Plant Physiol. (1 996) 11 2: 953-963

Lhcb Transcription 1s Coordinated with Cell Size and Chlorophyll Accumulation'

Studies on Fluorescence-Activated, Cell-Sorter-Purified Single Cells from Wild-Type and immutans Arabidopsis fhdiana

LeAnn Meehan, Kristi Harkins, Joanne Chory, and Steve Rodermel*

Department of Botany (L.M., S.R.), and Cell Sorting and Hybridoma Facility (K.H.), lowa State University, Ames, lowa 5001 1 ; and Plant Biology Laboratory, The Salk Institute, San Diego, California 921 38 (J.C.)

To study the mechanisms that integrate pigment and chlorophyll a/b-binding apoprotein biosynthesis during light-harvesting com- plex II assembly, we have examined P-glucuronidase (CUS) enzyme activities, chlorophyll contents, and cell sizes i n fluorescence- activated, cell-sorting-separated single cells from transgenic Arabi- dopsis thaliana wild-type and immufans variegation mutant plants that express an Lhcb (photosystem II chlorophyll a/b-binding polypeptide gene)/CUS promoter fusion. We found that CUS ac- tivities are positively correlated with chlorophyll content and cell size i n green cells from the control and immufans plants, indicating that Lhcb gene transcription i s coordinated with cell size in this species. Compared with the control plants, however, chlorophyll production i s enhanced in the green cells of immufans; this may represent part of a strategy to maximize photosynthesis in the green sectors to compensate for a lack of photosynthesis in the white sectors of the mutant. Lhcb transcription i s significantly higher in pure-white cells of the transgenic immufans plants than in pure- white cells from norflurazon-treated, photooxidized A. fhaliana leaves. This suggests that immufans partially uncouples Lhcb tran- scription from i ts normal dependence on chlorophyll accumulation and chloroplast development. We conclude that immufans may play a role in regulating Lhcb transcription, and may be a key component in the signal transduction pathways that control chloroplast biogenesis.

LHCII is composed of five distinct pigment proteins (reviewed by Thornber et al., 1993). The apoproteins, col- lectively termed CAB proteins, are coded by a small family of nuclear Lhcb (PSII CAB polypeptide) genes (Jansson et al., 1992) and imported into the organelle posttranslation- ally (reviewed by Keegstra et al., 1989). The carotenoid and

This work was supported by grants from the Department of Energy (DE-FG02-92ER20147) and the Iowa State Biotechnology Council to S.R., and by grants from the Department of Energy (DE-FG03-89ER13993) and National Science Foundation (MCB9206331) to J.C. This is journal paper no. J-16879 of the Iowa Agricultura1 Experiment Station (Ames, IA), project no. 2987, and it is supported by Hatch Act and State of Iowa funds.

* Corresponding author; e-mail rodermel8iastate.edu; fax 1-515-294-1337.

chlorophyll pigments that bind CAB are synthesized in the chloroplast by posttranslationally imported nuclear gene products (reviewed by Bartley and Scolnik, 1995; von Wettstein et al., 1995). It is thought that pigment binding occurs concurrently with or shortly after apoprotein inte- gration in the thylakoid membrane (Paulsen et al., 1993; Cohen et al., 1995; Kuttkat et al., 1995; Plumley and Schmidt, 1995).

The molecular mechanisms that coordinate CAB apopro- tein and pigment biosynthesis are not well understood, but integrative elements include light and phytohormones, as well as factors associated with circadian responses and the developmental state of the chloroplast (Mullet, 1988; Tay- lor, 1989; Thompson and White, 1991; Susek and Chory, 1992; von Wettstein et al., 1995). Analyses of mutants with apoprotein or pigment deficiencies have established that pigment cofactors, especially chlorophyll b, are required for the stabilization of many, but not all, CAB apoproteins in the thylakoid membrane (Somerville, 1986; Flachmann and Kiihlbrandt, 1995; KrÓ1 et al., 1995; Plumley and Schmidt, 1995; Preiss and Thornber, 1995; von Wettstein et al., 1995). This indicates that posttranslational mechanisms play a central role in coordinating pigment and CAB apo- protein biosynthesis. Although a positive correlation be- tween chlorophyll content and Lkcb mRNA accumulation has been noted in a number of studies (reviewed by Thompson and White, 1991), the question of whether the coordination of pigment and CAB accumulation is achieved, in part, at the leve1 of Lhcb transcription initiation has not been answered.

Variegation mutants constitute one prominent class of pigment-deficient mutants that have not yet been exploited for studies on LHCII biogenesis. We have been studying the immutans (im) variegation mutant of Arabidopsis thaliana as a tool to gain insight into chloroplast developmental processes (Wetzel et al., 1994). im has green-and-white- sectored leaves due to the action of a nuclear recessive

Abbreviations: CAB, chlorophyll alb binding; FACS, fluores- cence-activated cell sorting; FDA, fluorescein diacetate; LHCII, light-harvesting complex of PSII; MU, 4-methylumbelliferone; PDS, phytoene desaturase; PWTOF, pulse width time-of-flight.

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954 Meehan et al. Plant Physiol. Vol. 112, 1996

gene, and the extent of white-sector formation in the mu-tant is conditioned by light, temperature, and developmen-tal signals (Redei, 1963, 1967a, 1967b, 1975; Robbelen, 1968;Wetzel et al., 1994). Whereas the green sectors of im containphenotypically normal chloroplasts, the white sectors con-tain abnormal plastids devoid of pigments and lamellarstructures. The white sectors also accumulate phytoene, anoncolored C40 carotenoid intermediate, which indicatesthat im affects the activity of PDS, which converts phytoeneto f-carotene (Bartley et al., 1991; Pecker et al., 1992). How-ever, im is not the structural gene for PDS (Wetzel et al.,1994).

Molecular analyses have revealed that Lhcb mRNAs ac-cumulate to significant levels (approximately 20% of nor-mal) in white sectors of im (Wetzel et al., 1994). This standsin contrast to photooxidized, white-leaf tissues of A. thali-ana, in which Lhcb mRNAs are undetectable (Susek et al.,1993). However, one factor complicating the im studiesis that the white sectors of im are sprinkled with smallislands of green cells (Fig. 1A), which makes dissectionof visually white tissue difficult. In addition, some cellsin the white sectors are heteroplastidic and contain normalas well as abnormal chloroplasts, i.e. the mutation is "plas-tid autonomous" (Wetzel et al., 1994). Consequently, thequestion arose whether Lhcb mRNA accumulation is re-stricted to heteroplastidic cells in the im white sectors, orwhether Lhcb mRNAs are also abundant in homoplas-tidic (pure white) cells of these sectors.

The peculiar nature of pigment accumulation in immu-tans, coupled with our observations of Lhcb expression inthe white-leaf tissues of the mutant, suggested to us thatim might provide insight into the more general mecha-nisms that coordinate Lhcb expression and chlorophyllaccumulation might be instructive. As a first step towardunderstanding these mechanisms, the present experimentshad three main objectives: (a) to determine whether Lhcbtranscription is coordinated with chlorophyll contentin individual green mesophyll cells of mature wild-typeleaves, and if so, whether this coordination is perturbed ingreen-leaf cells of the mutant; (b) to examine the influenceof cell size on Lhcb transcription and chlorophyll accumu-lation; and (c) to ascertain whether significant Lhcb tran-scription occurs in pure-white (homoplastidic) cells of thewhite im sectors, or whether it is restricted to chlorophyll-containing (heteroplastidic) cells of these sectors.

To address these objectives, we took advantage of FACS(reviewed by Fox and Galbraith, 1990) to obtain homoge-neous populations of green and pure-white mesophyll pro-toplasts from transgenic immutans plants that expressan Lhcb promoter fused to the GUS reporter gene (Jeffersonet al., 1987). Transgenic Columbia ecotype plants thatexpress the promoter fusion were used as controls, andtreatment of these plants with norflurazon, which inhibitsthe PDS step of carotenoid biosynthesis (Sandmann et al.,1989; Chamovitz et al., 1991), provided a source of white,photooxidized leaf cells. After sorting, the sizes andchlorophyll contents of purified, individual cells from thevarious plants were determined, and GUS activities weremeasured to obtain estimates of Lhcb promoter activity.

Figure 1. im Lhcb/G\JS plant (A) and norflurazon-treated control(pOCA107-2) plant (B).

MATERIALS AND METHODS

Generation of Transgenic Plants and Plant Growth

pOCA107 is a Ti plasmid derivative in which 950 bp of aLhcbl*2 (C4B3) promoter from Arabidopsis thaliana (An,1987) drives the transcription of two different reportergenes: the uidA gene for GUS (Jefferson et al., 1987) and thehph (hygromycin phosphotransferase) gene (Susek et al.,1993). pOCA107 also contains between its left and rightT-DNA borders a transcriptional fusion between the cau-liflower mosaic virus 35S promoter and the nptll (kanamy-cin resistance) gene. pOCA107-2 is a transformed line of www.plantphysiol.orgon August 21, 2020 - Published by Downloaded from

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Coordination of Lhcb Transcription and Cell Size 955

A. thaliana that contains a single copy of the pOCAl07 insertion (Susek et al., 1993).

To generate a transgenic immutans line that expresses the LhcblGUS fusion, we crossed pOCA107-2 with the s p o t t y allele of im (Wetzel et al., 1994). The resulting F, progeny were self-fertilized, and homozygous im plants were iden- tified in the F, by growth under 30 pE m-’ spl continuous white light, which is optimal for im expression. Histochem- ical GUS assays (Jefferson et al., 1987) were performed on leaf samples of the F, im/ im seedlings, and a line was identified that gave rise in succeeding generations to 100% GUS-positive progeny plants. This line (designated im Lhcb/GUS) was used in subsequent studies. We have never observed segregation of non-GUS plants from this line.

A11 plants were maintained in potting soil under 30 pE of continuous white light using a mixture of m-2 s-7

fluorescent and incandescent bulbs. Some of the plants were watered with 5 X 10p6 M norflurazon (SAN 9789, Sandoz, Basel, Switzerland).

Protoplast lsolation

The protoplast isolation procedure was modified from Damm and Willmitzer (1988) and Harkins et al. (1990). Total leaves (1-2 g) from 4-week-old plants were sliced into approximately 1-mm strips, plasmolyzed in 0.5 M mannitol for 1 h, then transferred to a clean Petri dish containing 0.4 M mannitol, 8 mM CaCl,, and 1% cellulase (Calbiochem) (pH 5.5). A near-complete digestion occurred within 14 to 16 h in the dark at 22°C. Additional protoplasts were released from the tissue by 15 min of shaking at 40 rpm on a rotary shaker. The protoplasts were filtered through a 55-pm nylon mesh (Tetko, Briarcliff Manor, NY), and the filtrate was diluted 1:1 with W5 buffer (154 mM NaC1, 125 mM CaCl, 2H,O, 5 mM KC1, and 5 mM Glc, pH 5.6-6.0) (Negrutiu et al., 1987). After centrifugation for 5 min (ap- proximately 40g, or 200 rpm in a clinical centrifuge), the pellet was washed with W5 buffer and centrifuged again. W5 buffer was used because we found that white cells do not pellet well in mannitol-based solutions. The final pellet was resuspended in 25% SUC, overlaid with an equal vol- ume of W5 buffer, and centrifuged again. Enriched popu- lations of green protoplasts (which tend to accumulate at the Suc/W5 interface) and white protoplasts (which are less buoyant and found throughout the W5 phase) were collected by pipette and diluted with 0.5 M mannitol. The protoplasts were immediately subjected to flow cytometry to obtain pure populations of white and green protoplasts. A11 manipulations (after digestion) were performed on ice or at 4°C.

Samples of the protoplasts were stained for 12 min with 100 ng/mL FDA (Rotman and Papermaster, 1966; Larkin, 1976; Galbraith et al., 1988; Galbraith, 1990). Protoplast viability was monitored by fluorescence microscopy. FDA is hydrolyzed within the cell to produce fluorescein, which emits green fluorescence (emission maximum of 520 nm) when excited by 457-nm light; nonviable protoplasts do not fluoresce. In the present experiments, viability was nearly 100% for both white and green protoplasts. This leve1 of viability was maintained throughout the course of the sub-

sequent flow-sorting and GUS activity assays (described below).

Flow Cytometry

Protoplasts were sorted as previously described (Har- kins and Galbraith, 1987; Galbraith et al., 1988) on a flow cytometer (EPICS 572, Coulter, Hialeah, FL) containing a 200-pm tip and sorbitol/Mes/ CaC1, (pH 6.0) sheath fluid (Somerville et al., 1981). Chlorophyll-deficient protoplasts were detected by staining with FDA for 12 min (100 ng/ mL). To reduce background fluorescence, the stained sam- ples were centrifuged for 5 min at approximately 50g (300 rpm in a clinical centrifuge), then resuspended in 0.5 M

mannitol. Protoplasts that contained chlorophyll were de- tected by their red autofluorescence (mostly above 620 nm) following excitation at 457 nm of laser light (Galbraith, 1990).

To eliminate instrument-related variation, the cytometer was standardized prior to each experiment. First, a specific lot of fluorescent beads was placed at channel 154 on the red fluorescence histogram to allow daily comparisons of chlorophyll-based red fluorescence in intact protoplasts. This standardization was not used for chlorophyll deter- mination of individual protoplasts in the light-scatter ex- periments; comparisons were made between chloroplasts from the control and im LhcblGUS plants on the same day, and the instrument was adjusted to optimize positioning of the peaks at midscale for integral red fluorescence.

As a second standardization, aniline blue-stained pollen from Bvoussonetia papyrifeva (mulberry, 13.8 pm in diame- ter), Ambvosia elateior (ragweed, 20.9 pm), and Cavya illi- noensis (pecan, 51.1 pm) were analyzed for PWTOF, which defines the pulse width of the fluorescence signal as an object passes through the laser beam and corresponds to a unit of size (Leary et al., 1979; Galbraith et al., 1988). The Carya pollen were adjusted to channel 121 on the PWTOF scale, and the peak channel position of each pollen type was then plotted against the known diameter of the pollen grains. A linear regression was calculated (v > 0.95) and used to convert the mean channel value to a size value (in pm). This method was used to standardize size measure- ments for both green and white protoplasts.

White-protoplast populations were selected by setting sort windows on areas of a two-parameter histogram (in- tegral red fluorescence based on chlorophyll autofluores- cence versus PWTOF of FDA-based peak green fluores- cence). Green-protoplast populations were selected by setting sort windows on a one-parameter histogram. This histogram compiles data on PWTOF peak red fluorescence based on chlorophyll autofluorcscence. Protoplast size cat- egories were selected within a population by placing sort windows around 20-channel increments on PWTOF histo- grams, i.e. histograms of FDA-based peak green fluores- cence for white protoplasts and histograms of chlorophyll autofluorescence-based peak red fluorescence for green protoplasts. Chlorophyll fluorescence can be quantified by measuring the integral red fluorescence for cells within a particular sort window.

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956 Meehan et al. Plant Physiol. Vol. 112, 1996

Fluorometric Assays of CUS Activity

Approximately 150 to 300 protoplasts were sorted into individual wells of a 96-well microtiter plate; each well served as a separate data point. Protoplast numbers were confirmed by light microscopy prior to the assay for GUS activity (Jefferson et al., 1987). After adding 4-methyl um- belliferyl glucuronide, samples were taken at hourly inter- vals for a total of 4 h; the kinetics of MU production were linear with respect to time for pigmented and nonpig- mented protoplast populations (data not shown). The rate of MU production (in pmol MU produced h-l proto- plast-l) was calculated from the slope of each line. The duration of these experiments (commencing with proto- plast isolation and terminating with GUS activity measure- ments) was significantly shorter than estimates of GUS protein turnover in A. thaliana (40-50 h) (Jefferson et al., 1987; Harkins et al., 1990; Susek et al., 1993). Nontransgenic white protoplasts from immutans had no detectable GUS activity.

Chloroplast Size Comparisons

Protoplasts were isolated as described above. The cells were mechanically lysed by repeated flushing through a syringe containing a 20-pm, 100% polyamide nylon mesh (Tetko), and the resulting chloroplast preparations were subjected to flow cytometry. The relative chloroplast size correlates with the amount of forward light scatter detected in front of the laser beam (Melamed et al., 1990).

RESULTS

Ceneration of im Lhcb/GUS Transgenic Plants

A. thaliana may contain the full complement of Lhca and Lhcb genes for the CAB polypeptides of LHCI and LHCII,

respectively (Jansson et al., 1992; McGrath et al., 1992). The CAB polypeptides of LHCII are coded by three groups of genes (termed types 1, 2, and 3), and there are at least five Lhcbl (type 1) genes in A. thaliana (Jansson et al., 1992). One of these is Lhcbl*2, previously termed AB180 (Leutwiler et al., 1986) and CAB3 (Susek et al., 1993). Lhcbl*2 transcrip- tion is dependent on light, is restricted to chloroplast- containing cells, and is subject to circadian controls (An, 1987; Karlin-Neumann et al., 1988; Mitra et al., 1989; Millar and Kay, 1991; Susek et al., 1993). Lhcbl*2 also gives rise to a large fraction of the total steady-state Llzc mRNA pool in A. thaliana leaves.

pOCA107-2 is a transformed A. thaliana line (Susek et al., 1993) that contains a single copy of the full-length Lhcbl*2 promoter fused to the uidA structural gene coding for GUS (Jefferson et al., 1987). The LhcblGLIS transgene has the proper transcriptional start site in pOCA107-2, and it is expressed in this line in a light-, tissue-, and chloroplast- dependent manner, similar to the endogenous A. thaliana Lhcbl*2 gene (Susek et al., 1993). To assess Lhcb promoter activity in immutans, we crossed im with pOCA107-2. Self- ing of the F, yielded a line that was homozygous for both im and the Lhcbl*2/ GUS transcriptional fusion (see "Ma- terials and Methods"). The copy number and intactness of the transgene in this line were verified by genomic South- ern blot analysis (data not shown). This line (designated im LhcblGUS) is shown in Figure 1A; it and the parenta1 pOCA107-2 line (the control) were used in the analyses described below.

4 B

Flow Cytometry of Creen Protoplasts from Control Leaves

Protoplasts were isolated from pOCA107-2 leaves and subjected to PWTOF analysis using chlorophyll auto- fluorescence as the signal source (Fig. 2A). To eliminate

Cell Size O 50 1 O0 150 200 250

Chlorophyll Content

Figure 2. Flow cytometric purification of leaf protoplasts from control (pOCA107-2) plants. A, PWTOF cell-size analysis using chlorophyll autofluorescence (peak red fluorescence) as the source of signals. PWTOF is a measure of cell size (diameter). A, B, and C are sort windows. B, Integral red fluorescence (channel number) of the A, B, and C sort windows. Integral red fluorescence is a form of signal processing that provides a measure of the total chlorophyll autofluorescence (chlorophyll content) per protoplast (Galbraith et al., 1988).

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Coordination of Lhcb Transcription and Cell Size 957

instrument-related variation, the cytometer was stan- dardized prior to each experiment (detailed in ”Materials and Methods”). PWTOF is a time-duration measurement, and Galbraith et al. (1988) have shown that PWTOF is linearly correlated with protoplast size (diameter). The mean PWTOF of three different protoplast preparations from control plants was 90.1 2 8.4. When PWTOF values were converted to size values (in pm) using pollen of known diameters, the mean size of the three different protoplast populations was 34.9 ? 4.3 pm.

Because the yield of protoplasts from a single size class (a single PWTOF channel) is too small to obtain reliable esti- mates of GUS activity, we selected three size categories of protoplasts by setting sort windows around channels 41 to 61, 71 to 91, and 111 to 131 (A, B, and C in Fig. 2A). The integral red fluorescence of the protoplasts in each of these windows is illustrated in Figure 2B. These data were de- rived from the one-dimensional PWTOF histogram shown in Figure 2A, and they represent the chlorophyll autofluo- rescence profiles of the protoplasts in each of the three windows. The mean integral red fluorescence (chlorophyll autofluorescence) and PWTOF (cell size) values of three different protoplast preparations (Table I) are illustrated graphically in Figure 3A. Galbraith et al. (1988) have dem- onstrated that chlorophyll autofluorescence is an accurate measure of protoplast chlorophyll content, and thus, the data show that cell size is linearly related to chlorophyll content in protoplasts from the control leaves.

After obtaining information about cell sizes and chloro- phyll contents, the green protoplasts in each of the three windows were sorted into microtiter plates and assayed for GUS activities. Three different protoplast preparations from the control leaves were examined and replicate GUS assays were performed on the protoplasts in each window. The kinetics of MU production were linear during the 4-h sampling period for each assay (Y > 0.95). The data from these experiments are summarized in Table I. Figure 3B shows that the mean GUS activities of the control proto- plasts are linearly related to their size.

Flow Cytometry of Green immufans Protoplasts

Protoplasts were isolated from the leaves of the im Lhcbl GUS transgenic line and subjected to flow cytometry. Sort- ing of green protoplasts was based on PWTOF analysis of

chlorophyll autofluorescence (as in Fig. 2A). The popula- tion distributions of five independent protoplast prepara- tions and their mean PWTOF (89.6 2 3.9 or 34.6 +- 2.0 pm) were nearly identical to the control protoplasts in Figure 2A (data not shown). Sort windows were set using the same three size categories as in Figure 2A, and the mean PWTOF and integral red fluorescence values of the proto- plasts in each of the three windows were determined (Ta- ble I). Figure 3A shows that there is a linear relationship between PWTOF and integral red fluorescence (chloro- phyll content) in the green protoplasts from the im Lhcbl GUS leaves. However, the slope of the line that describes this relationship is steeper than that of the controls; we calculate that larger green protoplasts from im Lhcbl GUS leaves have approximately 25% more chlorophyll than sim- ilarly sized green protoplasts from control leaves. These increases are statistically significant.

GUS activity assays were next conducted on protoplasts from the three sort windows. As with the green protoplasts from control plants, the kinetics of MU production were linear during the 4 h of each experiment. Table I summa- rizes the results of up to 16 separate GUS determinations (per window) on the five different protoplast preparations. In Figure 38 the mean sizes of the protoplasts in each of the three windows are plotted against their mean GUS activi- ties. Variance analysis of these data revealed that devia- tions of the im LhcblGUS line from the control are due to chance, and that the slopes of the two lines and their deviation from linearity are not significantly different (data not shown). We reached similar conclusions from an ex- periment in which we performed GUS assays on proto- plasts from five (rather than three) sort windows of the green im LkcblGUS leaf protoplasts (data not shown).

Collectively, the data in Figure 3, A and B, indicate that GUS enzyme activities are linearly related to cell size in both the im Lhcb/GUS and control green-leaf protoplasts. In addi- tion, for a protoplast of a known size, GUS activities appear to be similar in the two sets of plants. Harkins et al. (1990) have demonstrated that GUS enzyme activities provide an accurate measure of Lhcb, rbcS (for the nuclear-encoded small subunit of ribulose bisphosphate carboxylase), and cauliflower mo- saic virus 35s promoter activities in flow-sorted protoplasts from transgenic tobacco leaves that express promoter-GUS fusions; this was true for photosynthetic as well as for non-

Table I. Cell sizes, chlorophyll contents, and CUS activities of green protoplasts n, Number of assays.

Protoplast Channels Isolations Sorted

Plants Mean PWTOF Mean Integral Red

(chlorophyll content)

CUS Activitf Mean (n)

Mean Cell Size Fluorescence

n Fm Control 3 41-61 (A) 54.1 2 0.3 16.2 % 0.1 24.1 i 3.5 1.19 2 0.38 (9)

(pOCA107-2) 3 71-91 (B) 81.2 2 0.6 30.2 i 0.29 58.2 i 4.3 1.63 5 0.26 (6) 3 111-131 (C) 11 8.9 i 0.5 49.7 ? 0.3 122.3 t 0.8 2.40 5 0.53 (6)

im L hcb/G U S 5 41-61 (A) 52.9 t 0.5 15.6 i 0.23 28.1 t 4.9 1.29 i 0.53 (14) 5 71-91 (B) 81.4 2 0.1 30.4 i 0.05 74.2 2 9.7 2.1 1 i 0.59 (1 6) 5 111-131 (C) 120.0 2 0.5 50.4 5 0.23 152.7 k 14.5 2.59 i 0.60 (14)

a Mean activity, Mean pmol of MU produced h-’ protoplast-’; r s 0.95.

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958 Meehan et al. Plant Physiol. Vol. 1 1 2, 1996

2w

Y

12 150

e B

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3 50

O O P

A * Contrnl T ........ ........ 0 im LhcblGUS

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Control B ........ ........ im LhcbfGUS 0 3.5 -

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Cell Size Figure 3. Sizes, chlorophyll contents, and CUS activities of green protoplasts from control and im LhcblGUS plants. The data are from Table I (means 2 SD). A, Mean cell size (cell diameter, PWTOF) versus mean chlorophyll content (integral red fluorescence or chan- nel number) . 6, Mean cell size (cell diameter, PWTOF) versus mean CUS activity (pmol M U produced h-’ protoplast-’).

photosynthetic (white) protoplasts. Thus, unless GUS mRNA and / or protein stabilities are markedly different in different- sized cells of A. thaliana, our data suggest that Lhcb promoter activities are correlated with cell size in im Lhcb/GUS and control A. thaliana plants.

Flow Cytometry of White immutans Protoplasts

The sorting of pure-white leaf protoplasts from the im Lhcb/ GUS transgenic line was based on PWTOF analysis of the green fluorescence signals of FDA. FDA-based peak green fluorescence has been shown to give accurate sizing of nonpigmented tobacco leaf protoplasts (Galbraith et al., 1988; Harkins et al., 1990). As with the green protoplast preparations, the cytometer was standardized prior to each experiment (see ”Materials and Methods”). The colorless protoplasts from im LhcblGUS leaves are represented by the boxed population of cells (Fig. 4A). Phase and fluores- cence microscopy confirmed that the protoplasts in this

population are not heteroplastidic and do not contain chlo- roplasts (data not shown). The population distribution of this sort window, determined by PWTOF analysis of FDA- based peak green fluorescence, is illustrated in Figure 4B. The mean PWTOF of three different protoplast prepara- tions was 63.8 ? 3.7 or 21.2 ? 2.0 pm. This value is significantly smaller than that of green leaf protoplasts from either the control (34.9 pm) or im LhcblGUS plants (34.6 pm) discussed above. This amounts to a reduction of approximately 39% in cell diameter or 77% in cell volume.

Sort windows were placed around the protoplasts in the two-dimensional histogram in Figure 4B and GUS assays were performed on the gated populations (Table 11). A total of four different protoplast isolations were assayed, and the kinetics of MU production were linear during the 4 h of the assay for each preparation. Table I1 reveals that there is a low amount of GUS activity per cell, and that the amount of activity does not vary with cell size. Nontransgenic white protoplasts from immutans had no detectable GUS activity (data not shown).

Flow Cytometry of Protoplasts from Norflurazon-Treated Leaves

Although normal leaves contain some nonpigmented cells, the vast bulk of the white cells in the box shown in Figure 4A are nonpigmented mesophyll cells from the im Lhcb/ GUS white sectors (data not shown). To determine whether Lhcb transcription in these cells resembles that in photooxidized cells, we treated control pOCA107-2 plants with norflurazon. When watered with this herbi- cide, newly emerged leaves are pure white (Fig. 1B). Figure 5A shows the results when protoplasts were iso- lated from norflurazon-treated leaves and sorted accord- ing to cell size (PWTOF) and chlorophyll autofluores- cence. Figure 5B shows the population distribution of nonpigmented protoplasts from this sort, as revealed by PWTOF analysis of FDA-based peak green fluorescence of the protoplasts illustrated in the box in Figure 5A. These cells were 100% viable throughout the duration of the experiment, and phase and fluorescence microscopy revealed that they lacked pigmented plastids (data not shown). The population distributions and mean PWTOF (68.1 2 0.3, or 23.5 ’. 0.15 pm) of two separate protoplast preparations were very similar to the pure-white proto- plasts from the im LhcblGUS plants. However, the levels of GUS activity in these protoplasts were appreciably lower (approximately 4-fold) than in the pure-white im Lhcbl GUS protoplasts (Table 11); these differences were statistically significant.

Chloroplast Sizes

The population distributions of chloroplasts from leaves of the control pOCA107-2 plants and from green leaf sec- tors of the im LhcblGUS plants were determined by for- ward light scatter. We found that chloroplast size distribu- tions were similar in both sets of plants (data not shown), confirming earlier phase and electron microscopic obser- vations (Wetzel et al., 1994). The mean chlorophyll contents

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Coordination of Lhcb Transcription and Cell Size 959

B

A

Cell Size Cell Size

Figure 4. Purification of white protoplasts from im LhcblGUS leaves. A, Biparametric analysis of the emission of red fluorescence (integral red fluorescence, chlorophyll content) and PWTOF (protoplast diameter or cell size) based on peak green fluorescence, using FDA fluorochromasia as the signal source. The box in A is the sort window for white protoplasts. B, PWTOF analysis of protoplasts in the box in A, based on peak green fluorescence.

(chlorophyll autofluorescence) of the chloroplasts from the control and im LhcblGUS plants were also similar (data not shown). These data indicate that the green cells of the mutant leaves are not detectably affected by alterations in chloroplast size or chlorophyll content per chloroplast.

DlSCUSSlON

Lhcb Transcription is Coordinated with Cell Size and Chlorophyll Accumulation

It is well established that chloroplast number, chloro- phyll content, and photosynthetic protein accumulation are positively correlated with cell size (Boffey et al., 1979; Ellis and Leech, 1985; Pyke and Leech, 1987, 1991; Pyke et al., 1994). However, whether controls at the leve1 of transcrip- tion initiation play a major role in regulating protein abun- dance in cells of different sizes is poorly understood. Our

examination of flow-sorted protoplasts from transgenic control (pOCA107-2) and immutans (im LhcblGUS) green- leaf sectors revealed that there is a positive, linear correla- tion between Lhcb transcription (as monitored by GUS activities), chlorophyll content, and cell size in both sets of plants. This not only reinforces the notion that Lhcb expres- sion and chlorophyll biosynthesis are normally highly co- ordinated events, but also suggests that this coordination may be achieved, at least in part, by factors relating to cell size.

Our conclusions rest on two major assumptions. The first is that the chlorophyll contents and sizes of individual protoplasts can be ascertained by FACS. This has been demonstrated by Galbraith and co-workers (Galbraith et al., 1988; Galbraith, 1990). The second assumption is that GUS enzyme activities provide an accurate measure of in vivo promoter activities. This notion forms the theoretical

Table II. Cell sizes and GUS activities of white protoplasts n, Number of assays.

Plants Protoplast Channels Mean Isolations Sorted PWTOF

Mean Cell Size

CUS Activitya Mean (n )

n Pm

im LhcblGUS 3 41-71 59.8 2 6.5 19.2 Z 3.3 0.27 2 0.06 (4) 2 61-81 69.1 ? 0.8 24.0 2 0.4 0.28 t 0.04 (6) 2 81-1 11 90.7 -C 7.0 35.3 ? 3.7 0.32 2 0.07 (5)

Control (norflurazon) 2 15-1 88 68.1 t 0.3 23.4 i 0.15 0.08 -C 0.01 (3) a Mean activity, Mean pmol of MU produced h-' protoplast-'; r 2 0.95.

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Plant Physiol. Vol. 11 2, 1996

50 100 ' ' 150 ' ' 200 ' ' 250 '

Cell Size Figure 5 . Purification of leaf protoplasts from norflurazon-treated control (pOCA107-2) leaves. A, Biparametric analysis of the emission of red fluorescence (chlorophyll content) and PWTOF (protoplast diameter or cell size) based on peak green fluorescence. The box i n A i s the sort window for white protoplasts. B, PWTOF analysis of protoplasts in the box in A, based on peak green fluorescence.

underpinning of a large number of plant studies that have used the GUS reporter system to examine various promot- ers in transgenic plants or in transient expression assays. A critical assumption of many of these experiments is that rates of GUS mRNA stability, GUS mRNA translation, and GUS protein turnover are similar in the different tissue or cell types under study. This may not be true in all cases (e.g. Hensgens et al., 1992). In the present study we made 'the assumption that these parameters were similar in the green and white mesophyll cells of control and immutans leaves, and that they did not vary appreciably as a function of cell size.

There are severa1 lines of evidence suggesting that these are reasonable assumptions. Susek et al. (1993) used the 'pOCA107-2 GUS-fusion construct (the control in the present experiments) to assess Lhcbl*2 transcription in green versus white, norflurazon-treated A. thaliana leaves and observed that GUS activities are high in green leaves and barely detectable in white leaves. These findings mir- rored the results of in vitro run-on transcription experi- ments showing high levels of Lkcb transcription in nuclei from green cells and a lack of detectable Lhcb transcription in nuclei from the white cells. Nuclear run-on transcrip- tion assays are thought to provide a measure of transcrip- tion elongation by already engaged RNA polymerases (Gallagher and Ellis, 1982). Second, the GUS activities in the present experiments parallel Lhcb steady-state mRNA levels in the various tissue-types: (a) Lhcbl*2 mRNA levels are high in green leaves but nondetectable in white, norflurazon-treated leaves (Susek et al., 1993; data not shown), reflecting the GUS activities in Tables I and 11; (b) Lhcb mRNA abundances are similar in green leaf tissues from wild type and immutans, reflecting the GUS

activities in Table I; and (c) the white-leaf sectors of im have approximately 20% of the Lkcb mRNA levels found in green sectors (Wetzel et al., 1994), again reflecting the GUS activities in Tables I and 11. Third, Harkins et al. (1990) demonstrated that GUS activities in flow-sorted photosyn- thetic and nonphotosynthetic tobacco protoplasts that ex- press rbcS and Lhcb promoter/GUS fusions are directly correlated with the transcription rates of the endogenous genes, as monitored by transient expression assays. And fourth, it has been estimated that GUS protein turnover is approximately 50 to 60 h in A. thaliana (Susek et al., 1993), mirroring results from tobacco in which GUS activities were measured after treatment of green and white flow- sorted protoplasts with cycloheximide (Harkins et al., 1990). In both types of cells, turnover rates of approxi- mately 50 h were observed. Unfortunately, a direct exam- ination of GUS mRNA pool sizes and/or GUS mRNA turnover rates would be technically challenging, especially for cells from the im white tissues, since such analyses would require isolating a large number of protoplasts. Nevertheless, the four lines of evidence mentioned above support the notion that the GUS activities in our experi- ments accurately reflect in vivo Lhcb promoter activities.

Another objection to our experiments that could be raised concerns the observation that A. thaliana leaf cells are endoreduplicated and have ploidy levels ranging from 2C to 32C, with levels greater than 4C predominating in cells from older leaves (Galbraith et al., 1991). Consequently, it might be argued that transcription rates on a per gene basis do not change as a function of cell size, but that larger cells have higher GUS activities because they have higher ploidy levels and more Lhcb genes. To challenge this proposal, we used leaf tissues from young plants (cells with mainly 2C

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Coordination of Lhcb Transcription and Cell Size 961

and 4C amounts) and observed via 4’,6-diamidino-2- phenylindole staining that there was no correlation be- tween ploidy leve1 and cell size in green protoplasts from either the control or immutans plants (data not shown).

A final point worth considering is that there is a circa- dian component to Lkcb transcription in A. tkaliana (Millar and Kay, 1991). This component is not reflected in the GUS activities in the present experiments because the transgenic control and im plants were maintained under conditions of continuous illumination, i.e. when circadian controls are minimal if not nonexistent.

Our conclusion that Lkcb transcription is directly corre- lated with cell size and chlorophyll content suggests that Lhcb transcription may be sensitive to factors related to cell size (or vice versa). One possibility is that the transcription of nuclear genes for chloroplast components is modulated by chloroplast numbers, which increase with cell size in both the control and im LkcblGUS plants (data not shown). This may occur via the elusive “plastid signal” (reviewed by Taylor, 1989; Susek and Chory, 1992) or by some other feedback mechanism relating to chloroplast metabolism. Consistent with this hypothesis, Huang et al. (1994) re- ported that the transcription of nuclear genes for respira- tory proteins is influenced by mitochondrial numbers dur- ing tobacco flower development. In this context it will be of interest to determine whether the transcription of other photosynthetic genes, as well as nonphotosynthetic genes, is influenced by cell size.

The Creen-Leaf Cells of immufans Have Enhanced Levels of Chlorophyll

Although green cells in the im LkcblGUS plants have normal size distributions and GUS activities, chlorophyll levels are enhanced up to 25% in larger cells of the mutant. This conclusion rests on the assumption that variation is minimal between the mutant and control lines. Genetic variation was minimized by crossing the control (pOCA107-2), which is in a Columbia background, with immutans, which is also in a Columbia background, to obtain the transgenic im LkcblGUS plants. We also mini- mized variation by maintaining the transgenic control and im lines under identical growth conditions, and by collect- ing protoplasts from pooled leaf samples from 4-week-old plants of both lines. Under these conditions, both sets of plants had about the same number of leaves.

The enhanced chlorophyll per unit volume in the larger im cells raises the question of whether these cells have more chloroplasts per unit volume, and hence whether im affects, either directly or indirectly, the chloroplast division pro- cess. The forward light-scatter experiments suggested that this may be the case, because chloroplasts from the control and im LhcblGUS leaves were found to have the same size distributions and chlorophyll contents, despite the higher- than-normal amounts of chlorophyll in the mutant. Al- though this question needs to be examined more directly, the elevated chlorophyll production in im suggests that the mutant employs a strategy to maximize growth that in- volves an increase in light-harvesting capacity and photo- synthesis in the green-leaf sectors to compensate for a lack

of photosynthesis in the white-leaf sectors. This may be a physiological response, e.g. involving nitrogen allocation, and / or a response of the green cells to signals generated by the white cells, perhaps signaling their metabolic status.

The “Plastid Signal” and the Primary Lesion in immutans

The white sectors of immutans accumulate phytoene, in- dicating that the IM product controls, either directly or indirectly, the activity of PDS (Wetzel et al., 1994). If this is a direct effect, then it might be anticipated that cells in the white tissues contain photobleached plastids as a conse- quence of the blockage in carotenoid biosynthesis; in the absence of colored carotenoids, oxygen radicals generated by nonquenched, triplet-state chlorophyll lead to the pho- tooxidation of plastid contents (reviewed by Oelmüller, 1989; Taylor, 1989; Susek and Chory, 1992). Although we have ruled out the possibility that IM is the structural gene for PDS, other direct effects are still possible. For example, IM could be a factor required for PDS stability in the thylakoid membrane, a factor required for its activity, or a transcription or translation factor required for its synthesis. An alternative hypothesis is that phytoene buildup is a secondary effect of the im mutation and that chloroplast development is blocked at an early stage, generating plas- tids that are nonpigmented, but not photooxidized.

In the present experiments we found that Lhcb transcrip- tion is barely detectable in photooxidized A. thulianu leaf tissues, confirming earlier results on whole-leaf tissues (Susek et al., 1993). Such observations are consistent with the “plastid signal” hypothesis that the transcription of some nuclear genes for chloroplast proteins is dependent on a signal that requires a developing or mature chloro- plast either for its generation or transmission (reviewed by Taylor, 1989; Susek and Chory, 1992; Hess et al., 1994). If this hypothesis is correct, our data and those of Susek et al. (1993) suggest that the dependence of Lkcb transcription on the plastid signal is nearly complete in A. tkaliana. In con- trast, we observed that GUS activities in pure-white (ho- moplastidic) cells of the im Lkcb/GUS plants are approxi- mately 20% of those in green cells from either the im LkcblGUS or control plants, and 3- to 4-fold higher than in white cells from norflurazon-treated leaves. This indicates that the pure-white im cells are capable of appreciable Lkcb transcription (compared with photooxidized cells), and that Lkcb mRNA accumulation in the white sectors is not likely limited to chlorophyll-containing (heteroplastidic) cells in these sectors.

One possibility is that immutans partially uncouples the requirement for the plastid signal from Lkcb expression. For example, it may allow more production of the signal. An alternative explanation is that the putative plastid sig- na1 is absent or nearly so in the im. white tissues, but another mechanism partially bypasses the requirement for this signal. Regardless of the precise mechanism, the fact that Lkcb transcription is partially derepressed in the white im cells suggests that im may be a component of one or more signal transduction chains that control Lkcb transcrip- tion and chloroplast development. To clarify the primary

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962 Meehan et al. Plant Physiol. Vol. 1 1 2, 1996

mode of immutans action, we are using map-based methods to clone the gene.

The Effects of Photooxidation May Not Be Limited to the Plastid Compartment

Previous analyses have suggested that the effects of plas- tid photooxidation are limited to the plastid compartment and have little impact on cell and leaf developmental pro- cesses (reviewed by Oelmiiller, 1989; Taylor, 1989; Susek and Chory, 1992). We have systematically examined the sizes of cells f rom norflurazon-treated tissues, and our results clearly show that the cells in these tissues are smaller than normal. Compared with the green protoplasts from either the control or im LhcblGUS lines, the white protoplasts f rom norflurazon-treated leaves have average reductions of approximately 35% i n cell diameter (average PWTOF values), o r nearly 75% reductions i n cell volume. W e do not know whether the reductions i n cell size are a consequence of plastid photooxidation, or alternatively, to another, unknown affect of norflurazon treatment. What- ever the mechanism, the immutans mutation appears to mimic this response because the size distributions a n d mean sizes of white im Lhcb / GUS leaf protoplasts are iden- tical to those of the protoplasts from norflurazon-treated leaves.

ACKNOWLEDCMENTS

The authors wish to express their appreciation to John Mullet (Texas A&M University) for critica1 review of the manuscript, and to David Cox (Department of Statistics, Iowa State University) and Marc Anderson (Department of Botany, Iowa State University) for help with the statistical analyses.

Received May 13, 1996; accepted August 7, 1996. Copyright Clearance Center: 0032-0889/96/ 112/0953/11.

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