that upregulation of hliA by light is primarilya transcriptional response but conditions that cause ribosomesto stall on the transcript (e.g., a shift to darkness) can helpstabilize hliA mRNA and affect expression levels.

INTRODUCTION

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Introduction

Like all photosynthetic organisms, cyanobacteria must carefullybalance their absorption and utilization of light energy withthe ambient light conditions. Absorption of light energy beyondwhat can be used in photosynthesis, such as during periods ofhigh-intensity light (HL) exposure at midday, can be extremelydamaging to a photosynthetic cell. This can lead to overexcitationof the photosynthetic apparatus, the generation of dangeroustriplet chlorophyll molecules, and damaging oxygen radicalsthat can ultimately lead to photoinhibitory oxidative damage.Therefore, acclimation mechanisms have evolved to allow an organismto avoid, compensate for, dissipate, or repair damage causedby excess light energy, including producing protective pigmentsor altering the activity and composition of the photosyntheticapparatus (for a recent review, see reference 33). Many of theacclimation responses to light stress necessitate alterationsin gene expression and are controlled through mechanisms thatallow the cell to sense changes in the light environment.

One way that plants can perceive changes in their light environmentis through sensing shifts in light color (quality). Light qualitycontrols development and many other processes in plants andis perceived through the use of photoreceptors that respondto specific light wavelengths (9, 36). Arguably the best-characterizedphotoreceptors are the phytochromes, with photointerconvertiblered-light and far-red-light forms. Although best studied withhigher plants, phytochrome-like proteins have been found incyanobacteria, photosynthetic bacteria, and recently in nonphotosyntheticbacteria (29, 44). For cyanobacterial phytochromes, the targetgenes and processes that are controlled remain largely unknown(31). Two blue/ UV-A light photoreceptors have been identifiedin higher plants, the cryptochromes and phototropin. Despitethe presence of blue-light-triggered processes in cyanobacteriaand the presence of a gene with similarity to cryptochrome inthe Synechocystis strain PCC 6803 genome sequence (20), thecyanobacterial cryptochrome has not yet been demonstrated toact as a blue-light photoreceptor and no blue-light photoreceptorhas yet been definitively identified in cyanobacteria (31).

Control of gene expression can take place at the transcriptionaland posttranscriptional levels. One posttranscriptional processis the decay of mRNA, the detailed mechanism of which has onlyrelatively recently become understood, and it has become wellaccepted that mRNA stability plays an important role in theexpression of many genes in all organisms, including bacteria(37). There are numerous examples of transcriptional controlof gene expression by light in photosynthetic organisms (e.g.,see references 14 and 41). In cyanobacteria, as in other photosyntheticorganisms, there are also examples of genes that are regulatedby light at the posttranscriptional level, including throughalterations in mRNA stability (13, 23, 24, 27, 38). In bacteria,mRNA decay proceeds through a combination of endonuclease andexonuclease activity, and it can be controlled by altering theability of degradation factors to access mRNA sites by suchmeans as the presence of mRNA secondary structures, protectionby translating ribosomes, and polyadenylation of transcripts(37).

One response in cyanobacteria that is triggered by light stressis the production of small polypeptides termed high-light-inducibleproteins (HLIPs) that are encoded by the hli genes (also calledscp genes). The HLIPs are localized in the thylakoid membranesof cyanobacteria (12, 19). They have been shown to be importantin photoprotection during exposure to HL (18, 19), and it hasbeen proposed that they function directly or indirectly in thedissipation of excess absorbed light energy (18, 21, 28). Ithas also been proposed that the HLIPs could serve as transientcarriers of chlorophyll (12) and that they may play a role inthe regulation of tetrapyrrole biosynthesis (47). HLIPs aresingle-helix members of the Lhc (light-harvesting complex) extendedgene family (16, 28). They exhibit close sequence similarityto the early light-inducible protein (ELIP) members of the familyin higher plants. The transcription of ELIP genes is inducedin response to light stress (3, 35). In addition, in etiolatedpea seedlings, ELIP transcription is induced by blue or redlight (1), while in adult tissues it is induced by blue or UV-Alight (2, 3). In Arabidopsis, phytochromes were found to mediatethe red-light and far-red-light regulation of ELIP gene expression,while blue-light control of the ELIP genes in this organismappeared to be mediated by an as of yet unidentified blue-lightphotoreceptor (not cryptochromes or phototropin) (17). Throughthe use of a GUS reporter fusion, expression of the hliA genefrom Synechococcus strain PCC 7942 was found to increase inresponse to high-intensity white-light or low-intensity blue-or UV-A-light exposure, with little expression in low light(LL) and red light (8). In a previous study, the sensor kinaseNblS that controls hliA upregulation by HL and UV-A light wasidentified (43). NblS was found to control the expression ofa number of other photosynthesis-related genes in HL and UV-Alight and also to control, during starvation for nitrogen orsulfur, expression of the nblA gene, the product of which isinvolved in degradation of the light-harvesting phycobilisomecomplex (43). This study was undertaken in order to understandthe transcriptional and posttranscriptional aspects of the lightcontrol of hliA expression towards a further understanding ofthe signal transduction mechanisms involved in controlling light-mediatedgene expression in cyanobacteria.

MATERIALS AND METHODS

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Materials and Methods

Culture conditions and light treatments. Synechococcus elongatus strain PCC 7942 was grown (25) at 30°Cin BG-11 medium under incandescent light (50 µmol of photonsm^-2 s^-1), and the cultures were bubbled with 3% CO₂ in air duringgrowth and during treatments with light and inhibitors. HL (800µmol of photons m^-2 s^-1) was obtained by using incandescentwhite-light bulbs. UV-A light (350 to 400 nm with a peak at366 nm) was supplied from black-light blue bulbs at 27 µmolof photons m^-2 s^-1. Other light qualities, all delivered tothe cultures at 30 µmol of photons m^-2 s^-1, were obtainedby wrapping fluorescent light bulbs with the following Lee Filters:blue (bright blue narrow-band filter [ ${lambda}$ _max, 440 nm]); green (darkgreen narrow-band filter [ ${lambda}$ _max, 520 nm]); red (deep golden ambercutoff filter [ ${lambda}$ _max, 640 nm]); and far red (medium red cutofffilter [ ${lambda}$ _max, 750 nm]). Prior to the various light treatments,cultures were grown to an A₇₅₀ of approximately 1.0, dilutedto an A₇₅₀ of 0.2 with fresh BG-11 medium (to avoid self-shadingof cells during exposure to light), and adapted to LL (10 µmolof photons m^-2 s^-1) for 18 h. Inhibitors, when used, were addedin the last 5 min of LL adaptation (to allow time for the cellsto absorb the inhibitor), and then the exposure to the lightconditions proceeded for the designated amounts of time beforecells were harvested for RNA. Rifampin was used at a concentrationof 200 µg/ml; chloramphenicol was used at 250 µg/ml;puromycin was used at 500 µg/ml; and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]was used at 5 µM.

RNA isolation and RNA blot hybridizations. Following the various treatments, cell suspensions were harvestedin darkness, samples were swirled in flasks on liquid nitrogen,cells were placed in centrifuge tubes on ice and immediatelycentrifuged for 10 min at 4°C, and cell pellets were storedat -80°C. RNA was isolated from cells as previously described(6). For RNA blot hybridizations, equal amounts of RNA (determinedspectroscopically) were resolved by electrophoresis in formaldehydegels. A fragment of hliA (extending from 26 bp upstream of theATG start codon of the hliA gene to 3 bp downstream of the translationtermination codon) was amplified by PCR and cloned into thepGEM-T Easy vector (Promega) to form the plasmid pTHL. Transcriptionof NcoI-digested pTHL with SP6 RNA polymerase and the Strip-EZRNA probe synthesis kit (Ambion) with [ ${alpha}$ -³²P]UTP generated theriboprobe used to detect hliA-encoding transcripts. A 303-bpinternal fragment of the rnpB gene, which encodes the constitutivelyexpressed RNA component of RNase P, of Synechococcus strainPCC 7942 was amplified by PCR (by use of primers 5'-AAAGTCCGGGCTCCCAAAAGACand 5'-CGGGTTCTGTTCTCTGTCGAAG) and cloned into pGEM-T Easy (Promega)to form the plasmid pTRP. As a control to confirm equal loadingof RNA samples, Northern blots were stripped of the hliA probeand hybridized with an rnpB DNA probe prepared by using thernpB-bearing NotI fragment of pTRP and labeled by using theStrip-EZ DNA probe synthesis kit (Ambion) with [ ${alpha}$ -³²P]dATP. Gelelectrophoresis of RNA was performed by using standard protocols(39). Northern hybridizations were done by using ULTRAhyb hybridizationbuffer (Ambion) per the manufacturer's protocol with hybridizationsand washes at 60°C for RNA probes and at 42°C for DNAprobes.

RESULTS

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Results

Evidence for early blue/UV-A-light photoperception triggering increases in hliA mRNA levels. hliA expression is upregulated by low-intensity blue or UV-Alight and by HL; the sensory mechanism that controls the HL-mediatedincrease in hliA transcript levels may wholly or partially operatethrough the same system as the blue-light response. In orderto help explore the relationship between the two responses,we monitored hliA mRNA levels in HL and UV-A light. We usedUV-A light in these studies rather than blue light because previousstudies had shown that hliA promoter-driven GUS activity wassomewhat higher after several hours in UV-A light than it wasin blue light of the same intensity (8). Figure 1A shows thatlevels of the hliA transcript, 300 nt in size, over time rapidlyincrease in both HL and UV-A light (with both showing peak expressionat the first time point, 10 min, at similarly high levels) butdrop off dramatically after 30 min with further UV-A exposure,whereas transcript levels decrease at a much slower rate inHL. This timing would be consistent with a model in which asensory system responds to the blue/UV-A light component ofHL in the early stages of HL-mediated hliA expression, whereasother factors are involved in maintaining high levels of expressionupon longer-term exposure to HL. In order to help determinethe nature of the blue/UV-A photoperceptive event involved inhliA expression, we monitored transcript levels following exposureto short pulses of UV-A light. Figure 1B shows that a pulseof low-intensity UV-A light (27 µmol of photons m^-2 s^-1)as short as 3 min is enough to cause a visible increase in hliAtranscript levels. (A spectrum of the UV-A light source usedis shown in Fig. 1C.)

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FIG. 1. (A) Northern hybridization analysis of hliA transcripts over time during HL or UV-A light exposure. LL (10 µmol of photons m^-2 s^-1)-adapted cells were exposed to HL (800 µmol of photons m^-2 s^-1) or UV-A light (27 µmol of photons m^-2 s^-1) for the indicated amounts of time before being harvested for RNA. A densitometric analysis of the signal from the hliA hybridization is presented. Hybridization of RNA blots with an rnpB-specific probe serves as a loading control for all the Northern hybridization analyses presented in this work. (B) Effects of short pulses of UV-A light on hliA transcript levels. LL-adapted cultures were transferred to UV-A light at 27 µmol of photons m^-2 s^-1 for the indicated amounts of time. (C) Light spectrum from the UV-A light source used in this study.

The increase in hliA transcript levels is a transcriptional response, and transcript stability can be important in determining final hliA mRNA levels. To see if hliA mRNA upregulation in HL and UV-A was a resultof an increase in transcription or a result of a light-mediatedincrease in stability of the message, we treated cells withthe transcriptional inhibitor rifampin prior to exposure toHL or UV-A. Rifampin blocked the HL- and UV-A-mediated increasesin hliA mRNA levels, indicating that accumulation of the messageis due to transcriptional induction (Fig. 2A). To determineif hliA induction involved the production of a protein factorthat is upregulated in HL and UV-A light, we treated cells withthe translational inhibitor chloramphenicol before shiftingcells to HL or UV-A light. Figure 2A shows that, rather thanblocking hliA induction, as would be the case if a light-inducedprotein transcription factor were involved in hliA upregulation,treatment with chloramphenicol resulted in dramatically elevatedhliA mRNA levels. Densitometry analysis of the RNA blot in Fig.2A shows that hliA mRNA levels in chloramphenicol-treated samplesare 2, 4, and 7.5 times greater than in nontreated samples inLL, HL, and UV-A light, respectively. This suggests that thefactors responsible for hliA induction are already present inthe cells (and their activity is altered in response to HL andUV-A light), and they do not have to be synthesized de novoin response to these light conditions. Another explanation forthe results with chloramphenicol is that, rather than a transcriptionfactor being synthesized in response to light, a protein degradationfactor involved in hliA mRNA turnover is upregulated by light.However, treatment with another inhibitor of translation, puromycin(which blocks the initiation of translation), did not causean increase in hliA mRNA (Fig. 2B). Thus, the increase in hliAmRNA in the presence of chloramphenicol is not likely due tolack of translation of a light-induced degradation factor; noris it due to some indirect effect caused by a general inhibitionof protein synthesis. Some transcripts have been found to bestabilized by antibiotics that block translational elongation,chloramphenicol in particular, by stalling ribosomes on thetranscripts and protecting them from degradation (34). The effectseen with chloramphenicol may be due to this phenomenon, andthe dramatic difference in transcript levels observed in thepresence and absence of chloramphenicol suggests then that factorsthat perturb RNA stability can have a significant effect onthe ultimate, steady-state levels of hliA mRNA.

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FIG. 2. Effects of inhibitors of transcription and translation on the HL- and UV-A light-mediated changes in hliA expression. LL-adapted cultures were exposed to chloramphenicol (Cm) or rifampin (Rif) (panel A) or puromycin (P) (panel B) or no inhibitor (-) before shifting to HL or UV-A or maintenance in LL. Light exposures proceeded for 30 min before cultures were harvested for RNA. (Neither ethanol nor methanol, in which Cm or Rif, respectively, was dissolved, was found to affect hliA induction [data not shown].)

Red light triggers a low-level, transient increase in hliA mRNA levels. We tested to what extent various qualities of light affect expressionof hliA. Figure 3 shows analysis of RNA from samples exposedto 20 min of light of different qualities (all presented at30 µmol of photons m^-2 s^-1). UV-A causes the largest increasein transcript levels, followed closely by blue light. Red lightcaused a very slight increase in message levels, while greenlight and far-red light caused no significant increase overLL levels. We then monitored transcript levels over time inred light (Fig. 4). A red-light-induced transcript was seenonly at 10 min of exposure and dropped to approximately LL levelsthereafter.

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FIG. 3. Effects of spectral quality on hliA expression. LL-adapted cells were exposed for 20 min to LL of various wavelengths. A densitometric analysis of the signal from the hliA hybridization is presented. BL, blue light; GL, green light; RL, red light; FR, far-red light.

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FIG. 4. Time course of hliA mRNA levels in red light. LL-adapted cells were exposed to red light (RL) for the indicated amounts of time. Samples exposed to 10 min of UV-A light or HL were included on the blot for comparison. A densitometric analysis of the signal from the hliA hybridization is presented.

The HL and UV-A light responses are not apparently reversible by any particular wavelength of light. As phytochrome is red and far-red reversible, some other wavelength-specificphotoreceptive events are specifically reversible by light ofanother wavelength. For example, certain blue-light-mediatedevents are specifically attenuated by another wavelength oflight, including the blue-light-induced opening of plant stomata,which is reversed by green light (11), and the blue-light inductionof the psbAII and psbAIII genes in Synechococcus strain PCC7942, which is specifically attenuated by red light (42). Therefore,we next tested to see if upregulation of hliA by UV-A or HLwould be reversed by subsequent exposure of cells to any otherspecific light wavelength. Cells were exposed to 10 min of HLor UV-A light and then shifted to light of various qualitiesor left in HL or UV-A light for 20 min (Fig. 5). hliA mRNA levelswere lower in all samples shifted to a different wavelength(rather than left in HL or UV-A or shifted to the dark). Theseresults suggest that the induction by HL and UV-A is attenuatedby light in general but is not photoreversible by any otherspecific light quality in particular. (However, we did not testshorter times after the light shift and thus would not be ableto detect more rapid changes.)

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FIG. 5. Effects of various light wavelengths in attenuation of the HL- and UV-A-mediated increase in hliA mRNA levels. Cells were exposed to 10 min of HL or UV-A light and then either harvested for RNA (HL and UV-A) or shifted to light of various qualities or the dark (D) for 20 min before being harvested for RNA. (Other abbreviations are the same as in the legend to Fig. 4.) The sizes of the two mRNA bands (the usual, full-length 300-nt transcript and a smaller, 265-nt fragment) seen in samples that have been shifted to the dark are indicated.

hliA mRNA turnover is slowed in the dark, and two mRNA species appear when photosynthesis is inhibited. As shown in Fig. 5, detectable levels of transcript were observedonly in those samples shifted to red light or to the dark. Likelywhat is visualized in the samples shifted to red light is theresult of a general decay of transcripts that have been removedfrom HL- or UV-A light-inducing conditions superimposed witha low-level induction by red light.

Regarding samples shifted to the dark, since darkness does notinduce hliA expression (data not shown), the increase in hliAtranscripts in dark-shifted samples over those exposed to noninducinglight qualities could be due to increased stability of the mRNA.Thus, the hliA transcript half-life was examined under differentlight conditions to determine the contribution of light-inducedchanges in mRNA stability to hliA transcript accumulation andto quantitate the stability of transcripts in the light versusthe dark (Fig. 6). Cells were exposed to HL or UV-A light for10 min, and then rifampin was added to block transcription andsamples were exposed to different light conditions, with samplestaken at various time points after the shift for Northern blothybridization analysis. Modest but significant differences werefound between half-lives calculated for messages maintainedunder inducing conditions and those shifted to the dark, withthe half-lives as follows: UV-A light, 7 min, versus UV-A lightto the dark, 10 min; and HL, 7 min, versus HL to the dark, 14min. We also explored whether the relative instability of thetranscripts in HL and UV-A light versus the dark was specificto the light conditions under which the transcripts were inducedor if light in general might cause the same phenomenon by calculatingthe half-lives of induced samples shifted to red light (30 µmolof photons m^-2 s^-1). The half-lives calculated were as follows:UV-A to red light, 5 min, and HL to red light, 8 min. The half-lifeof hliA is longer in the dark than in continued HL or UV-A orred light, indicating that some amount of light in general isrequired for optimal hliA mRNA turnover. Another phenomenonthat occurred in induced samples that have been shifted to thedark is the appearance of two hliA probe-hybridizing mRNA bandson RNA blots (Fig. 5A)—a typical, full-length transcriptof approximately 300 nt and a smaller, approximately 265-ntspecies. Since the hliA probe used is a riboprobe, both mRNAspecies are transcripts off the coding strand of hliA. Examinationof the hliA sequence (8) shows that the 265-nt species, evenif starting from the usual transcriptional start site of hliA,would end before the termination codon of this small polypeptidesequence. Alternatively, if the smaller species ends at thepredicted transcriptional termination site for hliA, its beginningwould be within the coding region of the gene. Therefore, thisspecies is not likely to be a transcript starting from a second,downstream initiation site. Thus, the smaller species is notlikely a message in and of itself but instead may be an mRNAprocessing intermediate of the 300-nt transcript. Decay of thehliA message is slowed in the dark versus in various light conditions(Fig. 5A and 6), and exposure to darkness causes the appearanceof two fragments. These results suggest that it is the absenceof light that causes the hliA mRNA decay to slow, and this resultsin the appearance of a decay intermediate. To test this idea,we treated samples with the photosynthesis inhibitor DCMU beforeshifting them to light-inducing conditions. Figure 7 shows thatboth fragments appear in the presence of DCMU in both HL andUV-A light and supports this hypothesis.

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FIG. 6. Effects of darkness on the stability of hliA transcripts. LL-adapted cells were treated for 10 min with HL (A) or UV-A (B) light, and then rifampin was added. Incubation was continued under the same light conditions for an additional 5 min, at which time zero time point samples for RNA were taken, and cultures were either maintained under the same light conditions or shifted to the dark (/D samples) or to red light (/R samples). Subsequent time point samples for RNA were taken at the indicated times following the light shift. RNA extracted from each of the samples was subjected to Northern hybridization analysis with an hliA-specific probe. Data were obtained by densitometry of the autoradiograms. (In the dark-shifted samples, which contain both the 300-nt transcripts and small amounts of the 265-nt transcripts, only the 300-nt transcripts have been included in the quantitation, since we view the 265-nt transcript as being inactive. The half-lives calculated for these samples with or without both transcripts included are very similar.) The average values of two independent repetitions of the experiments were subjected to exponential regression analysis and are plotted; the range between repetitions is indicated.

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FIG. 7. Effects of treatment with DCMU on the appearance of two hliA mRNA fragments. LL-adapted cells were exposed to DCMU (C) and then shifted to HL or UV-A light for 25 min before being harvested for RNA. The sizes of the two hliA-hybridizing fragments (the usual, full-length 300-nt transcript and a smaller, 265-nt fragment) seen in samples that have been exposed to the photosynthesis inhibitor DCMU are indicated. A densitometric analysis of the signal from each of the resulting fragments in the hliA hybridization is presented.

DISCUSSION

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Discussion

Possible photoperceptive mechanisms involved in hliA expression. This work explores details of the transcriptional and posttranscriptionalresponse of hliA to light. By comparing the timing of hliA inductionunder inducing light conditions, it appears that blue/UV-A lightand perhaps RL photoperception are involved in the early stages(the first 10 to 30 min) of HL-induced hliA expression. If thisis the case, what maintains high hliA transcript levels in HLand causes hliA transcript levels to drop in UV-A light afterthe first 30 min is not clear. One possibility is the actionof a sensory system monitoring the photosynthetic redox state,as we have found that exposure to photosynthetic inhibitorsperturbs hliA expression in both HL and UV-A light (K. Salemand L. G. van Waasbergen, submitted for publication). Moreover,the NblS sensor kinase that controls hliA expression also controlsexpression of the nblA gene under nutrient stress—a conditionthat, like HL, can result in overexcitation of the photosyntheticapparatus—raising the possibility that NblS may be sensingsome aspect of photosynthetic redox in the control of thesegenes (43). A number of other genes in cyanobacteria and plantsare regulated by both HL and blue light, including the ELIP(2), chalcone synthase (10), and psbA (42) genes, further supportinga role for blue light in HL gene control.

A 3-min pulse of low-intensity UV-A light at 27 µmol ofphotons m^-2 s^-1 was enough to cause hliA transcript accumulation.This result is consistent with blue/UV-A photoreceptor involvementin hliA induction. A number of other processes in cyanobacteriaare known to be affected by blue light. These include such activitiesas phototaxis in the motile strain of Synechocystis strain PCC6803 (32, 45); light-activated heterotrophic growth of Synechocystisstrain PCC 6803 (5); and, as mentioned above, the regulationof specific genes. Similar to our findings with hliA, the psbAIIand psbAIII genes of Synechococcus were found to be inducedby a 5-min pulse of blue light and may involve the action ofa blue-light photoreceptor (42). Like hliA, the psbAII and psbAIIIgenes are transcriptionally upregulated by HL and blue light(24, 42). The psbAIII gene (but not the psbAII gene), like hliA,is upregulated somewhat by red light, but unlike hliA, is alsoupregulated somewhat by far-red light (42). Moreover, red lightwas found to attenuate blue-light induction of psbAII and psbAIII(42). We have not found hliA induction to be attenuated by anyspecific wavelength of light. Thus, the hliA and psbA genesappear to be regulated by somewhat different photocontrol systemsthat may partially overlap. It should be noted that the NblSsensor kinase that controls hliA expression also controls expressionof the psbA genes in HL and UV-A light (43). It is not clearif NblS itself is a redox sensor and/or a blue/UV-A photoreceptoror if it acts in conjunction with a separate blue/UV-A photoreceptorin controlling the expression of the genes.

A survey of various light qualities showed that blue light andUV-A light cause the most profound increase in hliA mRNA levels.The so-called blue-light photoreceptors are typically responsiveto UV-A light as well (22). Red light also caused a slight increasein transcript levels. It may be that both a red light and ablue/UV-A light photoreceptor are involved in hliA expression,as has been seen for the ELIPs (17). Another possibility isthat the red-light and blue-light responses in hliA upregulationinvolve the same photoreceptor. Such may be the case for thecyanobacterial phytochrome Cph2 in Synechocystis strain PCC6803, which, when inactivated, generated a phototaxis towardblue light (45). The authors suggest that this phytochrome maybe involved in blue-light as well as red-light photoperception,since one of the two bilin lyase domains had been found to exhibita high blue spectral absorption maximum (46), while the othershows typical phytochrome red and far-red photoconversion invitro (46). Even so, as the strain inactivated for Cph2 is ableto sense blue light for taxis, The involvement of Cph in taxislikely involves interaction with a separate blue-light photoreceptor(45). An intriguing third possibility for hliA light qualitycontrol, given that photosynthetic redox is involved in hliAregulation during UV-A light as well as HL exposure (Salem andvan Waasbergen, submitted), is that photosynthetic pigmentsare involved as photoreceptors in mediating the blue- and red-lightresponses. However, it is equally possible that hliA is controlledby the activities of separate but interacting blue or UV-A photoreceptorand photosynthetic redox-monitoring systems. It should be noted,nevertheless, that the fluence of red light in which we observedslight hliA upregulation (18,000 µmol of photons m^-2)was approximately fourfold higher than the fluence of UV-A lightthat we found to cause hliA induction (4,860 µmol of photonsm^-2). Such a small (and transient) response to red light suggeststhat red light does not play a major role in HL-mediated hliAinduction by itself; but further studies are necessary to determinehow the red-light response interacts with the blue and UV-Alight and photosynthetic redox responses.

Posttranscriptional events involved in hliA expression. We have found that light affects hliA expression mainly at thetranscriptional level. However, the results showing that hliAexpression is greatly upregulated by treatment with chloramphenicolsuggest that mRNA stability can have a significant effect onthe ultimate mRNA level; as has been seen for some other transcripts(34), chloramphenicol (which blocks translational elongation)may act to stall ribosomes on the hliA transcript and protectit from decay, thus providing a level of control linking transcriptionwith translation.

We found the half-life of the hliA transcript to be slightlygreater in the dark than in the light. Likewise, in anotherstudy, transcripts of the psbA genes of Synechocystis strainPCC 6803 were found to be highly stable in the dark; this waslinked to the lack of photosynthetic electron transport (27).It could be that light energy is required directly for somestep of hliA mRNA decay and that there simply is not as muchenergy for efficient processing of hliA transcripts in the dark.However, most of the enzymes involved in RNA decay do not requireenergy input. Alternatively, it may be that in cyanobacteria,as in chloroplasts (30), light-dependent formation of a photosyntheticproton gradient across the thylakoid membrane is required fortranslational elongation (the process being slowed in the darkor by DCMU treatment); thus, in the dark, translational elongationis slowed and ribosomes stall and protect the hliA transcript,in a process similar to that which may be happening upon theaddition of chloramphenicol (above).

In darkness the half-life of hliA approximately doubles (atmost) over that seen under light conditions (Fig. 6). In thestudy mentioned above, in Synechocystis strain PCC 6803 thehalf-life of psbA transcripts in darkness was found to be dramaticallyhigher in the dark (7 h) than in the light (15 min), and itwas theorized that stabilization of the mRNA in the dark wouldallow its ready availability upon transition to a light periodand would benefit the cells during dark-to-light cycles in thenatural environment (27). It is not clear whether the relativelymodest increase in hliA mRNA stability observed in the darkover that in the light has a similarly meaningful ecologicalor physiological relevance.

A 265-nt mRNA fragment appeared in samples that had been shiftedto the dark or treated with DCMU. This fragment may representa decay intermediate that is visible when mRNA turnover is slowed.A similar result was seen for Synechocystis strain PCC 6803psbA transcripts, for which darkness or treatment with DCMUslowed their turnover and led to the appearance of a specificdegradation product (4, 26, 27). mRNA fragments that representdecay intermediates have also been observed for the psbA genesof Synechococcus strain PCC 7942 (15, 40) and photoinhibitedSynechocystis strain PCC 6714 (7). The present model for RNAdegradation in prokaryotes is that it involves a series of 5'-to-3'endonuclease cleavages that generate 3' ends that are degraded3' to 5', resulting in decay in a net 5'-to-3' direction (37).The 265-nt hliA mRNA fragment may be the result of an initial5' cleavage of the 300-nt transcript at a site apparently unprotectedby ribosomes as translation is slowed by darkness or DCMU. Thesmaller 35-nt product either may be completely degraded or istoo small to visualize by Northern hybridization analysis, whilethe 265-nt fragment is protected by stalled ribosomes and the3' terminator hairpin from subsequent endonuclease cleavagesand degradation. The 265-nt fragment is not as prominently visiblein samples treated with chloramphenicol (Fig. 2A). It may bethat chloramphenicol, perhaps by having a more pronounced effecton translational elongation and ribosomal stalling than darknessor DCMU, allows the entire transcript to be protected. The factthat inhibition of translational elongation by chloramphenicoltakes place under conditions where hliA induction is still occurring(i.e., light) could explain the much higher increase in messagelevels caused by the presence of chloramphenicol than by darknessor treatment with DCMU (compare Fig. 2A, 5, and 7). Future analysisof this cleavage site in the hliA transcript and sites in other,similarly cleaved transcripts (e.g., psbA) may yield insightinto site specificity for cyanobacterial (and possibly chloroplast)endonucleases and other events involved in the processing oflight-regulated messages.

ACKNOWLEDGMENTS

This work was supported by National Science Foundation GrantMCB-0003151 to L.G.V. and start-up funds and a Research EnhancementProgram grant to L.G.V. from the University of Texas at Arlington.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biology and Converging Biotechnology Center, Box 19498, The University of Texas at Arlington, Arlington, TX 76019. Phone: (817) 272-5239. Fax: (817) 272-2855. E-mail: Lorivw{at}uta.edu.

REFERENCES

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