detection of spatially- and stage-specific proteins in extracts … · and enough mse adde tod...

Development 103, 665-674 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

665

Detection of spatially- and stage-specific proteins in extracts from single

embryos of the domesticated carrot

R. H. RACUSEN1 and F. M. SCfflAVONE

Plant Development Laboratory, Department of Botany, University of Maryland, College Park, MD 20742 USA

Summary

Single embryos, representing each of four distinctmorphological stages, were selected from cultures ofthe domesticated carrot for analysis of total [3SS]meth-ionine-labelled proteins. Following exposure to radio-label for 12 to 18 h, embryos were individually dis-rupted in a 3 mm diameter, precisely-matched, plasticmortar and pestle. Radiolabelled proteins extractedby this procedure were separated by two-dimensionalelectrophoresis procedures, consisting of isoelectricfocusing in 1 mm tubes, followed by SDS-PAGE in asmall slab gel. Comparisons of autoradiographs ofthese gels revealed that the levels of a number ofproteins were modulated during the conversion ofdisordered callus cells into maturing embryos. Inaddition, miniature surgical techniques were used to

separate the apex (cotyledon end) from the base (rootend) of late-stage embryos, for extraction of proteinsand analysis of spatial differences in protein distri-bution. About five proteins in extracts from eachsection were observed to be synthesized at differentrates in the two halves, indicating that there aremolecular correlates for early polarized growth.About half of the proteins, whose appearances wereunique to apical and basal sections of embryos, werealso observed to fluctuate in comparisons of autoradio-graphs of two-dimensional protein separations fromembryos at different developmental stages.

Key words: somatic embryo, carrot, two-dimensionalelectrophoresis.

Introduction

In contrast to the conspicuous molecular changes thataccompany developmental transformations in otherwell-characterized systems, such as Caenorhabditis(Johnson & Hirsh, 1979; Sulston et al. 1983), Dictyo-stelium (Alton & Lodish, 1977; Barklis & Lodish,1983) and sea urchin (Davidson et al. 1982; Brand-horst et al. 1983), the present literature on geneexpression in somatic embryos of carrot suggests thatperhaps only 1-2 % of the proteins detected on two-dimensional electrophoretic gels appear or disappearwhen one compares extracts of disorganized cellularclumps (callus) and whole embryos (Sung & Oki-moto, 1981, 1983). This paucity of detectable shifts intemporal gene expression has fostered the notion thatthe majority of genetic events that regulate carrotembryogenesis may occur in advance of visiblechanges in morphology; the changes in form beinghypothetically coordinated by physical or metabolicfactors that do not require new gene expression (Sunget al. 1984). Evidence for the direct involvement of

such mechanisms in development is lacking; however,measurements of electric fields around carrot em-bryos (Brawley et al. 1984), which appear to bealigned with the axis of embryo elongation, indicatethat nonmolecular modes of information transferclearly exist in these organisms.

An alternative explanation for the absence of asizeable number of stage-specific proteins in theearlier studies is that the design of these experimentsmay have militated against the detection of novelpolypeptides. For example, examinations of totalproteins contained in whole-organism extracts weretypically conducted as comparisons between undiffer-entiated cells and a mixed population of variousstages of embryos. Such an experimental designpresumes that callus cells will uniformly exhibit aprotein complement typical for disorganized growthand that, once initiated, all embryos will displayproteins characteristic of ongoing differentiation. Itdoes not provide for the possibility that groups ofundifferentiated cells may synthesize overlappingportions of the total protein complement necessary

666 R. H. Racusen and F. M. Schiavone

for ordered growth, nor is it possible to be certain thatembryos, which may have aborted in development,have not reverted to the production of some callus-type proteins. Further, the use of mixtures of em-bryos to prepare extracts precludes comparisons be-tween the conventional embryo stages, which arepresently defined on the basis of anatomic features.

Some of these problems have been alleviated bythe extraction of proteins from embryos that havebeen sorted by stage following successive passesthrough graded sieves (Giuliano et al. 1983). Thisrefinement has permitted the characterization of aspecific antibiotic resistance (Pitto et al. 1985) and thecloning of three cDNAs which are purportedly corre-lated with transitions between embryo stages (Choi etal. 1987). The criteria for separating embryos in thesestudies, however, continues to rely on rather rudi-mentary differences in embryo shape; thus, there isno direct assurance that the mixture of organismssustains comparable homogeneity at the molecularlevel. Considering the impressive lower limits ofdetection which contemporary procedures in molecu-lar biology are capable of delivering, even small,unsuspected amounts of contaminating materialcould produce misleading results.

In this paper, we report a reexamination of stage-specific differences in gene expression during carrotembryo development by comparing two-dimensionalgel separations of total proteins extracted from singleembryos. To ensure that each embryo was accuratelycategorized by morphological stage, we followed atwo-phase selection protocol. From subcultures ofembryos that had been sorted into size ranges bypassage through screens, we manually selected rep-resentatives according to a schedule of geometricmeasurements that we found in earlier work (Schia-vone & Cooke, 1985) and which provides moreprecise demarcations between adjacent stages. Sincewe also wished to explore the possibility that certaingenes might be expressed in a tissue-specific manner,we analysed extracts of portions of single embryosthat had been removed by microsurgery.

Materials and methods

Carrot cell cultureCells, originally derived from young hypocotyls of Daucuscarota L. cv. Danvers, were maintained in shaking 125 mlsuspension cultures at 25°C in dim room light. The me-dium, traditionally used for growing these cells in anundifferentiated state (Murashige & Skoog, 1962), con-tained 5/jM-2,4-dichlorophenoxyacetic acid (2,4-D) andwas changed at weekly intervals. Embryo formation wasinitiated by procedures detailed in Schiavone & Cooke(1985), in which the cells were transferred to the samemedium without 2,4-D (MSE medium). Embryo cultures,

8-10 days old, were passed through first a 380pm sieve andthen a 117um sieve. In this procedure, heart- and torpedo-stage embryos are captured on the 117/an sieve, whileoblong- and globular-stage embryos and a small amount ofundifferentiated cells were rinsed through. Heart- andtorpedo-stage embryos were transferred to a dry Petri dishand enough MSE added to dilute the embryos to approxi-mately 50 embryos ml"1. From this dish, single embryoswere selected with a Pasteur pipette while being viewedunder a dissecting microscope at xlO.

Selection and transfer of individual embryos andcallusTo determine the stage of a particular embryo, we madecamera-lucida drawings of each embryo, tracing the em-bryo's periphery using methods and criteria developed bySchiavone & Cooke (1985). Briefly, any embryo whoseoutline also corresponded to a complete circle was termed aglobular-stage embryo. Embryos that had undergone polar-ization along the future root-shoot axis (thus having anaxial length greater than the embryo width), but stillmaintaining a smooth apical end were considered to be inthe oblong stage. Heart-stage embryos, like the oblong-stage, are clearly longer than wide, but are in the process ofcotyledon formation. These embryos have an apical endwhich is not part of a circle, but contains two prominentcotyledonary bulges at the apex. Since we were unable toprovide a clear morphological distinction between heart-and torpedo-stage embryos in the earlier studies, wedenned torpedo-stage embryos as those cotyledon-bearingembryos greater than 400/jm in axial length. Callus wasmaintained in medium containing 2,4-D and two or threeclumps of these undifferentiated cells, ranging from 50 to100/im were utilized for protein extracts. Embryos andcallus were transferred to 200/il of fresh MSE medium inwells of an Elisa plate for use in the surgical and proteinextraction procedures that followed.

Application of radiolabel, surgery and tissueextractionA. 5Ou\ droplet of sterile medium was placed in a shallowparafilm well, which had been formed by pressing a sterilestrip of this pliable material with a gloved index fingeragainst the 0-5 cm square openings of a 0-5 cm thick plasticgrid. With a 5x7cm section of the grid, it was possible toform 16 of these shallow wells, such that each was separatedon all sides by unused cells. There were several advantagesin using these multiwell plates. First, the hydrophobicnature of parafilm kept the droplet spherical so thatsubsequent microlitre additions and withdrawals could beperformed with minimal wetting of the well or the transfertip. Second, the entire grid was nested in a covered plastictray containing a few ml of water, which slowed evaporationof the droplets during labelling procedures. Third, the gridcould be placed under a dissecting microscope for removalor addition of embryos to the droplets. Because thedroplets protruded above the sides of the well and tended toremain centred in the depression, it was relatively easy tomanoeuvre the pipette tip in from any angle to retrieve aparticular embryo. Finally, the parafilm surface could be

Proteins in carrot embryos 667

simply stripped and discarded following the labelling pro-cedure.

Individual embryos were sterilely transferred in 2/il ofmedium with a microlitre pipetting device to the 50 y\droplet in the shallow, parafilm wells. Generally 1/jl of L-[35S]methionine (Amersham; 50-lTBqmmol"1), consistingof 555 kBq total activity was added to 50^1 droplets,containing 1-4 embryos. The tray was then covered, placedin darkness and allowed to incubate for 18 h at 25 °C.Labelled embryos were washed by nonsterile transfer in a2fi\ volume to a fresh 50 jA droplet of culture medium in ashallow well identical to the ones used for labelling. Thisprocedure was repeated two more times, with the finaltransfer being made into an extraction buffer consisting of25 mM-Tris-HCl, pH7-8; lOOmM-KCl; 0-5mM-MgCl2;10 % Triton X-100; 200mM 2-mercaptoethanol and 0-lmM-phenylmethylsulphonyl fluoride. The number of counts inthe final wash was typically less than 0-1% of the totaladded.

To separate apical and basal sections for protein analysis,embryos, viewed at xlO under a dissecting microscope,were severed at the midpoint along the longitudinal axis,using a tiny scalpel that was fabricated according tomethods from Lowry & Passonneau (1972). The cuttingedge of this instrument is a shard of a double-edged razorblade about 0-5 mm in length, which was glued to the end of0-5 cm long, single toothbrush bristle. The other end of thebristle fibre was glued to a dissecting needle. For the cuttingoperation, selected embryos were transferred with a Pas-teur pipette in about 20 t̂l of medium to the bottom of asterile, 100mm Petri dish. We found that embryos could besectioned with a single, clean cut by first positioning theblade edge parallel to the bottom surface of the plastic dish.While maintaining the parallel orientation as closely aspossible, the blade was then positioned over an embryo atthe site where the cut was to be made, and the bladebrought straight down to separate the tissues.

Under a dissecting microscope at x25, individual washedembryos or sections were transferred in a 2^1 volume to thesurface of a 3 mm diameter, conically shaped, teflon pestle.The pestle with droplet was pushed into a tightly fitting,1 cm long, polypropylene mortar, the opposite end of whichwas formed to precisely mate with the conical pestle. Thetip of the conical end of the mortar possessed a 0-5 mmopening. Using moderate finger pressure and two or threerotations of the pestle, embryos were crushed between theclosely matched surfaces of this apparatus. The pestle wasthen slowly withdrawn and 3-8 y\ of additional cold extrac-tion buffer from a microlitre pipette were allowed to bedrawn in through the hole in the tip of the mortar. Theextract fluid of 5-10 jA was efficiently removed by lmincentrifugation in a cold Eppendorf centrifuge; fewer than0-1% of the total counts incorporated into embryosremained in the components of the extracting device.Extracts were sonicated for 2 min by placing the tips of theEppendorf tubes in a bath sonicator filled with ice water.The tubes were again centrifuged for 1 min and the extractsfrozen or analysed immediately.

Two-dimensional gel electrophoresisTo determine total [35S]methionine uptake into embryos,

protein extracts were occasionally added to 5 ml of ascintillation cocktail (Scintiverse II, Fisher Scientific) andcounted in a Beckman model LS 7000 scintillation counter.Without disturbing the pellet in the Eppendorf tube, 3-8 /ilof most extracts were loaded onto 6 cm long, 1 mm diam-eter, isoelectric focusing gels. The polyacrylamide-basedgel mixture has been described previously (O'Farrell, 1975)and was made with 2 % of 3/10, 4/6 and 5/7 ampholytes(Bio-lyte; Biorad, Richmond, CA, USA). The gels wereprefocused at 120 V for 1 h and were run with extracts for 12to 18 h at 120 V. Completed IEF runs were always followedimmediately by electrophoresis in the second dimension ona 8cm wide x 6cm long x 0-75 mm thick, 2% SDS, 10%polyacrylamide slab gel (Laemmli, 1970). The pH gradientformed in the focusing tube was determined by measuringthe pH of degassed solutions of 0-025 M-KCI, containingequilibrated, 0-5cm sections of a focused gel. The relativemolecular masses of separated proteins were determinedfrom MW standards (Bio-rad) that were loaded in aseparate lane, adjacent to the well containing the first-dimension tube gel. Completed SDS-polyacrylamide slabswere stained in Coomassie Blue solution, destained anddehydrated between cellophane sheets in a commercial geldrier. The dried gel was then sandwiched against KodakX-O MAT X-ray film in a standard cassette and exposed for3 to 10 days at -80°C. Comparisons of autoradiographsfrom two-dimensional gels of different embryo extractionswere done by side-by-side visual inspection. To standardizethis somewhat subjective procedure, we used a type of'constellation analysis' in which we drew interconnectinglines between suggestive groupings of spots on x3 photo-graphic enlargements of the autoradiograph films. Sincethere is a certain amount of variability in the degree ofseparation in each dimension on the gels and in the amountof radioactivity supplied from each extract (see resultssection on incorporation, below), we registered only thosespots where a similar change in intensity occurred in threeseparate gels of extracts from embryos and callus of thesame age and approximate size.

Results

Incorporation of radiolabelTable 1 shows total amount of [35S]methionine takenup by callus and embryos at different stages. Ingeneral it appeared that there was a direct relation-ship between the size (measured as axial length ortotal protein) of an embryo and the amount of labelincorporated. The transport of methionine was highlydependent on the presence of a carbon source in themedium; incubation of heart embryos in growthmedium lacking sucrose resulted in 95 % loss inaccumulation of the isotope (data not shown).

Two-dimensional gel electrophoresis of embryoextractsAnalysis of the total sulphur-containing proteins bythis technique revealed, in the more heavily loadedgels, about 200 spots, which are catalogued in Fig. 1.


21-5

Fig. 1. Two-dimensional gel electrophoresis of [35S]methionine-labelled polypeptides from a single torpedo-stage carrotembryo. The autoradiograph has been purposely overexposed to reveal proteins that were synthesized in loweramounts. All the spots that were reproducibly seen to appear in the course of running many such gels with extracts ofembryos were numbered with a single digit-single letter designation, starting from the high molecular weight, basiccorner of the slab gel. Since certain spots, which are apparent in other embryos, are not visible in autographs oftorpedo-stage proteins, the '+ ' sign is used to indicate their positions.


Table 1. Total incorporation of [SJmethionine into callus cells and somatic embryos of the domesticatedcarrot (means ± S.E.)

Somatic embryos

Callus Globular Oblong Heart Torpedo

Axial length (jim) NATotal protein (//g) 8-88 ± 103Total incorporation (cpm x 10*) 2-3 ± 0-4

113 ±50-83 ±0060-3±01

178 ±71-86 ±0-291-5 ± 0 1

430 ±185-91 ±0-982-5 ±0-2

899 ±578-88 ±1 003-6 ±0-2

About 50 of these proteins produced the most intensespots, and we estimated that these represented about90% of the protein extracted from the embryo, asfollows. First, we summed the areas of the darkerspots by tracing them on a tablet digitizer (JandelScientific, Sausilito, CA, USA), coupled to a micro-computer. We then compared the optical density ofone of the darker spots with a lighter one, and usingBeer's law deduced that the darker regions wereproduced from the conversion of about 10 times asmany silver grains in the film. Assuming that theremaining 10% of silver grains, reduced by isotopedecay, were equally divided between the 150 lighterspots would imply that individual spots detected onthis film comprised as little as 0-05 % of the totalprotein extracted from the embryo. This is about 25times higher than the apparent threshold for detec-tion that has been experimentally determined (John-son & Hirsh, 1979), which suggests that proteinanalysis by these methods may be extended down tothe extremely small extraction volumes used in ourassays without unacceptable losses in resolution.

Fig. 2 shows an example of a two-dimensional gelobtained with an extract of callus, containing about5 /ig total protein as determined by the Lowry method(Lowry et al. 1951). In this autoradiograph, threespots are highlighted with arrows which, using thecatalogue in Fig. 1, correspond to numbers 4d, 6u and7c. These proteins were produced at much lowerlevels following the transition to early stage embryos,but reappeared in certain later stage embryos. Asdescribed below, synthesis of these polypeptides alsoappeared to be restricted to basal regions of the laterstage embryos. Autoradiographs of two-dimensionalgels from single globular embryos were lighter inappearance owing, most likely, to the tiny size andlower radiolabel uptake of embryos at this stage(Fig. 3A). As a consequence, we felt that the loss of aparticular spot from these autoradiographs would notbe a reliable indicator of a decline in synthesis of aprotein. The oblong-stage autoradiograph showedthe appearance of three polypeptides that were not asactively synthesized in callus cells (Fig. 3B). Theseare highlighted with arrows and correspond to num-bers 2m, 3a and 6b. The appearance of a fourthprotein, which was incompletely resolved, is also

21-5 -

Fig. 2. Two-dimensional autoradiograph ofundifferentiated callus cells, containing about 5 /̂g totalprotein. The autoradiograph has been overexposed toreveal proteins that were synthesized in lower amounts.Three polypeptides, indicated with arrows, disappear insubsequent autoradiographs of protein extracted fromembryos (see results).

indicated (number 2a). Three other proteins (arrows)later declined in synthesis, either at the heart stage(number 4v), or the torpedo stage (numbers 3a and6b). In autoradiographs of heart stage gels, we notedthree proteins that exhibited an increase in intensityover those seen in the oblong-stage films (Fig. 3C).Two of these, which did not resolve into well-definedspots (numbers 2a and 2i), subsequently decreased inintensity in the torpedo stage. The appearance of theother enhanced protein (number 4d) apparentlybracketed the oblong stage, being found additionallyin callus and the base-section extracts of torpedoembryos. Two other proteins (numbers 6a and 6b)


i

B

Fig. 3. Two-dimensional autoradiographs of extracts of individual embryos from each of the four recognizable stages ofembryo development: (A) globular, (B) oblong, (C) heart and (D) torpedo. Autoradiographs of globular-stage embryoswere typically as light as the one shown here, perhaps due to the lower rate of radiolabel incorporation and smaller sizeof these embryos. Autoradiographs of separations of proteins from the other three stages are marked with arrows toindicate those proteins whose levels increased or decreased between adjacent stages (see Results). Vertical andhorizontal tick marks correspond to approximate molecular weights and pH shown in Figs 1 and 2.


were seen to be synthesized at much lower ratesfollowing the passage into the torpedo stage. Inaddition to these declines in synthesis of certainproteins, extracts of torpedo-stage embryos (Fig. 3D)showed the return of two proteins that were last seenin callus (number 6u) and oblong-stage embryos(number 4v). A small amount of synthesis of aprotein (6w), not seen in earlier stages, also appearedin torpedo-stage embryo extracts.

Gels from extracts of sectioned embryosDifferences in the spatial distribution of a number ofproteins were evident in autoradiographs of two-dimensional gels from apical and base extracts oftorpedo-stage embryos. In surgically bisected em-bryos, three proteins were restricted to the halfcontaining the apical (cotyledonary) region (Fig. 4A;numbers 4k, 7n and 7o) and six proteins weresynthesized in higher amounts in sections with thesuspensor (root/hypocotyl) pole (Fig. 4B; numbers4d, 4v, 7a, 7c, 8q and 9m). Interestingly, certain ofthese spatially distinct proteins were identical to thosewhose rates of synthesis were modulated duringtransitions between developmental stages. For in-stance, apical end protein, number 6a, first appearedin heart-stage embryos. Similarly, base end proteinnumber 7c appeared in callus, but was synthesized in

• * ~

lower amounts in all embryo stages until torpedo; andprotein number 4d, synthesized in callus, was absentin radiolabelled form in extracts of embryos until theheart stage. A summary of stage- and spatiallyspecific changes in protein synthesis are shown dia-grammatically in Fig. 5.

Discussion

Developmental processes such as plant embryogen-esis are undoubtedly coordinated by transfer ofinformation between different cells or between com-partments in individual cells. The passage of charac-teristic form between parent and offspring as aheritable trait implies that the ultimate store ofinstructions resides in the genome, but it is not certainif the successive expression of particular genes is themechanism whereby the temporal framework for adevelopmental transition is established. In certainsystems, for example, there is evidence that cellularlyderived electrical fields (reviewed in Jaffe & Nucci-telli, 1977), diffusing chemical 'morphogens'(reviewed in Meinhardt, 1982) or physical stresses ina cellular matrix (Lintilhac, 1984) may be the primaryeffecters of observed changes in shape.

In interpreting these, or other experiments, utiliz-ing sensitive techniques in protein or nucleic acid

B

Fig. 4. Two-dimensional autoradiographs of extracts from an apex (A) and a base (B) of a surgically bisected torpedo-stage embryo. Arrows indicate polypeptides that were unique to the arrays of proteins from each tissue section. Verticaland horizontal tick marks correspond to approximate molecular weights and pH shown in Figs 1 and 2.


I E F -

7

xicr3

92-5-

66-2

45-

21-5

C,G,O. H

o5k

H

C,G,O,H,Ta

8oO

Fig. 5. Diagrammatic summary of the changes in the levels of synthesis of polypeptides extracted from carrot callus andembryo tissues. This tracing of an autoradiograph of a two-dimensional electrophoretic separation of proteins fromcarrot cell extracts includes many of the more intense spots, but omits the lighter ones. All spots are numberedaccording to the system described in Fig. 1. The protein spots that were observed to change during embryodevelopment, or were found only in apex or base sections of embryos, are indicated by lines connecting them to letterdesignations of the stages, or positions, in which they were present. In this scheme, C, callus; G, globular; O, oblong;H, heart, T, torpedo; Ta, apex of torpedo; Tb, base of torpedo.

analyses to perform broad molecular comparisonsbetween organisms which differ in outward appear-ance, one must take care not to overextend thepostulates of gene activation, which were originallyput forth to explain the directional biasing of metab-olism through the synthesis of key enzymes. There-fore, we issue the following caveats in advance ofconsidering these findings. First, the detection ofproteins by the methods used in this investigation islimited to sulphur-containing proteins with isoelectricpoints between 4-5 and 7-5. Second, the differences in

intensities of spots on autoradiographs of electro-phoregrams only provide information about the rela-tive abundance of proteins synthesized during theapplication of label, and furnish no direct measure ofthe total abundance of any protein in the tissueextracts. Third, the search for stage- or tissue-specificchanges in polypeptide composition, following incor-poration of radiolabel, is prejudiced to identify onlythose events that are accompanied by appearance ofnew proteins; the possibility that a developmentaltransition might be cued by the degradation of an


existing polypeptide is not accommodated in theexperimental design. Finally, the identity and func-tion of proteins detected in two-dimensional gels areunknown, as is their role, if any, in promoting theprogression of morphogenesis.

The changes in the appearance of proteins in ourexamination of extracts of single embryos fall intothree general classes: (1) proteins that were observedin one or two stages of embryos, (2) proteins thatwere observed in callus cells and certain embryostages and (3) proteins that were apparently localizedto apical or basal portions of a sectioned embryo. Atthe level of detection in these experiments, we did notidentify any proteins that were unique to extract ofcallus cells. Of the 15 proteins that were determinedto be either stage- or tissue-specific, 9 were present incallus. Interestingly, 3 of these 9 proteins were notfound in autoradiographs of early embryo stages(globular, oblong and, in one case, heart) but re-appeared in extracts of basal portions of torpedo-stage embryos. These observations raise the possi-bility that the expression of certain genes might occurin a polarized fashion, at least in later stages ofembryo development. Whether they are similarlyexpressed with respect to position in earlier stages, orperhaps in regions of callus that are to become thesuspensor end of the embryo, are intriguing questionsthat may ultimately be approached by analysis ofextracts from surgically removed tissues fromyounger organisms.

Nine of the radiolabelled proteins that were foundin extracts of various stages of embryos were alsodetermined to be asymmetrically distributed intoapical or basal halves of sectioned torpedo embryos.Since the establishment of polarity is the pivotalmorphological event which signals the conversion toorganized growth, it is tempting to consider thepossibility that early, spatially polarized gene ex-pression gave rise to these protein distributions,which then might serve as molecular determinants ofthe ensuing polarized morphology. It is equally poss-ible, of course, that these protein differences simplyrepresent fundamental biochemical differences be-tween the cell types in the apical and base regions.Again, analyses of extracts from earlier stage em-bryos that have been surgically sectioned would benecessary to determine the onset of such differences,in turn reinforcing or repudiating the notion that theyhave causal significance.

The most provocative changes in newly synthesizedpolypeptides that we observed were ones in which aprotein appeared in one stage, was not detected inone or two following stages and then reappeared in aneven later stage. There were four examples of suchepisodic synthesis in our survey, three of which beganin the callus cells. Assuming it can be shown that

these gaps in the synthesis of proteins are usefulpreindicators of specific embryogenic transitions, itwill be important in future experiments to determinewhat happens to the level of these polypeptides whenthey are not being synthesized. If they are notdegraded during the stages when they are absent fromautoradiographs, then each ensuing period of syn-thesis would raise the total level, creating a step-wiseaccumulation of the polypeptide as embryosmatured. If, on the other hand, the protein isdegraded in the intervening stages of low synthesis,then an oscillation in the level of the peptide wouldoccur. Either of these two possibilities could, intheory, serve as an effective means of signalling a shiftin the activities of cells in the embryo.

Whatever may be their role in development, it isclear that the modulation in the levels of synthesis of anumber of polypeptides in carrot embryos is morecomplex than has been previously appreciated. Com-parisons between extracts of callus and extracts of amixture of embryos, as has been done previously,would not have permitted about 50 % of the stage-specific differences to be detected, leading us toconfirm the earlier conclusions that carrot embryo-genesis is accomplished with the addition of only afew new gene products. Microsurgery of individualembryos proved to be a useful addendum to the singleembryo extraction procedures; the combined meth-odologies provide a means for directly identifyingproteins that are spatially segregated into differentregions of a developing embryo.

The authors wish to thank Drs Gary R. Pasternack andFrank P. Kuhajda of the Johns Hopkins University, Schoolof Medicine, for the use of their laboratories and forguidance in some of the techniques.

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(Accepted 11 April 1988)

detection of spatially- and stage-specific proteins in extracts … · and enough mse adde tod...

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