diurnal rhythmicity in the pattern of mrnas in the leaves of

Plant Physiol. (1990) 94, 1590-15970032-0889/90/94/1 590/08/$01 .00/0

Received for publication February 28, 1990Accepted July 10, 1990

Diurnal Rhythmicity in the Pattern of mRNAs in theLeaves of Sinapis alba'

Frederic Cremer*, Jacques Dommes, Claude Van de Walle, and Georges BernierLaboratory of Plant Physiology, Department of Botany, University of Liege, B22, Sart Tilman, 4000 Liege, Belgium

ABSTRACT

Previous studies have shown that certain specific leaf mRNAsexhibit a diumal rhythmicity in their quantity in higher plants. Todetermine whether this situation is restricted to a few mRNAs, oraffects a large number, we have used in vitro translation and two-dimensional polyacrylamide gel electrophoresis to analyze themRNA complement in leaves of Sinapis alba at different timesduring an 8-hour/16-hour day/night cycle. A method for the visualanalysis of two-dimensional polyacrylamide gel electrophoresiswas also developed. This method selected, at each samplingtime, spots that were significant. It then selected, between twosampling times, intensity changes that were significant at the0.02 confidence level. During a day/night cycle, complex rhythmicchanges affected about 10% of the mRNAs. Nineteen differentrhythm pattems were found. These 19 patterns fell into four mainclasses: mRNAs that increase during the light period and de-crease during the dark, mRNAs that increase and then decreaseduring the light period, mRNAs that decrease during the lightperiod and increase during the dark period, and mRNAs thatincrease and then decrease during the dark period.

represents a large part of them, we have used in vitro transla-tion and 2D2-PAGE according to the method of O'Farrell(23) to analyze the mRNA complement in the leaves of adultvegetative plants of Sinapis alba at different times during aday/night cycle. Because we were not interested in analyzingonly a few spots, but rather all the polypeptides found in thegels, we started by developing a reliable procedure to analyzethe electrophoretograms.

MATERIALS AND METHODS

Plant Material

Plants of Sinapis alba L. were grown as described byLejeune et al. ( 15).The plants used were 65 d old, grown in 8-h SD. Sampling

was done every 4 h during a single day/night cycle, startingat the beginning of the light period. At each sampling time,the three youngest fully expanded leaves were collected fromeach of five plants. The 15 leaves were pooled to make onesample.

In leaves of higher plants submitted to the alternation ofday and night, numerous processes exhibit a diurnal rhyth-micity (1 1). A variety of enzymes have been shown to followa diurnal or circadian rhythm of activity: sucrose phosphatesynthase (24), nitrate reductase (27), protochlorophyllide ox-

ydoreductase (10), etc.In one effort to determine if such variations are the result

of rhythmic changes in a synthesis/degradation balance, Brul-fert et al. (3) used immunotitration to measure phosphoenol-pyruvate carboxylase extracted from leaves of Kalanchoeblossfeldiana. They showed that during the day/night cycle,the rhythm in enzyme capacity was not due to a rhythm inprotein synthesis or degradation but rather to a rhythmicinterconversion between active and inactive forms of theenzyme.On the other hand, several recent studies have demon-

strated diurnal or circadian rhythmicity in the level of specificmRNAs (8, 9, 13, 18, 21, 25, 28). To determine in one case

whether this latter situation is restricted to a few mRNAs, or

'Supported by the Belgian Government through the "Action deRecherche Concertee" No. 88/93-129. F. C. and J. D. were fellowsof IRSIA during part of this work. C. V. is a senior researcher fromthe FNRS.

RNA Extraction

RNA was extracted from leaves (about 4 g fresh weight)according to the method of Martin and Northcote (17) withminor modifications: NaCl was replaced by LiCl, and 0.1%heparin was added to the extraction buffer.

Fractionation of Poly(A)+ RNA

Poly(A)+ RNA was separated by affinity chromatographyon poly(U)-Sepharose (4). Poly(A)+ RNA concentration wasmeasured by spectrophotometry.

Cell Free Protein Synthesis

Poly(A)+ RNA was translated in a message-dependent rab-bit reticulocyte lysate (26) in the presence of [35S]methionine(2.8 x 107 Bq ml-'). We determined experimentally that theoptimum concentration of added K+ was 150 mm, whileaddition of Mg2+ was unnecessary. To ensure that incorpo-ration was proportional to the amount of mRNA, thepoly(A)+ RNA concentration was 10 ,ug ml-'. Samples wereincubated for 1 h at 30C. The amount of radioactive proteinwas estimated by TCA precipitation.

2Abbreviations: 2D, two-dimensional; poly(A)+RNA, polyadenyl-ated RNA.

1590

DIURNAL RHYTHMICITY in mRNAs in LEAVES of SINAPIS ALBA

Two-Dimensional Gel Electrophoresis

Samples were treated 15 min with RNase A (100 ,ug ml-'),then mixed with two volumes of loading buffer (80 mm Tris-HCl [pH 6.8], 0.1 M DTT, 20% glycerol, 4% SDS, 0.0005%bromophenol blue) and heated 5 min in boiling water. Elec-trophoresis was as described previously (6). Gels were pre-pared for fluorography using Amplify (Amersham, UK) andexposed to preflashed Kodak X-Omat AR film, 48 h for400,000 cpm.Due to inherent variability, electrophoresis was carried out

at least twice and the whole experiment was duplicated 3months later. Four gels were thus available at each samplingtime. Comparison procedures are described in the first sectionof "Results."

RESULTS

Changes in Intensity of Spots between Two SuccessiveSampling Times

Leaves of S. alba were harvested and their mRNA analyzedat the beginning (0 h), middle (4 h), and end of the day (8 h)as well as three times during the following night (12, 16, and20 h). The gels corresponding to the successive sampling timeswere compared in pairs, that is 0 h with 4 h, 4 h with 8 h,and so on.A method was developed to achieve this with maximal

reliability. Using a photographic enlargement of one gel (Fig.IA), a map of all spots appearing on the 24 gels was drawn.To eliminate poorly reproducible spots and some artifacts, we

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Plant Physiol. Vol. 94, 1990

discarded all spots that did not appear on at least four of the24 gels. The other 528 spots were kept and constituted a new

map called "master" (Fig. lB). Spots corresponding to trans-lation products synthesized in the absence of added mRNAdid not appear on the master due to their very faint intensity.

Differences in spot intensity were determined after a very

careful visual examination of each gel. Differences betweentwo sampling times were considered valid only when theywere found in all of the 16 possible comparisons between thefour gels of one sampling time and the four gels of the nextsampling time. Practically, when comparing two batches offour gels, we searched for polypeptides present in the four gelsof one batch, each having a greater intensity than the greatestintensity reached in the other batch. This rule allowed us toavoid considering any spot that was not present four times inat least one of the two batches involved. Furthermore, a spotwhose intensity on one gel of one batch was equal to itsintensity on one gel of the other batch was not considered toshow a variation of intensity according to our rule. These twocorollaries greatly diminished the work involved in comparingtwo batches of gels.

This method of gel analysis has a high threshold to char-acterize "change." It was not able to determine with goodreliability those spots that are basically constant. For example,a spot present four times in the batch of gels A and four timesin the batch of gels B, with the three higher intensities in Aand only one common intensity, was not classified as chang-ing, but this did not imply that it was constant. We consideredthat a spot was really constant if its intensity was similar inthe four gels of each batch and if, after the intensities were

arranged in ascending order in the eight gels of both batches,the intensities in the two batches overlapped for more thanone value. Undetected spots were counted as very faint spots,because they could vary from just above to just below the

detection limit. Spots that did not comply with this rule wereclassified as doubtful.The results of pair comparisons at six consecutive times are

presented in Table I. Spots showing a variation of intensityare presented in Figure 2.As an example, we will describe the comparison between 8

h and 12 h. From the 528 spots present on the master, 517were present in these two batches. There were 365 spotspresent four times at 8 h and 282 at 12 h; 396 spots were

present four times in at least one of the two batches. Amongthe 365 spots present four times at 8 h, 21 showed a decreasedintensity at 12 h. Among the 282 spots present four times at12 h, three showed an increased intensity. Thus, of the 396spots possibly showing a variation of intensity, 24 did show a

changed intensity. These 24 spots were subtracted from the517 spots present. The 495 remaining spots were examinedand, among them, 375 spots (76%) were classified as constant.The 24 spots changing by our criteria represented 6% of the399 spots classified.Because searching for constant spots was very tedious, and

because constant spots were less interesting in this work, we

determined the percentage of constant spots only once andused this percentage in all other comparisons.

Diumal Rhythmicity in Spot Intensity

From Figure 2, it appears that only 41 spots were involvedin the 98 changes in spot intensity detected. These 41 spotswere among the 445 spots analyzed, these being present fourtimes at one sampling time at least. Each of these 41 spotsshowed, at least over one interval, an increasing intensity and,over another, a decreasing intensity. The intensity of thesespots therefore showed diurnal rhythmicity. The patterns ofchanges for these clearly varying 41 spots fell into 19 classes.

Table I. Number of Spots Showing a Variation of Intensity at the Different Sampling TimesSampling Times (h)

0 (A)- 4 (A)- 8 (A)- 12 (A)- 16 (A)- 20 (A)-4 (B) 8 (B) 12 (B) 16 (B) 20 (B) 0 (B)

Number of spots present at least once in 514 516 517 519 524 504one series

Number of spots showing a decreased 9 12 21 3 5 0intensity in time B as compared totime A and number of spots present 4times at time A 296 301 365 282 322 281

Number of spots showing an increased 22 3 3 9 10 2intensity in time B as compared totime A and number of spots present 4times at time B 301 365 282 322 281 296

Number of spots present 4 times at time 354 392 396 362 367 344A and/or B

Number and percent of spots showing a 31 (8) 15 (4) 24 (6) 11(3) 15 (4) 2 (1)variation of intensity

1 592 CREMER ET AL.


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1593


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17 18 19

Figure 3. Schematic representations of the 19 observed diurnalrhythms in spot intensity. Time is along abscissa, together with a

representation of the light and dark periods. Upper horizontal linecorresponds to maximum intensity. Lower horizontal line correspondsto minimum intensity. Central line corresponds to an intermediateintensity when applicable. Spots following the different rhythms are

indicated in Table II.

These are presented in Figure 3. Table II shows which patterneach spot followed. Some of these patterns of changes are

illustrated in Figure 4.We also produced a more synthetic analysis by grouping

the patterns in only four classes. The first class consisted ofpatterns that showed an increase over some intervals duringthe light period and a decrease over some intervals during thedark period (rhythm patterns 1, 2, 5, 6, 7, and 8 [Table II]).The second class had patterns that showed a decrease over

some intervals during the light period and an increase over

some intervals during the dark period (rhythm patterns 11-19 [Table II]). The third class consisted of patterns thatshowed an increase and a decrease during the light period(rhythm patterns 3 and 4 [Table II]). And the fourth class hadpatterns that showed an increase and a decrease during thedark period (rhythm patterns 9 and 10 [Table II]).

DISCUSSION

Analysis of 2D-PAGE

Variability in 2D-PAGE has three components: variabilityin spot detectability, spot position, and spot intensity (16k.Even if top-quality 2D-PAGE are produced, a good protocolfor gel analysis is needed to deal with these three components.

Variability in spot detectability appears when differentnumbers of spots are visible on two gels of the same proteinextract or on two gels of two protein extracts of the same

sample. For example, between 539 and 785 spots have been

detected on 12 gels of the same extract (2). To overcome partof this problem, we decided to keep for analysis only thosespots that appeared on at least four gels. Most of the elimi-nated spots were very faint spots at the limit of detectability.

Variability in spot intensity appears when the same spot ontwo gels from the same extract differs in intensity. Thiscomponent of variability is partly related to variability in spotdetectability, because faint spots can vary from just above tojust below the limit of detectability. But darker spots alsoshow the same type of variability. For example, Hruschka( 12) has shown that, of the 100 darker spots on one gel, only90 were in common with the 100 darker spots on a secondgel of the same protein extract. When looking for changes inspot intensity between two different situations, it is absolutelynecessary to overcome this variability. We have therefore usedfour gels for each sampling time, with two gels coming fromone experiment, and two from another. In each experiment,the extract was derived from three leaves collected on each offive plants. This extract was used for two gels. Moreover,differences were considered only when detected in all of the16 possible comparisons between the four gels of sample Aand the four gels of sample B (14, 19). According to thestatistical test of Mann-Whitney, the probability that twosamples, one with the four smallest values and the other withthe four greatest values, belong to the same population is0.02.

Variability in spot position appears whenever it is virtuallyimpossible to superimpose two gels of the same extract so as

to obtain good alignment of each common spot. The relativeposition of spots is kept, but not their absolute position. Thelonger the distance between two spots, the greater the varia-bility. Authors have therefore often superimposed small areas

of gels in order to obtain a good matching (20). During visual

Table II. Number of Spots Following the Diurnal Rhythms Describedin Figure 3

Rhythm patterns are numbered as in Figure 3.Number of

Rhythm Pattern Spot Number

1 7, 8, 9,11,12, 13,14,16,17,19,20,21,22,38,39,40

2 33 24 15 56 237 48 10,159 6,25

10 1811 37,4112 2413 3214 33,34,3615 2816 2617 29,3018 3119 27,35

1 594 CREMER ET AL.


21 22

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Figure 4. Details of 2D-PAGE showing changes of selected spots during one light/dark cycle. Spot numbers are indicated. Sampling times areindicated for each line.

inspection of the gels, we have also used this technique toimprove difficult spot matching. However, it is usually suffi-cient to construct constellations joining small patterns ofspotsto identify the spots (7).Our method for gel comparison deals with each type of gel

variability and therefore provides quite reliable results.

Changes in Spot Intensity at Different Times of the Day/Night Cycle

Of the 528 spots kept in the master, 41 spots showed achange in intensity over one or another interval during theday/night cycle. From the 487 remaining spots, 370 could beconsidered constant, i.e. 76%. The 41 changing spots thereforerepresent about 10% of the 411 spots that were classified aseither constant or changing.

O'Farrell (23) has shown that, with a very high probability,each spot corresponds to a single polypeptide. In our study,the change in intensity of a spot corresponded to a change inthe relative quantity of the corresponding mRNA. Strictlyspeaking, it corresponded to a change in the relative translat-

ability of the corresponding mRNA. The experiment did notdiscriminate between a diurnal change in mRNA quantityand a time-specific variation in the selective degradation orin the premature termination of translation of some mRNAs.Despite this uncertainty, we will adopt below the term 'relativequantity' as the simplest explanation of the phenomenon.The changes observed in the relative quantity of some

mRNAs are not evenly distributed during the 24-h cycle.Changes are prominent at two specific times: the first 4 h ofthe light period and the first 4 h of the dark period.Of the 41 mRNAs showing a variation in their relative

quantity, 22 showed an increase after light was turned on.The level of many mRNAs is known to rise when light isgiven. This has been particularly well-illustrated with etiolatedplants transferred to light as reviewed by Tobin and Silver-thorne (29). In particular, in S. alba, the quantity of themRNAs coding for the two subunits of ribulose 1,5-bisphos-phate carboxylase is greater in seedlings grown 48 h in thelight than in seedlings grown 48 h in darkness (22).Nine other mRNAs are present in smaller quantity after 4

h of light. This situation is also well documented, e.g. boththe mRNA for NADPH-protochlorophyllide oxydoreductase

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1 595


in barley and the mRNA for phytochrome in several speciesdecrease when etiolated plants are exposed to light (1, 5).The second time of important changes is at the end of the

light period. Four h after the light was turned off, 21 mRNAsshowed a decreased and three an increased relative quantity.

Diumal Rhythmicity in the Quantity of Some mRNAs

Each mRNA that showed an increased relative quantity atone time during the light/dark cycle also showed a decreasedrelative quantity at another time. The relative quantity ofsuch mRNAs thus displayed a diurnal rhythm. Due to thelack of an appropriate experiment, it is not known if therhythms are circadian. The patterns ofchanges observed differamong them by the time that the relative quantity increases,the duration of the increase, the time that the relative quantitydecreases, and the duration of the decrease. Nineteen types ofpatterns have been descjibed.The most frequent pattern (pattern 1, 16 mRNAs) is char-

acterized by an increase at the start of the light period and adecrease at the start of the dark period. Light seems to act asa direct positive control of the quantity of these mRNAs.

Patterns 14 and 16 are characterized by a maximum quan-tity at the end of the night, a decrease during the day, and asubsequent increase at the end of the night. They are similarto those found for light-harvesting complex protein and theearly light induced protein in pea (13) and for nitrate reductasein tobacco and tomato (8).

Patterns 3 and 4, characterized by an increase at the begin-ning of the day and a decrease in the second part of the day,are similar to those found for the small subunit of ribulose1,5-bisphosphate carboxylase in pea (13), for light-harvestingcomplex protein in tobacco (25), for cab in tomato (9, 18),and for cab91 R in Petunia (28). All of these previouslydescribed rhythms are circadian.Some of the 19 patterns described are probably small vari-

ations of the same basic pattern. The existence of thesevariations is strongly linked to the method used for thecomparison of the 2D-PAGE. The 19 patterns have thereforebeen grouped into four classes showing strong differencesbetween them.

It is possible that some small changes have not been de-tected using our method. A more sensitive approach is thecloning of the mRNAs and the measurement of their abun-dance using molecular hybridization techniques. This methodhas been used until now only to measure the quantity ofknown mRNAs, not for the analysis of hundreds ofunknownmRNAs.

This work shows that foliar diurnal rhythmicity is notrestricted to a few mRNAs. During a light/dark cycle therelptive quantity of about 10% of the mRNAs of the leaf ofS. alba follows a diurnal cycle. The pattern of changes is verycomplex: 19 types of diurnal rhythms have been detected andgrouped into four classes.

ACKNOWLEDGMENTS

We are indebted to Professor P. B. Green for his critical reading ofthe manuscript and to Professor R. Moors for his valuable suggestions.

We are also grateful to P. Ongena for his technical assistance inrunning the 2D-PAGE.

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1 596 CREMER ET AL.


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diurnal rhythmicity in the pattern of mrnas in the leaves of

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