Light Differentially Regulates Cell Division and the mRNAincrease in the growth rate of leaves....

Light Differentially Regulates Cell Division and the mRNAAbundance of Pea Nucleolin during De-Etiolation1

Stuart A. Reichler 2, Janneke Balk, Margaret E. Brown, Kathryn Woodruff, Greg B. Clark, andStanley J. Roux*

Section of Molecular Cell and Developmental Biology, University of Texas, Austin, Texas 78713

The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between themRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whetherthe effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we comparedthe kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolinmRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 hafter a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistentwith separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. Theseincreases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are,like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasingS-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis.Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNAsynthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulatedby light than by mitosis.

As part of the characterization of a pea (Pisumsativum) cDNA with homology to yeast and animalnucleolin, Tong et al. (1997) found that the abun-dance of the pea nucleolin mRNA was regulated byphytochrome in etiolated pea shoots. The exposure ofa dark-grown seedling to light leads to a major de-velopmental transformation known as de-etiolation,which is in part mediated by the red light- (R) sen-sitive photoreceptor, phytochrome. Relative to light-grown plants, etiolated plants are thinner, taller, andlack photosynthetic pigments. Phytochrome media-tion of de-etiolation begins in the first few secondsafter exposure to R and continues for up to a day ormore. Phytochrome effects that occur within the firstfew seconds after R irradiation include changes inelectrical potentials (Smith, 1975) and changes in cy-tosolic free calcium levels (Shacklock et al., 1992).Some R-induced gene expression changes occur inminutes, and other photomorphogenic responses takea few hours to be detected. For instance, increases inleaf growth rate and decreases in stem growth ratehave been measured in peas 4 h after R irradiation(Smith, 1975). Full greening and leaf formation may

not occur until 12 to 24 h after R and require constantlight.

In etiolated plants, changes in growth are amongthe earliest measurable whole-plant responses medi-ated by phytochrome. Phytochrome activation causesa decrease in the elongation rate of epicotyls and anincrease in the growth rate of leaves. These changesin growth are known to involve shifts in elongationrates, as well as long-term changes in mitotic rates(Cosgrove, 1994). Changes in elongation and cell di-vision occur at two distinct regions of the plant.Elongation rates are mediated in cells that have al-ready divided and are in the process of maturing,whereas changes in the rate of cell division occur inthe meristems. For de-etiolation, the elongation re-sponse has been studied most intensely. In peas,inhibition of epicotyl elongation (as measured bychanges in the zone of elongation) has been detectedas rapidly as 1 h after R exposure (Behringer et al.,1990). Studies of changes in cell division induced byphytochrome are much less exact. What is known upto now is that phytochrome does cause changes incell division rates. The data about R-induced celldivision changes in flowering plants come from stud-ies measuring the differences in cell size many hoursafter irradiation (Cosgrove, 1994). The only detailedstudy of phytochrome-mediated cell division kineticsis in germinating fern spores (Furuya et al., 1997).Prior to the work presented here, the relative kineticsof light-induced changes in cell division that occurduring de-etiolation were not well documented.

1 This work was supported by the U.S. National Science Foun-dation (grant no. IBN–9603884 to S.A.R.) and by the NationalAeronautics and Space Administration (grant no. NAG2–1347).

2 Present address: Instituto de Biotecnologıa, P.O. Box 510 –3,Cuernavaca, Morelos 62250, Mexico.

* Corresponding author; e-mail [email protected]; fax512–232–3402.

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In contrast, differential gene expression during de-etiolation has received much attention. The regula-tion of genes that are required for greening and pho-tosynthesis have been well characterized. There aremany genes that have been identified as being up-regulated by the activation of phytochrome in etio-lated seedlings (Batschauer et al., 1994). One of theearliest studied phytochrome regulated genes wasrDNA. In higher plants the photoreversible increasein accumulation of rRNA has been measured at 2 to6 h after R irradiation (Tobin and Silverthorne, 1985).In peas specifically, Koller and Smith (1972) showedthat plumules reacted to an R pulse by transientlyincreasing rRNA synthesis beginning 2 to 3 h post-irradiation. In addition, phytochrome mediates anincrease in polysomes (Pine and Klein, 1972). Thisphytochrome-mediated increase in ribosomes andprotein synthesis is presumably acting to increase thepopulation of regulatory, enzymatic, and structuralproteins as the plant undergoes de-etiolation. To up-regulate ribosome levels, proteins involved in ribo-some synthesis must also be up-regulated.

Nucleolin is one of the best-characterized proteinsinvolved in ribosome synthesis (Tuteja and Tuteja,1998; Ginisty et al., 1999). The initial steps in ribo-some synthesis, such as the transcription of the rRNAand its subsequent splicing, occur in the nucleolus.Although the involvement of nucleolin in the processof ribosome synthesis has been established, the exactstep or steps in which it is involved are still unclear.In an in vitro experiment using mouse cell extracts,nucleolin was shown to be a vital component forsplicing of the 59-external transcribed spacer regionof the initial rRNA transcript (Ginisty et al., 1998).Other studies using yeast mutants deleted in the geneencoding the yeast nucleolin homolog, NSR1, haveshown nucleolin to be involved in the maturation of

the rRNA from a single transcript to the individualsubunits (Kondo and Inouye, 1992; Lee et al., 1992).Although participation of nucleolin in ribosome syn-thesis appears to be its primary function, other func-tions such as helicase activity, binding to laminin,transcriptional repression of the AGP gene (Tutejaand Tuteja, 1998; Ginisty et al., 1999), binding to theMyb transcription factor (Ying et al., 2000), and bind-ing to telomeres (Pollice et al., 2000) have been pro-posed. In addition to functional data about nucleolin,in animals (Bugler et al., 1982), yeast (Kondo et al.,1992), and plants (Bogre et al., 1996), nucleolin hasbeen characterized as being up-regulated duringtimes of increased cell proliferation.

The correlation of increased nucleolin expressionwith increased cell proliferation led to the postulatethat the up-regulation of nucleolin could serve as amolecular marker for the onset of mitosis in plants(Bogre et al., 1996). We wondered whether this cor-relation would hold in the case of de-etiolation, inwhich phytochrome regulates nucleolin expression(Tong et al., 1997) and mitosis (Cosgrove, 1994). Us-ing the nucleolin-like cDNA described in Tong et al.(1997), we performed experiments designed to deter-mine the relationship between nucleolin and mitosis,as well as to learn whether plant nucleolin, like ani-mal and yeast nucleolin, functions in ribosomesynthesis.

RESULTS

S-Phase Kinetics Show an Increase 9 h afterR Irradiation

A comparison of the S-phase index of plumules ateach time point after R irradiation to its green safe-

Figure 1. S-phase index of R-irradiated plu-mules. Seven-day-old etiolated pea plumuleswere irradiated with an R pulse and returned todarkness. S-phase index was measured by thenumber of nuclei that had incorporated 59-bromo-29-deoxyuridine (BrdU) relative to the to-tal number of nuclei per time point. A significantincrease (P , .05) between R and G plumuleswas found only at 9 h postirradiation, markedwith asterisks. Cross-hatched bars represent theG control plumules, and the solid bars repre-sent the R-irradiated plumules. The error barsare 61 SD.

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light (G) control shows that there is a statisticallysignificant (P , 0.05) increase 9 h after the irradiation(Fig. 1). At 6 and 12 h after R irradiation, the S-phaseindex was lower than at 9 h. In addition, there was nostatistically significant difference between R and Gplumules at 6 or 12 h post-irradiation. These dataindicate that there is an increase in the number of peaplumule cells replicating their DNA 9 h after an Rpulse.

Measurement of M-Phase Kinetics after an R Pulse

Figure 2A shows a typical excised pea plumule andFigure 2B shows the same plumule after the outerleaves have been removed. The two tissue types areshown with the inner leaves (IL) and the apical re-gion (A) marked. The mass of the plumule is domi-nated by the leaves so that the A only comprises a

small portion of the plumule with the apical meri-stem being an even smaller region contained withinthe apex. Figure 2C shows various cells in M-phasewith a background of non-mitotic cells. The mitoticcells are indicated by arrows and were counted toobtain the M-phase index.

The mitotic index for each time point after R irra-diation was compared with a G control to account forany inherent pattern of mitotic rates that might existin the plumules. Beginning at 12 h post-irradiationand continuing through 15 h post-irradiation there isa significant increase (P , 0.01) in mitotic rates forthe R-irradiated leaves (Fig. 3A).

Similar measurements were performed with theapical tissue. Because the A is small and difficult towork with, time points were only taken every 6 h. Asignificant decrease (P , 0.001) in the mitotic rate ofthe apex is indicated at 24 h after the R irradiation(Fig. 3B).

mRNA Abundance of Cell Cycle-Regulated Genes

The mRNA abundance of genes known to beregulated in a cell cycle-dependent manner, pro-liferating cell nuclear antigen (PCNA) and cyclin B,was compared with that of nucleolin after an Rpulse (Fig. 4A). Nucleolin rises rapidly at 6 h post-irradiation, stays steady through 9 h, and thenbegins to decrease at 12 h. The abundance of PCNAstays relatively steady through 6 h, increases at9 h, and then begins to decrease at 12 h. Cyclin Babundance rises at a relatively constant rate begin-ningat 6 h post-irradiation and continuing through12 h.

Figure 4B shows the pattern of control plants thatwere exposed only to G. The cell cycle-regulatedgenes, PCNA and cyclin B, in agreement with theresults seen for M-phase and S-phase measure-ments, showed little or no change in expressionwhen exposed to G. In contrast, nucleolin did in-crease when exposed to G. Nucleolin mRNA abun-dance in G-exposed plumules began to rise at 6 hpost-irradiation, and reached saturation at 9 h. Aswith the S-phase index of R-exposed plumules, theabundance of nucleolin mRNA began to markedlydecrease at 12 h.

Two patterns emerge from this experiment. Whenexposed to R, the cell cycle-regulated genes increasetheir mRNA abundance slowly, peaking at 9 or morehours post-irradiation. In contrast, the pattern of ex-pression for nucleolin is notably different, character-ized by a strong initial increase at 6 h post-irradiation.In addition, although the cell cycle-regulated genesshow no change when exposed to G, nucleolin mRNAlevels do increase, although more slowly than whenexposed to R.

Figure 2. Dissected etiolated pea plumules and the mitotic figuresobtained by staining the nuclei. A, An etiolated pea plumule afterexcision from the shoot. The outer leaves surround the IL and theapex. B, The outer leaves have been removed and the inner leavesand apex are ready to be separated and then stained. The mass of theapex is less than one-tenth of the inner leaves. The IL and the apex (A)are marked for reference. C, Example photos of mitotic figures, asvisualized by Feulgen staining, from plumules that were counted toobtain the mitotic indices in Figure 3. The arrows point to cellsundergoing mitosis. On the left side are two cells in metaphase, andon the right two cells in anaphase. All of these cells were from IL ofthe plumule. A and B are shown at a magnification of 303. In C, thebar 5 10 mm.

Light Regulation of Cell Division and Nucleolin in Pea

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Photoreversibility of the R-Induced Increases in mRNAAbundance of Cell Cycle-Regulated Genes

The expression of the cell cycle-regulated genescompared with the S- and M-phase measurementsindicated that northern blots could be used as anaccurate determination of cell proliferation levels.Therefore, to determine if phytochrome is the photo-receptor for the R-induced increases in cell prolifer-ation that we detected in etiolated pea plants, north-ern blots of known cell cycle-regulated genes wereperformed with the following light regimens. Plantswere exposed to either R alone, an R pulse followedby a far-R (FR) pulse, or a FR pulse was given with-out R. PCNA and histone H2A, another cell cycle-regulated gene, show similar increases to R at 10 h asin Figure 4A, and both show little-to-no increase inmRNA abundance after R followed by FR or with FRalone (Fig. 5). Thus changes in the mRNA abundance

of cell cycle-regulated genes are photoreversible, im-plicating phytochrome as the photoreceptor for thesechanges.

Rescue of a Yeast nsr1 Mutant with Pea Nucleolin

To determine if the nucleolin-like cDNA from peaswas functionally similar to nucleolins from animalsand yeast, the pea nucleolin-like cDNA was ex-pressed in a yeast nucleolin mutant (WYL353), whichhas the yeast nucleolin homolog, nsr1, deleted. Themost complete data about the function of nucleolinhave come from these yeast deletion mutants. Thesemutants splice the rRNA transcripts less efficientlythan wild type (Lee et al., 1992), and they grow moreslowly than wild type (Kondo and Inouye, 1992).

The first indication that the pea nucleolin couldrescue the nsr1 mutant was the difference in growth

Figure 3. Mitotic index of R-irradiated pea plu-mules. A, The mitotic index of the IL of R irra-diated plumules. The mitotic index of inner plu-mule leaves significantly increased (P , 0.01) incomparison to G control IL starting at 12 hpostirradiation and continuing through 15 hpostirradiation. B, The mitotic index of the As ofR-irradiated plumules. The mitotic index ofR-irradiated apices significantly decreased (P ,0.001) in comparison to G control apices at 24 hpost-irradiation. Each time point shows the mi-totic index of R-irradiated plumules and G con-trol plumules picked at the same time. The timecourse of G is shown in dashed lines, and thetime course of R is shown in solid lines. Theerror bars represent 61 SD, and the asterisksindicate time points at which G and R are sta-tistically different as noted in the text.

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rates of the nsr1 mutant transformed with the peanucleolin or with vector alone. When grown onplates with inducing media, the nsr1 mutants ex-pressing the pea nucleolin grow faster than thosecontaining the vector only (Fig. 6A). These differ-

ences in growth results were demonstrated morequantitatively by growing these yeast strains in liq-uid inducing media. In this liquid culture assay, thelogarithmic phase of growth lasted from 7 to 12 hafter inoculation. OD600 readings taken 9, 10, and 11 h

Figure 4. The mRNA abundance of cell cycle-regulated genes compared with the mRNAabundance of nucleolin. A, The expression ofthe mRNA levels of nucleolin, PCNA, and cyclinB after 2 min of R at time zero as measured bynorthern blots. The densitometry units are val-ues assigned by digitizing the blot on a phos-phoImager (Molecular Dynamics). The mRNAabundance of nucleolin increases and rapidlyreaches saturation at 6 h postirradiation fol-lowed by a gradual increase of cyclin B begin-ning at 6 h postirradiation and then an increasein PCNA at 9 h postirradiation. Nucleolin andPCNA are decreasing at 12 h postirradiation,whereas cyclin B is continuing its increase. B,The expression of the mRNA levels of nucleolin,PCNA, and cyclin B after an exposure of approx-imately 10 min of G at time zero as measured bynorthern blots. This exposure was during thenormal handling of the peas in the growth room.The densitometry units are values assigned bydigitizing the blot on a phosphoImager (Molec-ular Dynamics). The mRNA abundance ofnucleolin rises at 6 h postirradiation peaking at9 h and then decreasing at 12 h. Neither cyclinB nor PCNA shows much change when exposedto G. C, Northern blot that was used to obtainthe values in A and B. The numbers across thetop indicate the hours after exposure to either Gor R, and the identification of the probe used foreach blot is shown on the left side. Along thebottom is shown the ethidium bromide stainingof the large and small subunits of the rRNA thatwas used as an equal loading control. The RNAat each time point used for these blots was froma single RNA isolation. The lines and pointsrepresenting the abundance of the various genesare as follows: solid line with diamonds, nucleo-lin; dashed line with squares, PCNA; and smalldashed line with triangles, cyclin B.





after inoculation showed that during logarithmicgrowth the yeast nsr1 mutant expressing the peanucleolin gene grew between 1.4- and 1.6-fold morerapidly than the mutant containing vector alone.

Polysome analysis was performed on the sametransformed lines (Fig. 6, B and C). When the vectorwith the pea nucleolin gene was induced, the amountof the 60S subunit increased (Fig. 6B). The vectoralone did not show this increase in 60S subunit (Fig.6C). The polysome analysis was corroborated by den-sitometry of large subunit rRNA abundance in aga-rose gels. When the expression of pea nucleolin wasinduced in the transformed yeast, there was a statis-tically significant (P , 0.01) increase in large subunitrRNA (data not shown). This increase was not seen inthe vector-only controls.

These three experiments show that expressing thepea nucleolin gene in NSR1-deficient yeast cells in-creases the relative amount of large subunit rRNAand leads to an increased growth rate of the trans-formed yeast. This increase in large subunit rRNAoccurs only when the pea nucleolin cDNA is presentand confirms the classification of the pea nucleolin asfunctioning in ribosome synthesis.

DISCUSSION

Initial experiments performed during the charac-terization of the pea nucleolin-like cDNA demon-strated that the mRNA abundance of nucleolin inetiolated pea plumules increases following an Rpulse and that this increase was photoreversible, im-plicating phytochrome as the photoreceptor for thisresponse (Tong et al., 1997). This led to the questionof whether this light-mediated response was a down-stream effect of increased mitotic rates, as might beconcluded from previous work on nucleolin (Bugler

et al., 1982; Bogre et al., 1996), or whether nucleolinmight be regulated independently of proliferationrates.

As etiolated plants proceed through de-etiolation,they shift the focus of their growth from upward tooutward. More specifically, the leaves of the plantbegin to expand, whereas the upward growth at theshoot apex decreases. These shifts are accomplishedby a change in elongation rates and cell division rates(Cosgrove, 1994). Although the kinetics of the shiftsin elongation rates has been well studied, the kineticsof cell division changes has not, until this report,been determined. By using several different methodsto determine changes in cell proliferation during de-etiolation, the different stages of cell proliferationand their kinetics have been definitively resolved andtracked. The first measurable change in cell prolifer-ation after an R pulse is an increase in cells proceed-ing through S-phase at 9 h post-irradiation. Next isan increase in M-phase in the inner plumule leaves at12 h post-irradiation, followed by a decrease inM-phase in the apices at 24 h post-irradiation. Thedifferent and opposite reactions to R of the leavesand apices correspond to the opposite directions thattheir growth rates are changing. By combining thesedirect measurements of S- and M-phase, the kineticsof cell proliferation changes following an R pulsehave been established.

Even though the cell proliferation kinetics werewell established by these direct measurements,northern-blot analysis was employed to be able todirectly compare R-induced differences in the mRNAabundance of nucleolin with that of other known cellcycle-regulated genes and to more rigorously assessthe photoreversibility of these proliferation events.Although each gene has its own pattern of expressionafter R, the mRNA abundance of the cell cycle-

Figure 5. Photoreversibility of the R inductionof the cell cycle-regulated mRNAs. Plumuleswere irradiated by R alone, R immediately fol-lowed by FR, or by FR alone and the accumu-lation of cell cycle-regulated genes was thenmeasured at 10 h postirradiation. R followed byFR and FR alone did not induce the mRNAaccumulation as R did, thus indicating that thisresponse is regulated via phytochrome. Cross-hatched bars indicate histone H2A, and solidbars indicate PCNA.

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regulated genes increases later and more graduallythan that of nucleolin (Fig. 4A). The differences inexpression patterns for the cell cycle-regulated genesmay be due to the part of the cell cycle in which theyare expressed. PCNA is expressed during S-phase(Kodama et al., 1991), it peaks at 9 h postirradiation,and it is noticeably decreasing at 12 h, which mirrorsthe peak measured for S-phase index (Fig. 1). CyclinB is expressed during M-phase (Shimizu and Mori,1998), and its expression increases more graduallyand is still increasing at 12 h when M-phase in the

leaves is increasing as in Figure 3A. According tothese results, during de-etiolation nucleolin is up-regulated prior to increases in cell proliferationchanges.

Although nucleolin has been commonly character-ized as being regulated in a cell proliferation-dependent manner (Bugler et al., 1982; Bogre et al.,1996), there is at least one other example in whichnucleolin is induced by a stimulus without an in-crease in cell proliferation. Kondo et al. (1992)showed that NSR1, the yeast nucleolin homolog, in-creased not only with increasing cell proliferation,but also when yeast cells are subjected to a coldshock, a stimulus that suppresses the rate of cellproliferation.

It has been previously shown that increases in themRNA abundance of nucleolin during de-etiolationare mediated by phytochrome (Tong et al., 1997). Inthis report we have shown that increases in S-phase,M-phase, and cell cycle-regulated genes are all in-duced by R and that the direct measurements of S-and M-phase match changes in cell cycle-regulatedgenes. Therefore, we used northern blots of knowncell cycle-regulated genes to determine whether thesecell proliferation changes like the mRNA abundanceof nucleolin were photoreversible, which implies reg-ulation by phytochrome. Our results (Fig. 5) showthat the increases in the cell cycle-regulated genes arephotoreversible. So even though the mRNA abun-dance of nucleolin and changes in cell proliferationhave different kinetics during de-etiolation, they areboth mediated by phytochrome.

Further analysis of the R-induced cell proliferationdata reported here yields additional valuable in-sights. The first is that these results parallel those ofKaufman et al. (1986). They used whole, etiolated peaplumules and measured the kinetics of fresh weightchanges after an R pulse. Their findings show thatfresh weight began to significantly increase between8 and 12 h post-irradiation and that this R-inducedeffect was reversible by FR. The increase in plumuleweight measured by Kaufman et al. (1986), accordingto our data, would be partially due to the increase incell division in the plumule leaves. Although elonga-tion rates in pea plumule leaves begin to increase at4 h post-irradiation (Smith, 1975), significant in-creases in plumule weight are not recorded until thetime that M-phase increases are detected. As Cos-grove (1994) points out, changes in growth cannotinvolve only changes in cell division, as this wouldincrease the number of cells, but not affect the size ofthe tissue unless the dividing cells elongate. It isknown that the shift in growth that accompaniesde-etiolation involves changes in elongation and celldivision. But even though elongation rates increasesoon after R irradiation, it appears that major growthchanges, those that can be measured by an increase infresh weight, accompany an increase in cell division

Figure 6. Rescue of the yeast nsr1 mutant with pea nucleolin. A,Yeast transformed with pea nucleolin or vector alone were grown oninducing media plates, and the relative growth rates were observed.Transformed yeast expressing the pea nucleolin grow faster thanthose with the vector only. B and C, Polysome profiles of yeastcultures in noninducing or inducing media transformed with vectorcontaining pea nucleolin (B) or vector alone (C). The peaks arelabeled according to which ribosomal subunits they represent. Theprofiles read from left to right as from top to bottom of the gradients,and the peaks are in relative OD254 units. When the pea nucleolin isexpressed there is an increase in large subunit ribosomes.





in the plumule leaves at around 12 h after Rirradiation.

Another valuable insight from our data is that Rinduces some level of synchronization in the numberof cells replicating their DNA. There is a significantdifference between R- and G-irradiated plants intheir S-phase index at 9 h postirradiation, but notbefore or after. The data presented for the S-phaseindex are an average of apices and leaves, and, as ourM-phase data show, the leaves are increasing theirproliferation, whereas the apices are, at a much latertime, decreasing their proliferation rate. Althoughthis mixture of apices and leaf tissue could increasebackground noise, the effect would be small becausethe mass of the apex is so much smaller than that ofthe leaves, and the apices are comprised of only asmall number of mitotically active cells. Further sup-porting the idea that light helps to synchronize thenumber of cells replicating their DNA is the peak inPCNA mRNA abundance at 9 h postirradiation (Fig.4A). PCNA is known to be up-regulated duringS-phase (Kodama et al., 1991), and its peak at 9 hpost-irradiation mirrors the peak measured for theS-phase increase. So data from direct measurementsand northern blots show some synchrony to the in-crease in cells entering S-phase following an R pulse.

The fact that R induces an increased abundance ofthe mRNA for nucleolin somewhat before it stimu-lates increased rates of cell division could be inter-preted to mean that these two processes are regu-lated differentially by phytochrome, but they do notresolve the question of whether nucleolin expressionis independent of cell proliferation. This question ismore directly addressed by the results that show Gcan induce a significant increase in nucleolin expres-sion without having any measurable effect on eithercell proliferation or on the expression of cell-cyclerelated genes. It is clear that in this case, nucleolingene expression is independent of the onset of celldivision.

G is sufficiently actinic to induce the expression ofa number of phytochrome regulated genes, via thelight hypersensitive-signaling pathway known as theVery Low Fluence response (Thompson et al., 1985;Hsieh et al., 1996). Rigorous demonstration that theeffect of G on nucleolin gene expression is mediatedthrough phytochrome would require additional ex-periments such as repeating these experiments usingvarious phytochrome deficient mutants. If this G in-duction of nucleolin mRNA expression is via a phy-tochrome pathway, then because phytochrome canphotoreversibly regulate nucleolin expression (Tonget al., 1997) and Very Low Fluence responses aretypically not photoreversible, the nucleolin genewould be classified among other genes that can beregulated by R under low fluence (photoreversible)and Very Low Fluence conditions (Thompson et al.,1985). Independent of the question of the photore-ceptor for the G response, the fact that G can up-

regulate the expression of nucleolin in etiolated peaseedlings without increasing cell division demon-strates conclusively that the expression of nucleolinis regulated differently from cell division.

Phytochrome is known to regulate several differentresponses such as germination, de-etiolation, shade-avoidance, and flowering. Which response is trig-gered by phytochrome activation is often controlledby what developmental stage the plant is in. Forresponses that occur concurrently, however, such asdifferent genes being regulated during de-etiolation,it is known that some of these responses are regu-lated with very different kinetics and that there are atleast two signal transduction pathways that regulatesome of these genes (Neuhaus et al., 1993). For in-stance, phytochrome-mediated up-regulation of themRNAs for the small subunit of Rubisco and chloro-phyll a/b-binding protein requires only a short burstof R, whereas chalcone synthase up-regulation re-quires a longer R exposure (Peters et al., 1998). Phy-tochrome can also act through different signal trans-duction pathways. The mRNA abundance forchlorophyll a/b-binding protein is regulated througha calcium-dependent pathway, whereas chalconesynthase is regulated via a calcium-independentpathway (Neuhaus et al., 1993; Batschauer, 1999).These three genes are all up-regulated during de-etiolation, whereas the mRNA for phytochrome A isdown-regulated (Colbert et al., 1983). Thus duringthe single developmental process of de-etiolation,several different regulatory events are occurring si-multaneously, which are all regulated via phyto-chrome. The different pathways through which phy-tochrome regulates responses, which are possiblymediated by different members of the phytochromegene family, can explain how nucleolin and rRNAare up-regulated with different kinetics than the up-regulation of cell proliferation, even though they areall induced by phytochrome.

Knowing when and how nucleolin is regulatedduring de-etiolation does not resolve what is its func-tion. Much information has been learned about theyeast nucleolin homolog, NSR1, through analysis ofdeletion mutants. By transforming the pea nucleolin-like cDNA into one of these mutants, we hoped todetermine if it had similar functions to other nucleo-lins. Our findings that yeast deletion mutants ex-pressing the pea nucleolin gene show increases inlarge rRNA subunit and an increased growth ratedemonstrate a rescue of the yeast mutant phenotype.The pea nucleolin shares about the same alignmentsimilarity at the amino acid level with the yeastand animal nucleolins (pea:yeast, 50% similarity,pea:Xenopus, 45% similarity), however, the animalnucleolin does not rescue the yeast nsr1 mutants (Xueet al., 1993). The ability of the plant nucleolin torescue nsr1 mutants may be related to the fact thatthe pea and yeast nucleolin share the structural fea-ture of having only two RNA recognition motifs,

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whereas the animal nucleolins have four (Tong et al.,1997). These RNA recognition motifs are likely re-sponsible for the interaction of nucleolin with rRNA(Serin et al., 1996), and changes in these motifs couldbe expected to have functional consequences. Al-though these results do not indicate a precise role forthe pea nucleolin in ribosome synthesis, they dodemonstrate that the pea nucleolin, like other nucleo-lins, functions in the synthesis of ribosomes.

The function of nucleolin in ribosome synthesiscould help explain why its R-induced expression iscoordinated with the light-induced up-regulation ofrRNA synthesis that has been repeatedly demon-strated in etiolated seedlings. Several studies havelooked at rRNA abundance and polysome formationafter phytochrome activation. Increases in rRNA havebeen measured at around 2 to 6 h post-irradiation,depending on the species (Koller and Smith, 1972;Tobin and Silverthorne, 1985), which is similar tochanges in the abundance of the mRNA for peanucleolin after a pulse of R (Fig. 4A). The observationthat nucleolin and rRNA increase with similar kineticswould be predicted by the fact that the primaryfunction of nucleolin has been identified as the pro-cessing of rRNA into pre-ribosomes. However,nucleolin is known to be multifunctional (Tuteja andTuteja, 1998; Ginisty et al., 1999), so this coincidenceof up-regulation for rRNA and nucleolin does notpreclude other possible functions that nucleolin mayhave during de-etiolation.

The process of de-etiolation involves a massivechange in expressed proteins. This increased needfor new protein synthesis mandates precedent up-regulation of nucleolar activity and accumulation ofribosomes for increased translation. Our results in-dicate that nucleolin up-regulation is likely to be akey event in the overall build-up of molecular ma-chinery leading to increased ribosome synthesis.Because it appears that pea, like yeast and Xenopus,has only one gene encoding nucleolin (Tong et al.,1997), this gene would be a prime target for knock-out studies to assess the relative importance ofnucleolin in plant rRNA synthesis and in R-inducedde-etiolation.

The study of pea nucleolin and its regulation hasyielded three main findings. The first is that phyto-chrome partially synchronizes and alters the timecourse of cell division during its induction of de-etiolation. Second, the fact that the pea cDNA rescuesa yeast nsr1 mutant confirms that the pea gene en-codes a functional nucleolin homolog and that thepea nucleolin, like animal and yeast nucleolins, isimportantly involved in the synthesis of ribosomes.Third, nucleolin expression, although linked to cellproliferation, is independent of it since it can beinduced without an accompanying change in the rateof DNA synthesis or mitosis.

MATERIALS AND METHODS

Plant Material and Irradiations

Pea (Pisum sativum cv Alaska) seedlings were grown inthe dark for 7 d at 22°C 6 3°C. They were the source for thepea plumules used in S- and M-phase determinations andfor isolation of the RNA used in the northern analyses.

The R irradiations were for 2 min; FR irradiations werefor 4 min. The R source produced a fluence of 43 mW/m2

through an interference filter with a lmax of 660 nm and aband width of 20 nm. The FR source produced a fluence of26 mW/m2 through an interference filter with a lmax of 730nm and a band width of 20 nm. The dark-grown seedlingswere irradiated by R alone, FR alone, or by R followed byFR. All other manipulations of the pea seedlings were doneunder G, described in Hsieh et al. (1996). After each irra-diation treatment, seedlings were returned to the dark andthen harvested at different time points as indicated in thefigure legends.

Determination of S-Phase Index

After the 7-d-old seedlings were irradiated, an apicalsection including the plumule 1 2 cm of epicotyl was cutoff and placed in a Petri dish with 10 nm BrdU (Amersham,Buckinghamshire, UK) for 2 h. At the end of the incubationperiod, the basal 2 cm of epicotyl was removed and theplumules were fixed in 4% (w/v) paraformaldehyde in Trisbuffer (50 mm Tris, 10 mm EDTA, and 100 mm NaCl, pH7.8) with 0.01% (w/v) Triton X-100 for 1.5 h. The fixativewas rinsed out of the plumules by three washes in the Trisbuffer. The plumules were then “teased” apart using for-ceps and a scalpel to release intact cells from the tissue, andthe released cells were immobilized on a poly-Lys-coatedmicroscope slide. The cells were washed in the Tris bufferplus 0.01% (w/v) Triton X-100 for 30 min, treated with 2 nHCl for 20 min, and washed twice for 5 min each time inthe Tris buffer. The slides were blocked in 1% (w/v) bovineserum albumin in Tris buffer for 20 min followed by a shortwash in a buffer (Tris buffer, pH 7.8, 0.1% [w/v] bovineserum albumin, and 0.1% [w/v] Triton X-100). All subse-quent washes of the sample used this buffer. The sampleswere then incubated with mouse anti-BrdU (Amersham)overnight at 4°C. The samples were washed three times for5 min each prior to incubation with secondary antibody.Goat anti-mouse IgG conjugated to Bodipy was used di-luted 1:100. This incubation was performed at room tem-perature for 2 h. The samples were then washed three timesfor 5 min each followed by a 10-min incubation with 49,6-diamino-phenylindole (to allow identification of nuclei).One final wash was followed by the addition of citifluor-glycerol and overlay of the sample with a coverslip. Thecells were then scored for BrdU incorporation by fluores-cence of the secondary antibody, and this number wascompared with the total number of nuclei to obtain theS-phase index. A minimum of 500 nuclei were counted perdata point, and each individual experiment was performedtwice. The Student’s t test was used to determine statisticalsignificance between light treatments at each time point.





Determination of M-Phase Index

The irradiated plumules were excised from the seedlingsand immersed in Farmer’s fixative (3 parts ethanol:1 partglacial acetic acid) for 30 to 45 min. The plumules were thenwashed for 5 min in 100% (w/v) ethanol and stored in 70%(w/v) ethanol at 4°C. The plumules were washed in tapwater immediately prior to Feulgen staining. The Feulgenstain binds to DNA, and therefore allows visualization ofnuclei. The stained plumules were then prepared for mi-totic index counting.

The outer leaves were removed (very little mitosis couldbe detected in this tissue) and discarded, whereas the ILand A were separated. The As were teased apart as de-scribed in the S-phase determination. The IL were teasedapart in a small volume of water contained within a mi-crofuge tube using a Teflon grinder. The cells and teasedtissue were then placed on a microscope slide, excess waterwas evaporated on a slide warmer, and the samples werecovered with a coverslip. The samples were viewed undera light microscope. The number of cells undergoing celldivision was counted by identifying cells in prophase,metaphase, anaphase, or telophase, and this number wascompared with the total number of nuclei to obtain themitotic index. A minimum of 500 nuclei were counted perdata point. The Student’s t test was used to determinestatistical significance between light treatments at eachtime point.

RNA Isolation and Northern-Blot Analysis

Plumules from the G, R, R/FR, or FR light-irradiatedplants were frozen in liquid nitrogen and ground in amicrofuge tube with a Teflon grinder. The resulting frozenpowder was used for total RNA isolation according to themanufacturer’s instructions using TRIzol (Gibco-BRL,Cleveland). The RNA was electrophoretically separated ina 1.2% (w/v) agarose gel containing 6% (w/v) formalde-hyde, blotted by capillary action onto Zeta-Probe Nylonmembrane (Bio-Rad, Hercules, CA) or Hybond-N1 mem-brane (Amersham), irradiated with short-wavelength UVfor 2 min to crosslink the RNA to the membrane, andincubated in prehybridization solution (0.25 m Na2HPO4,pH 7.2, 1 mm EDTA, and 7% [w/v] SDS). Hybridization toa 32P-labeled probe was performed at 65°C or 60°C for 16 h.The washing procedure was carried out under high strin-gency conditions described in the supplier’s protocol. Thewet membrane was wrapped with plastic wrap and placedin a phosphoImager (model no. 445S1, Molecular Dynam-ics, Sunnyvale, CA). The different probes were used withtissue from same RNA isolation.

Probes Used in Northern Blots

Radioactive probes were made from full-length cDNAsfor the various genes using a Decaprime II kit (Ambion,Austin, TX) according to the manufacturer’s instructions.The pea nucleolin probe was made from the cDNA of cloneNA481–5 (Tong et al., 1997). The pea cyclin B and PCNAcDNAs were obtained from Dr. Hitoshi Mori (Shimizu and

Mori, 1998). The pea histone H2A was obtained from Dr.Joel Stafstrom (Devitt and Stafstrom, 1995).

Yeast Strains and Their Growth

The nsr1 mutant (WYL353) was obtained from Dr. TeriMelese (Lee et al., 1992). WYL353 (MATa ade2-1 can1-100ura3-1 leu2-3, 112 trp1-1 his3-11, 15 nsr1::HIS3) was trans-formed with pYES2 (Invitrogen, Carlsbad, CA), a yeastexpression vector containing the GAL1 promoter. WYL353was transformed with pYES2 alone or pYES2 containingthe full-length pea nucleolin cDNA. All transformed yeastwere grown at 30°C on either inducing media (5.7 g/Lyeast nitrogen base [Bio 101, Inc., La Jolla, CA], 0.77 g/LCSM-URA [Bio 101, Inc.] 10 mm potassium phosphate,0.1% [w/v] dextrose, 2% [w/v] Gal, and 5% [w/v] glyc-erol) or non-inducing media (5.7 g/L yeast nitrogen base[Bio 101, Inc.], 0.77 g/L CSM-URA, 10 mm potassium phos-phate, and 2% [w/v] dextrose).

For the liquid growth complementation experiments,WYL353 containing either pYES2 alone or pYES2 with thepea nucleolin cDNA was transferred from noninducingplates and grown in 5 mL of noninducing media overnight(each of these experiments was done in triplicate). Equalamounts of each of these yeast strains, as judged by OD600,were then transferred to another 5 mL of pre-warmednoninducing media and allowed to grow to early logphase. Equal amounts of each of these cultures, as judgedby OD600, were then transferred to inducing media andgrown to saturation. To monitor the growth rates in liquidinducing media, OD600 readings were taken hourly.

Polysome and Total RNA Isolation from Yeast

Yeast polysomes were isolated by growing cells to mid-log (A600 5 approximately 0.5) phase and then addingcycloheximide to a final concentration of 200 mg/mL. Thecultures with cycloheximide were placed on ice for 30 min,and then spun at 4,000g for 5 min. These cells were frozenat 270°C. The frozen cells were resuspended in 1 volumeof lysis buffer (10 mm Tris, pH 7.5, 50 mm KCl, 8 mmMgCl2, 6 mm 2-mercaptoethanol, and 200 mg/mL cyclohex-imide) and broken open by vortexing with glass beads. Thesupernatant was collected and spun 2 3 10 min in a mi-crofuge. This supernatant was collected and quantified byreading A260. Continuous Suc gradients from 7% to 47%were prepared in lysis buffer, and the equivalent of 10absorbance units were loaded per gradient. The sampleswere centrifuged at 40,000 rpm in an ultracentrifuge (SW-40, Beckman, Fullerton, CA) for 2.5 h. The gradients wereplaced in an ISCO 640 density fractionator and the poly-some profiles were produced by reading A254.

Total RNA from yeast in liquid culture was isolated bycentrifuging the cultures in a table-top centrifuge, pouringoff the supernatant, and resuspending the pellet in theleftover media. The pellets were frozen in liquid nitrogenand ground in a microfuge tube with a Teflon grinder. Theresulting frozen powder was used for total RNA isolationaccording to the manufacturer’s instructions using TRIzol

Reichler et al.




(Gibco-BRL). The total RNA was separated on a 1.2% (w/v)agarose gel, and the relative densitometry of the large andsmall rRNA subunits was determined by digitizing the gelimage on a gel reader (AlphaImager 2000, Alpha Innotech,San Leandro, CA). The densitometry of the large and smallsubunits was calculated as a percentage for each subunit ofthe total densitometry for both subunits. The relative den-sitometry of the large subunit was used to measurechanges in rRNA levels that occurred when expression ofthe vector was induced. Three independent transformantswere used along with three different experiments to gen-erate statistical data. The Student’s t test was used todetermine significance between samples.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Hitoshi Mori for thepea cyclin B and PCNA cDNAs and Dr. Joel Stafstrom forthe pea histone H2A cDNA. We would also like to thankDr. Arlen Johnson and George Kallstrom for assistance inperforming the polysome profiles, and Dorcena Deutsch,Yulin Zhang, William Hanson, and Adam Molofsky forvaluable research assistance.

Received January 12, 2000; modified March 23, 2000; ac-cepted August 3, 2000.

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