a biochemical model for the initiation and maintenance of the … · key words: quiescent center,...

2825Development 121, 2825-2833 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

A biochemical model for the initiation and maintenance of the quiescent

center: implications for organization of root meristems

Nancy M. Kerk* and Lewis J. Feldman

Department of Plant Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720-3102, USA

*Author for correspondence (e-mail: [email protected])

A new hypothesis for the formation of the quiescent centeris presented. Reported data support a mechanism for theestablishment and maintenance of the quiescent center.The quiescent center is located at the most distal part ofthe root proper, the most terminal location in the rootproper on the path of polar transport from the shoot. Ofthe many substances polarly transported in the root, auxinis one of the best studied and has been shown to affect rootmeristem organization. In our mechanism, polar auxin isdirectly linked to quiescence through the action ofascorbate oxidase and ascorbic acid. Immunolocalizationof auxin in the root tip of Zea mays showed that auxin levelsin the quiescent center were high compared to the levels inthe immediately surrounding meristematic cells. Isolatedquiescent centers were shown to have high levels ofascorbate oxidase mRNA and ascorbate oxidase activityrelative to proximal meristem tissue. Exogenous auxincaused an increase in ascorbate oxidase mRNA levels andascorbate oxidase enzyme activity in cultured root tissue.Immunolocalization of ascorbate oxidase in Zea root tips

showed high levels of the protein in the quiescent centerrelative to surrounding cells. This is the first report of apositive marker and activity for the quiescent center. His-tochemical detection of ascorbic acid in Zea root tipsshowed that quiescent center cells have low or undetectablelevels of ascorbic acid, presumably due to the high levels ofascorbate oxidase in the quiescent center. As ascorbic acidis a compound known to be necessary for the transitionfrom G1 to S in the cell cycle, its low levels in the quiescentcenter may be directly responsible for holding these rarelydividing cells in the extended G1 state in which they aremainly found. We propose that our mechanism comple-ments published mathematical modeling of the anatomicalstructure of root apices, and further propose that thecontrol of relative growth rates in this focal region of theroot apex by this mechanism is a determining aspect in gen-erating anatomical patterning in the root apex.

Key words: quiescent center, auxin, ascorbic acid, Zea mays, rootdevelopment

SUMMARY

INTRODUCTIONApical meristems give rise to all tissue and organ systems ofthe postembryonic plant. Not only do they generate the cellsfrom which the plant is constructed, but apical meristems alsofunction as the organizing centers for postembryonic morpho-genesis. The evidence for this conclusion has emergedgradually from many different studies addressing the initiation,organization, maintenance and function of apical meristems,especially root apical meristems (Steeves and Sussex, 1989).

The organization of root meristems has been studied throughanalyses of cell lineages, based on histological sections.Because cell lineages, or files, converge at the root pole,Hanstein (1868) postulated that a few cells, located at the polecould serve as the initials for the root, and through high mitoticactivity, generate all the cells that make up the root. In an effortto define these cells precisely, Clowes (1953, 1954) performedsurgical experiments on Zea mays root apices and showed thatif these cells were damaged or removed, the remaining sur-rounding cells could directly reconstitute a complete apex. Thisled Clowes to conclude that the functional initials were actually

located peripherally to the very central cells, in a region of themeristem later named the proximal meristem (Feldman andTorrey, 1975). Clowes’s (1956) use of thymidine labelingshowed for the first time that the most central cells of themeristem actually divide infrequently, or not at all, and henamed this population of cells the quiescent center (QC).

Since its discovery, much work has contributed to the char-acterization of the QC. While it is believed to be a feature ofall angiosperm root apices, the most extensive analysis of theQC has been done on roots of maize, in which the QC canattain a size of 1000-1500 cells (Feldman and Torrey, 1976).Dolan et al. (1993) have shown that in Arabidopsis the QCcomprises only four central cells derived from the hypophysisand is surrounded by cells that act as the initials for the filesof cells that make up the root. Average cell cycle times withinthe QC of Zea are in the range of 170 hours. In contrast, thecell cycle time of the root cap initials, the most rapidly dividingcells in the root, is only 10-16 hours, and for the proximalmeristem, 18-25 hours (Clowes, 1961).

Cells within the QC can be distinguished from surrounding

2826 N. M. Kerk and L. J. Feldman

Fig. 1. Autoradiograph of a median longitudinal section of a maizeroot that had incorporated [3H]thymidine. The silver grain deposits inthe darkly colored nuclei indicate those cells that were undergoingDNA synthesis during the labeling period. Note the prominentquiescent center at the apex. Superimposed on the section is anoverlay indicating relative amounts of the elements of the proposedmechanism for maintenance of the quiescent center at locations inthe meristematic and quiescent regions in the root tip. Auxin andascorbate oxidase are at relatively high levels in the quiescent centerwhile levels of ascorbic acid are relatively low. (IAA, auxin; AAO,ascorbate oxidase; AA, ascorbic acid). Magnification, ×110.

Polar IAA

IAA AAO

AA

meristem cells by their fainter histochemical staining (Goyaland Pillai, 1986), lower RNA content (Clowes, 1972), lowerprotein content (Jensen, 1958), and low RNA polymeraseactivity (Fisher, 1968). In situ hybridizations with a 3H-labeledhistone gene probe and with [3H]polyuridylic acid (poly U) toroot tissue sections have revealed populations of unlabeledcells which correspond precisely to the area defined as thedeveloping QC in rice and Capsella for each probe respectively(Raghavan and Olmedilla, 1989; Raghavan, 1990).

Prior to discernible apical organization, cells at the pre-sumptive root pole (the future root meristem) show uniformlyhigh rates of mitosis, and stain densely with a variety of his-tochemical stains. Establishment and elaboration of a QCoccurs gradually and precedes stable histological patterning inthe meristem. From experiments using uptake of radiolabelledprecursors, Clowes (1978) concluded that establishment of theQC preceded all histogenic events except establishment of aroot cap meristem layer. More recently, using in situ hybridiz-ation of radiolabelled poly U to Capsella, Raghavan (1990)was able to show that very early during embryogenesis, beforeany root apical organization was evident, one cell and subse-quently several were clearly distinguished by their relative lackof bound probe. He then demonstrated that these unlabelledcells were the origin of the QC. His results, in an elegant way,supported previous hypotheses that the organization of apicalmeristems in roots follows the establishment and elaborationof the QC. Other work concerning the establishment of histo-logical patterning in developing lateral root primordia and inadventitious root primordia has also provided evidence for theestablishment of a QC prior to histological organization of aroot meristem (Rondet, 1961; Feldman, 1977).

Using microsurgical techniques, earlier workers had shownthat it was possible to excise defined regions of the root apicalmeristem in maize, including the QC itself (Feldman, 1977).Results of these efforts showed that roots are able to regener-ate a new apical meristem following surgical excisions, but thatprior to the initiation of distinctive histological zonation, a QCreformed, appearing initially as a group of mitotically rela-tively inactive cells surrounded by the rapidly dividing cellsremaining at the cut root stump (Feldman, 1977; Rost andJones, 1988). From this work it was also concluded that thereformation of a QC precedes and is requisite for organizationof root meristems.

Despite these many studies we do not have definitive infor-mation about the factors that initiate and maintain quiescencein these cells nor do we have a convincing functional role forthese cells. Past workers have suggested that the QC and the‘ultimate initials’ that it contains are equivalent to ‘stem’ cellsin animals (Barlow, 1978). Torrey (1972), and later Feldman(1975, 1979), proposed that the QC may be a site of hormonebiosynthesis in the root and as a consequence of theselocalized, enhanced metabolic processes these cells wereinhibited with regard to many other physiological activities.Other possible explanations for the quiescent state havefocused either on the supposed nutritional status of the QC oron the possibility that physical constraints prevent cells of theQC from dividing (Clowes, 1972).

In this paper we present a new hypothesis for the formationof the quiescent center and provide data that address the causeand maintenance of the QC. We have used the perspective ofexamining the QC with regard to its position in a whole plant

context. The QC is located at the most distal part of the rootproper, the most terminal location on the path of polar transportfrom the shoot. Of the many substances polarly transported inthe plant, auxin is one of the best studied and has been shownto affect root meristem organization. Here we provide adetailed mechanism linking polar auxin transport with theestablishment and maintenance of the QC (Fig. 1). In thismechanism polar auxin is directly linked to quiescence throughthe action of ascorbate oxidase and ascorbic acid. Briefly, wereport that auxin and ascorbate oxidase levels are high in theQC relative to surrounding cells and that the QC cells have lowor undetectable levels of ascorbic acid, a compound known tobe necessary for the transition from G1 to S in the cell cycle.Having discussed the mechanism imposing quiescence wediscuss the implications that this mechanism has for the estab-lishment of pattern at the root apex.

MATERIALS AND METHODS

Plant growth conditions and tissue collectionCorn caryopses (var. Merit, Asgrow Seed Co., Kalamazoo, MI) wereimbibed and germinated in the dark at 25˚C for 2 days. Tissue wascollected by surgical removal of the cap and excision of the QC,(Feldman and Torrey, 1976). In this cultivar, the QC is separated fromthe proximal meristem by a weak, thin-walled junction makingpossible routine, clean dissections of isolated QCs. QCs werecollected in a moist environment, quick frozen on dry ice and storedat −80˚C for extractions as described below. Approximately 2 mm ofthe remaining root stump, the proximal meristem region, was alsocollected and stored in this manner. Where mature root tissue wasused, 1 cm segments located approximately 1 cm behind the tip werecollected. For root tissue cultured with or without exogenous auxin,mature root segments (1 cm) were cultured on Murashige and Skoog(MS) medium, 3% sucrose, 0.8% agar in the presence of 0 or 1.0 mg/l

2827Maintenance of the quiescent center

of 2,4-D. Material was incubated in the dark at 25˚C and washarvested and collected as above after 2 and 4 days (Esaka et al.,1992).

Indoleacetic acid transportCaryopses were germinated as above and grown for 3 days. Roots weresevered at the base, below the scutellar node and the cut end was placedin a 0.5 ml microfuge tube containing half-strength MS medium,10−8 M nonradioactive indoleacetic acid (IAA) and [14C]IAA (1µCi/ml, specific activity = 4.8 Ci/mM) in 0.8% low melting tempera-ture agarose (Feldman, 1981). After 24 hours of transport at room tem-perature in the dark, roots were removed from tubes and squashedbetween plastic wrap and Whatman blotting paper. A glass plate wasplaced over the plastic wrap and pressure was applied squashing theroot firmly and uniformly onto the paper. Squashed roots were exposedto X-ray film for approximately 2 weeks.

RNA isolation, northern blotting and in situ hybridizationRNA was isolated from frozen tissues collected as described above.RNA was isolated from material using a modification of the methodof Puissant and Houdebine (1990). Approximately 150 individualfrozen QCs were ground in a small grinding tube in 25 µl guanidiniumbuffer on ice. 2.5 µl 2 M sodium acetate, pH 4.0, and 25 µl watersaturated phenol/chloroform was added. The solution was transferredto a microfuge tube, vortexed and centrifuged at 12,000 g for 10minutes at 4˚C. The upper phase was recovered and precipitated withisopropanol. The pellet was resuspended in 25 µl 4 M LiCl, then cen-trifuged at 3000 g for 10 minutes at 4˚C. The resulting pellet wasredissolved in Dep-treated H20, 0.1% SDS, extracted with an equalvolume of chloroform, and the aqueous phase was adjusted to 0.2 Msodium acetate, pH 5.0, and precipitated again with isopropanol. Theresulting pellet was resuspended in Dep-treated H2O, and RNA wasquantitated and used for northern blot analysis. RNA was extractedfrom proximal meristems and other root sources by the same methodexcept the volumes were scaled up to process the tissues which weremore easily collected in larger amounts.

RNA electrophoresis, blotting and hybridization were performedessentially as described previously (Maniatis et al., 1989). 5 or 10 µgof total RNA was electrophoresed in a 1% agarose gel containingformaldehyde and blotted to Nytran (Schleicher & Schuell). Equalloading of RNA was confirmed by ethidium bromide staining of thegel before transfer to the membrane. RNAs were probed with the nearfull length cDNA clone for cucumber AAO, pASO11 (Ohkawa et al.,1989). The probe was radiolabeled using random hexamer primingwith the Prime-a-Gene method (Promega).

In situ hybridizations were done according to the method ofJackson (1991). Maize root tips were hybridized with 35S-labeledsense and antisense riboprobes (synthesized with Ribo Probe kit,Stratagene) coding for elongation factor-α cloned from radish (Kerk,1990). Slides were hybridized overnight, washed in 2× SSC, 50%formamide at 50˚C, dried and exposed to Kodak NTB-2 emulsion.

Assay of ascorbic acid oxidase activity Ascorbic acid oxidase (AAO) activity was assayed by following thedecrease in the spectrophotometric absorbance of ascorbic acid overtime in the presence of proteins extracted from QCs, proximalmeristems, and other root tissues using the method of Oberbacher andVines (1963). Tissue homogenates were prepared from freshlycollected quiescent centers, proximal meristems and cultured rootsegments. Tissues were ground in 5 parts (w/v) 0.1 M potassiumphosphate buffer, pH 7.0, on ice and centrifuged at 10,000 g for 15minutes at 4˚C. 10 µl samples of these homogenates were added tothe reaction mixture containing 0.05 M potassium phosphate buffer(pH 7.0), 0.5 mM EDTA, and 0.15 mM L-ascorbic acid in a volumeof 1.0 ml (Esaka et al., 1988). One unit of activity was defined as theamount of enzyme which oxidizes 1.0 µmol of L-ascorbic acid perminute and was converted from the change in A265 at 25˚C over time

(Oberbacher and Vines 1963). Protein concentration was determinedusing the Bio-Rad protein assay and units of activity were normalizedto this amount.

Ascorbic acid localizationThis procedure uses the unique ability of ascorbic acid to reduce silvernitrate to silver in acidic conditions (Chayen, 1953). Root tips werequick-frozen in isopentane, cooled in a dry ice bath and dehydratedin several changes of absolute ethanol at −30˚C. The ethanol wasreplaced with toluene and the tissue infiltrated with paraffin andsectioned at 10 µm (Jensen, 1962). Ascorbic acid was localizedaccording to the methods of Jensen and Kavaljian (1956). Blackdeposits of metallic silver indicate regions where ascorbic acid ispresent. The control for this procedure is to expose the sectionedmaterial to a copper sulfate solution that oxidizes all the ascorbic acidto dehydroascorbic acid, which does not react with the silver nitrate.

ImmunolocalizationTissue for immunolocalization was fixed in freshly prepared 2%aqueous ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochlo-ride (EDC) (Sigma) on ice for 30 minutes under vacuum (Shi et al.,1993), followed by postfixation in 2.5% paraformaldehyde/0.25%glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, at 4˚C overnight.Binding of antibody (Ab) was carried out essentially as described byChichiricco et al. (1989) using alkaline phosphatase for detection. Themonoclonal antibody (mAb) to auxin has been shown to be specificto free auxin in Zea root tips (Shi et al., 1993). Several controls werecarried out to confirm specificity. The controls included: (1) omittingprefixation with EDC, (2) omitting incubation with the primary mAband (3) incubation with the mAb previously exposed to an excess ofauxin in incubation solution. The AAO antibody was a polyclonal Ab(Esaka et al., 1988) and was used for localizations in tissue sectionsas above, with the omission of the prefixation in EDC.

BrdU incorporationCorn caryopses were imbibed and germinated for 3 days as describedabove. Caryopses were then placed upon parafilm covered deep Petridishes such that the root extended through holes punched in the filminto solution contained in the dish. Solutions were either water or 0.1mM ascorbic acid both maintained at pH 5.9. Roots were incubatedat 25˚C in the dark for 24 hours with gentle agitation. Bromo-deoxyuridine (BrdU) was added to 10 µM and roots were incubatedfor a further 24 hours. Root tips were then excised, fixed and sectionedas described above.

Hydrolysis and immunofluorescent stainingA modification of the procedure recommended by BoehringerMannheim for use with their Anti-BrdU antibody was used. Sectionswere hydrolyzed in 1 N HCl for 1.5 hours at 37˚C, then neutralized byimmersion in 0.1 M borate buffer, pH 8.5, washed with PBS, andincubated with anti-BrdU antibody (Developmental StudiesHybridoma Bank, NICHD) for 2 hours. Slides were washed in PBSand incubated with rabbit anti-mouse IgG-FITC (Southern Biotech-nology Associates, Inc.) overnight in the dark at room temperature ina humidified chamber. Slides were washed and covered with a drop of50% glycerol, 0.15% N-propyl gallate in PBS and a coverslip. Thestained material was observed with a Zeiss Axiophot microscopeequipped with a Zeiss ZVS-47DEC video camera. Video frames fromthe ZVS-47DEC were digitized and displayed on a Macintosh PowerMac 8100/80AV and arranged using Adobe Photoshop v. 3.0 software.

RESULTS

Localization of auxinWe established that auxin accumulates in the region of the QC


Fig. 2. Autoradiograph of a primary maize seedling root after 24hours of incubating the basal end with [14C]IAA. Arrow shows theaccumulation of IAA in the root tip. Also note the distribution oflabel in the vascular tissue and developing lateral root primordia(small arrowheads). Bar, 1 cm.

by using the following two methods: (1) by demonstratingregions of accumulation of polarly transported [14C]IAA usingautoradiography and (2) immunolocalization of IAA to root tiptissue sections. The first approach allowed visualization of thepath of polar IAA transport in 3-day old maize seedling rootsusing [14C]IAA. Roots were severed from the hypocotyl andexposed to [14C]IAA at the cut surface. Transport was allowedto proceed for 24 hours and the roots were processed andexposed to X-ray film. Fig. 2 shows a representative root.Radioactivity was localized in the vascular tissue and in theroot tip. A characteristic feature was the low level of signal in

Fig. 3. Auxin localization in longitudinal sections of maize root apices. staining in region of the quiescent center. In addition, staining is intenseelements. B and C are controls. B treated as A but without incubation wexcess of auxin before incubation with tissue sections. Magnification ×9

the region behind the tip, the region of cell elongation. Signalcan also be seen in vascular traces leading to developing lateralroot primordia.

Higher resolution of auxin localization in the root tip wasobtained using a monoclonal antibody to auxin. This antibodyhas previously been shown to have high specificity for freeauxin (Shi et al., 1993). Fig. 3A shows the alkaline phos-phatase staining pattern indicating antibody binding to auxin.There was distinctive dark staining in the region correspond-ing to the quiescent center. The root cap also showed highlevels of auxin, as did the outer cortical cells of the rootproper. The vascular tissue showed significant staining aswell. The root cap meristem region between the QC and thecap had much less auxin as did the inner cortex and epidermis.Two controls are also shown. First was the pattern seen whentissue sections were treated as above but without the primaryantibody in the dilution buffer (Fig. 3B). There was littledetectable staining in the QC. The other control shows theresult of incubating the antibody with an excess of auxin insolution prior to exposure of the antibody to the tissue section(Fig. 3C). With this control, sites antigenic for auxin shouldbecome saturated prior to exposure to auxin in the tissuesections, and hence should not be able to bind auxin duringimmunolocalization. This pattern showed some generalizedbackground staining but the marked differential distributionseen with the antibody alone was not detectable. The stainingin the quiescent center, root cap, outer cortical cells andcentral cylinder was very reduced when the antibody was pre-treated with auxin.

Effect of auxin on ascorbate oxidase levelsAuxin has an effect on AAO levels in the root. AAO activity

(A) Section incubated with monoclonal antibody to auxin. Note dark in the root cap and outer cortical files and in maturing vascularith the primary antibody; C as A but antibody pretreated with a molar0.


Fig. 4. Effects of auxin (2,4-D) on ascorbate oxidase activity andexpression in cultured roots. (A) Ascorbate oxidase activity afterculture with (m) or without (v)2,4-D. (B) Northern blot of totalmRNA from root tissues cultured in the presence, +, or absence, −,of 2,4-D after 0, 2, and 4 days of culture. Note message level ishighest after 2 days of culture with auxin.

Fig. 5. Expression of ascorbate oxidase and p34cdc2 in variousroot tissues (Q, quiescent center; P, proximal meristem; R, matureroot tissue). (A) Ethidium bromide stained gel to show RNAloadings of the blotted gel (M = molecular mass (×10−3) markers).(B) Northern blot of gel hybridized with ascorbate oxidase cDNAprobe. (C) Same filter as in B, hybridized with p34cdc2 cDNAprobe.

Fig. 6. Characterization of the quiescent center region of maize root apices. (A) Immunolocalization of ascorbate oxidase in the root apex. Note the dark staining in thequiescent center. (B) In situ hybridization of elongation factor-α to the root apex. Notice theprobe does not bind strongly to the region of the quiescent center, and in the root cap, bindsonly to the root cap meristem. Magnification, ×90.

increases in tissue cultured with auxin.Roots cultured with auxin for 4 daysshowed a ten-fold increase in activitycompared to control roots (Fig. 4A).Northern blots of RNA prepared fromportions of the same root tissue as thatused for the activity assays showmessage levels of AAO peak at day 2and decrease by day 4 (Fig. 4B). Esakaet al. (1992) also observed this samemRNA profile in pumpkin fruit tissuecultured with auxin. These data provideevidence that culture with auxin resultsin an increased level of AAO mRNAand enzyme activity in root tissue, andthat a continued increase in the mRNAlevel does not underlie the increasinglevels of enzyme activity, since AAOactivity levels continue to increase eventhough mRNA levels decrease beforeday 4.

Ascorbate oxidase localization inthe quiescent centerThe following experiments examinedAAO mRNA and protein distribution,and AAO activity in the different regionsof the root. The cultivar of maize used forthis work has a slightly weakened cellwall zone between the QC region and the

proximal meristem, making it possible to remove the root capand collect individual QCs. Northern blots of total RNAprobed with a full-length cDNA for AAO show high levels ofmRNA in the QC and lower levels in the proximal meristemand mature root region (an ethidium bromide stained gel isshown as a control for equal loading of the blotted gel, Fig.5A,B). The same filter was reprobed with a cDNA for a cell


0.00

0.01

0.02

0.03

0.04

0.05

0.06

Uni

ts o

f A

AO

/mg

of t

otal

pro

tein

QuiescentCenter

ProximalMeristem

Fig. 7. Ascorbate oxidase activity in two regions of the root tip.

cycle gene, p34cdc2, (Colasanti, J. et al., 1988) to demonstratethe different levels of these two messages in the total mRNAextracted from these tissues (Fig. 5C). No detectable mRNAwas present in the QC, while high levels were detected in theproximal meristem where a high rate of cell division occurs.There is lower signal in the mature root as would be expectedin tissues with fewer dividing cells.

The distribution of AAO protein in these regions of the tipis similar to the pattern of mRNA distribution. AAO islocalized very distinctly in the QC but occurs at much lowerlevels in the proximal meristem (Fig. 6A). The root capmeristem region has relatively low levels of AAO protein.When this pattern is compared to the in situ hybridizationpattern of elongation factor-α, a translation factor that has beenshown to be a marker for cells undergoing active proteinsynthesis, the two patterns can be seen as the inverse of eachother (Fig. 6B). While elongation factor-α is at very low levels

A

C

Fig. 8. Histochemical localization of ascorbicacid in the root tip of maize. (A) Region of theproximal meristem; note black dots of silverdenoting ascorbic acid and the mitotic figureindicating this is a region of high mitoticactivity. (B) The control for A; notice mitoticfigures but the absence of black dots in thesection. (C) Section from the same root as A,but from the region of the quiescent center.Note the absence of black dots and mitoticfigures. (D) Low power view of a longitudinalsection indicating the regions from which Aand C were photographed (QC, quiescentcenter; C, mitotically active cortex).Magnification, (A-C) ×450; (D) ×80.

in the QC, root cap, central vascular cylinder and outer corticalcells, AAO protein is relatively high in these tissues. The AAOpattern is also very similar to the pattern of auxin distributionpreviously shown with the auxin antibody (compare with Fig.3A). Spectrophotometric assays of AAO activity in proteinextracts made from QCs and proximal meristems show that thisincreased level of AAO protein in the QC corresponds to muchhigher activity levels of AAO in the QC (Fig. 7). Thus we haveshown that the QC has high levels of AAO mRNA, AAOprotein and AAO activity, compared to the immediately sur-rounding cells of the proximal and root cap meristems.

Ascorbic acid localizationThe primary substrate for AAO is ascorbic acid, which isutilized in several metabolic processes, and has been reportedto be necessary for the transition from G1 to S in the cell cycleof several plants (Chinoy, 1984). Others have also suggestedthat ascorbic acid may have a regulatory role in cell prolifera-tion (Innocenti et al., 1989; Arrigoni et al., 1989; Liso et al.,1984; Citterio et al., 1994). Moreover, it is known that cells inthe quiescent center have extended G1 phases and divide rarely(Clowes, 1975). Localizations of ascorbic acid show it to beabsent or present at undetectable levels in quiescent center cells(Fig. 8). At high magnification, the proximal meristem regionshows orthogonal cell files with a clearly visible mitotic figure.The silver deposits that indicate the presence of ascorbic acidin these cells appear as black dots. Cells in the QC region aremuch less regularly shaped, show no mitotic figures andcontain no silver deposits.

Effect of ascorbic acid on the quiescent centerRoots incubated for 48 hours in 0.1 mM ascorbic acid showedincreased levels of cell division activity compared to rootsincubated in water. Most striking was the activation of all cellsin the quiescent center as viewed in median sections throughthe root apex (Fig. 9). The immunofluorescent signal over the

B

DQC

C


nuclei indicates cells that had incorporated BrdU duringnuclear DNA replication and thus marked those that had passedfrom G1 through S of the cell cycle during the period of theexperimental treatment. The control roots in water showed aprominent quiescent center and low levels of signal in the areaof the stele. The root cap meristem was evident and appearedto be composed of two clearly labeled cell tiers. In contrast,the ascorbic acid-treated roots had an overall higher level ofBrdU incorporation, which is reflected not only by the brighterfluorescence of the majority of nuclei, but also by enhancedcytoplasmic fluorescence, perhaps indicative of increased ratesof organelle DNA replication (Fujie et al., 1993). No QC couldbe distinguished, but just distal to the root cap juncture, theroot cap meristem showed at least 4 prominent tiers of dividingcells. Thus, applying ascorbic acid directly to roots activatescell division in the majority if not all quiescent center cells.

In summary, the QC has relatively higher levels of auxin,AAO mRNA, AAO protein, and AAO activity, and lower orundetectable levels of ascorbic acid compared to the morerapidly dividing cells surrounding it. In addition, the generalpattern of auxin distribution in the root tip is coincident withthe pattern of AAO protein localization; both of which appearto be the inverse of the pattern of elongation factor-α, whichhas been used as an indicator of high levels protein synthesisand as a negative delineator of the quiescent center (Kerk,1990).

DISCUSSION

Establishment and maintenance of the quiescentcenterWe have proposed a new model for the establishment andmaintenance of the quiescent center that is derived from con-sideration of its position and possible function in a whole plantcontext. As a terminal region with regard to the transport of

Fig. 9. Cell division activity shown in median longitudinal sections of mhour experimental treatment. Immunofluorescence indicates nuclei that hwater. Note the prominent quiescent center. (B) Root was treated with 0Magnification, ×140.

many substances, the QC is located in a potentially dynamicregion of the plant root. Recent studies of phloem transport andunloading in Arabidopsis roots have shown that fluorescentdye tracers can accumulate in the quiescent center after phloemunloading in the elongation zone and symplastic transport tothe tip (Oparka et al., 1994). Our studies of transport of[14C]IAA in maize seedling roots showed accumulation ofradioactivity at the root tip and also in the region of cellelongation. This latter area corresponds anatomically to theregion of phloem unloading in Arabidopsis roots.

Antibody localization of auxin revealed that the root cap andquiescent center contained relatively higher levels of IAA thanthe immediately surrounding proximal meristem and root capmeristem cells. The vascular tissue, pericycle and outer corticalcells also showed high levels of antibody binding and thesetissues also correlate anatomically with the regions in whichOparka et al. (1994) observed phloem transport and unloading.Salamatova (1993) has also reported similar patterns of auxindistribution in Monstera roots, using color reagents.

The effects on growth and development of the balance ofhormone levels in plant tissues is well established. Localizedaccumulation of a hormone in a small population of cells couldcause them to have significantly different metabolic propertiesthan the cells surrounding them. Indeed, other investigators ofthe quiescent center have hypothesized that the hormonebalance in the quiescent center may be different from that inadjacent regions, but speculations as to the exact differencehave varied widely.

Many auxin responsive genes have now been identified andpromoters for some of these have been shown to drive tran-scription of reporter genes in response to auxin exposure. Herewe report that exogenous auxin can increase the level of AAOmRNA in root segments and that the mRNA level of AAO issignificantly higher in quiescent center cells than in proximalmeristem cells or mature cells of the root. Auxin was previ-ously shown to increase levels of ascorbate oxidase activity in

aize root apices supplied with 10 µM BrdU for the last 24 hours of a 48ad incorporated BrdU during DNA synthesis. (A) Control root kept in

.1 mM ascorbic acid for 48 hours. No quiescent center is evident.


tobacco pith tissue and cultured pumpkin fruit (Newcomb,1951; Esaka et al., 1992) and the present results show the sameeffect in segments of maize root tissue. It would be of interestnow to determine the mechanism for these higher levels ofAAO in the quiescent center, and to determine if this gene hasan auxin-inducible or auxin-sensitive promoter.

AAO is a plant-specific copper-binding protein that has beenlocalized mainly to the cell wall but there are reports of local-ization to other cellular compartments in many different celltypes. It is very effective at oxidizing ascorbic acid, acompound necessary for many metabolic reactions and for thetransition from G1 to S in the cell cycle. We have shown highlevels of AAO protein and AAO activity in QC cells relativeto proximal meristem cells, and we suggest this causes the lackof detectable ascorbic acid in the QC, and as a consequence,the reduced mitotic activity there. These results are consistentwith other reports of the involvement of ascorbic acid in theprogression of G1 phases in the cell cycle as well as in otheraspects of plant cell growth (Cordoba et al., 1994). Moreover,as a redox reaction, the oxidation and ratio of ascorbic acid todehydroascorbic acid in regions of the root suggests that theascorbate system could be part of a larger redox regulatorysystem. There is a growing body of data that show redoxpotential can influence the regulation of gene expression andthat modulation of the redox potential in cells is indeed animportant regulatory system for cellular functions (Allen,1993; Crane, 1994). When root tips are cultured in the presenceof ascorbic acid, cells in the QC are activated to divide, andwhen root tips are treated with an inhibitor of ascorbic acidbiosynthesis, cell division is inhibited and cells arrest in G1(Liso et al., 1984, 1988). Hence we conclude that the localizeddepletion of a substance that is essential for many cellularprocesses, and especially for the completion of the cell cycle,should be viewed as an important factor in maintaining thequiescent state of these cells.

Implications of the model for meristem organizationHow can this biochemical model be used to answer questionsof meristem organization? Hejnowicz and Hejnowicz (1991)have modeled the formation of root apical patterning using abiophysical perspective based on growth tensor analysis. Oneaspect of their modeling predicts the cellular patterngenerated when a focus of slow growth is imposed in anotherwise actively growing uniform growth field. Usinggiven rules for cell wall placement with respect to theprincipal directions of growth, the model quite strikinglypredicts the formation of a closed meristem cell pattern asshown in maize root apices (Hejnowicz and Hejnowicz,1991). When a different tensor is applied, such that the focusregion becomes the region of maximal growth, the modelpredicts the pattern in a pteridophyte root with a single apicalcell. This suggests that root apical patterning may be con-trolled by regulation of relative growth rates at this focalpoint in the developing root meristem.

Our model is essentially a mechanism for controlling thegrowth rate at this focal point. Therefore, experimental changesthat alter the steady state would be expected to have an effecton apical patterning. For instance, inhibition of polar auxintransport severely disrupts organization of the root meristem.This has been shown for embryonic roots, lateral roots, intactprimary roots, and regenerating roots in many families of

plants (Schiavone and Cooke, 1987; Liu et al., 1993; Hincheeand Rost, 1992; Stange, 1988; Kerk and Feldman, 1994).Hence alterations of other components of the model that relateto the growth rate at the quiescent center should also disruptnormal patterning of the root apex.

We suggest that our proposed biochemical model comple-ments this biophysical growth model by providing amechanism for regulating the relative growth rate in this focalregion of a developing root meristem. Our model (Fig. 1)accounts for the localized depletion of ascorbic acid in the QC,and hence for the rate of cell growth and division in thisspecific region of the root tip. In addition our work supportsthe models of Hejnowicz and Hejnowicz which predict thatlocalized control of growth rates here is a determining aspectin generating the pattern of cell walls in a given root tip. Thesegrowth rates may be different in different species and atdifferent times during development, and thus underlie thediversity of anatomical patterns that have been reported in roottips. A similar type of mechanism may even underlie the organ-ization of other kinds of meristems.

We are very grateful to Dr Ian Sussex for his critical reading of themanuscript and helpful discussions and suggestions. We are also verygrateful to Dr Steven Ruzin who was so generous with his help withmicroscopy and image processing in the NSF Center for Plant Devel-opment at Berkeley. Thanks also to Dr A. Shinmyo, Department ofFermentation Technology, Osaka University, Japan, for his generousgift of the ascorbate oxidase cDNA clone and antibodies that we usedin this study. We thank Prof. E. W. Weiler, Ruhr-Universität Bochum,for the monoclonal antibody to auxin, and we also thank Dr Sun-daresan, Cold Spring Harbor Labs for the p34cdc2 cDNA clone. Thiswork was supported by a USDA post-doctoral fellowship to N. Kerk,and an NSF grant to L. Feldman.

REFERENCES

Allen, J. (1993). Redox control of transcription: sensors, response regulators,activators and repressors. FEBS Lett. 332, 203-207.

Arrigoni, O., Bitonti, M. B., Cozza, R., Innocenti, A. M., Liso, R. andVeltri, R. (1989). Ascorbic acid effect on pericycle cell line in Allium ceparoot. Caryologia 42, 213-216.

Barlow, P. W. (1978). The concept of the stem cell in the context of plantgrowth and development. Stem Cells and Tissue Homeostasis (ed. B. I. Lord,C. S. Potten and R. J. Cole). Cambridge: Cambridge University Press.

Chayen, J. (1953). Ascorbic acid and its intracellular localization with specialreference to plants. Intern. Rev. Cytol. 2, 78-132.

Chichiricco, G., Ceru, M. P., D’Alessandro, A., Oratore, A. and Avigliano,l.. (1989). Immunohistochemical localization of ascorbte oxidase inCurcubita pepo medullosa. Plant Science. 64, 61-66.

Chinoy, J. J. (1984). The Role of Ascorbic Acid in Growth, Differentiation andMetabolism of Plants. (ed. N. J. Chinoy). The Hague: Martinus Nijhott/DrJunk Publishers.

Cittero, S., Sgorbati, S., Scrippa,. and Sparvoli, E. (1994). Ascorbic acideffect on the onset of cell proliferation in pea root. Physiol. Plant. 92, 601-607.

Clowes, F. A. L. (1953). The cytogenerative centre in roots with broadcolumellas. New Phytologist 52, 48-57.

Clowes, F. A. L. (1954). The promeristem and the minimal constructionalcentre in grass root apices. New Phytol. 53, 108-116.

Clowes, F. A. L. (1956). Nucleic acids in root apical meristems of Zea. NewPhytol. 55, 29-34.

Clowes, F. A. L. (1961). Apical Meristems. Oxford: Blackwell ScientificPublications.

Clowes, F. A. L. (1972). The control of cell proliferation within root meristems.The Dynamics of Meristem Cell Populations (ed. M. W. Miller and C. C.Kuehnert). New York: Plenum Press.

Clowes, F. A. L. (1975). The Quiescent Centre. In The Development and


Function of Roots (ed. J. G. Torrey and D. T. Clarkson). New York:Academic Press.

Clowes, F. A. L. (1978). Origin of the quiescent centre in Zea mays. NewPhytol. 80, 409-419.

Colasanti, J., Tyers, M., and Sundaresan, V. (1988). Isolation andcharacterization of complementary DNA clones encoding a functionalp34cdc2 homologue from Zea mays. Proc. Natl. Acad. Sci. USA 88, 3377-3381.

Crane, F. (1994). Minireview series: Ascorbate function at membranes. J.Bioenerget. Biomembr. 26, 347-421.

Cordoba, F. and Gonzalez-Reyes, J. A. (1994). Ascorbate and plant cellgrowth. J. Bioenerget. Biomembr. 26, 399-405.

Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts,K. and Sheres, B. (1993). Cellular organisation of the Arabidopsis thalianaroot. Development 119, 71-84.

Esaka, M., Fujisawa, K., Goto, M. and Kisu, Y. (1992). Regulation ofascorbate oxidase expresson in pumpkin by auxin and copper. Pl. Physiol.100, 231-237.

Esaka, M., Hattori, T., Fujisawa, K., Sakajo, S. and Asahi, T. (1990).Molecular cloning and nucleotide sequence of full-length cDNA forascorbate oxidase from cultured pumpkin cells. Eur. J. Biochem. 191, 537-541.

Esaka, M., Uchida, M., Fukui, H., Kubota, K. and Suzuki, K. (1988).Marked increase in ascorbate oxidase protein in pumpkin callus by addingcopper. Pl. Physiol. 88, 656-660.

Feldman, L. J. (1975). Cytokinins and quiescent center activity in roots of Zea.The Development and Function of Roots (ed. J. G. Torrey and D. T.Clarkson). New York: Academic Press.

Feldman, L. J. (1977). The de novo origin of the quiescent center inregenerating apices of Zea mays. Planta 128, 207-212.

Feldman, L. J. (1979). Cytokinin biosynthesis in roots of corn. Planta 145,315-321.

Feldman, L. J. (1981). Effect of auxin on acropetal auxin transport in roots ofcorn. Pl. Physiol. 67, 278-281.

Feldman, L. J. and Torrey, J. G. (1975). The quiescent center and primaryvascular tissue pattern formation in cultured roots of Zea. Can. J. Bot. 53,2796-2803.

Feldman, L. J. and Torrey, J. G. (1976). The isolation and culture in vitro ofthe quiescent center of Zea mays. Am. J. Bot. 63, 345-355.

Fisher, D. B. (1968). Localization of endogenous RNA polymerase activity infrozen sections of plant tissues. J. Cell Biol. 39, 745-749.

Fujie, M., Kurowia, H., Suzuki, T., Kwano, S., and Kuroiwa, T. (1993).Organelle DNA synthesis in the quiescent centre of Arabidopsis thaliana. J.Exp. Bot. 44, 689-693.

Goyal, V. and Pillaai, A. (1986). Anatomical and histochemical studies of rootapical meristems of some angiosperms. Beitr. Biol. Pflanzen 62, 167-178.

Hanstein, J. (1868). Die Scheitelzellgruppe im Vegetationspunkt derPhanerogamen. Fetschr. Niederrhein. Gessell. Natur-und Heilkunde 1868,109-134.

Hejnowicz, Z. and Hejnowicz, K. (1991). Modeling the formation of rootapices. Planta 184, 1-7.

Hinchee, M. A. W. and Rost, T. L. (1992). The control of lateral rootdevelopment in cultured pea seedlings. II. Root fasciation induced by auxininhibitors. Bot. Act. 105, 121-126.

Innocenti, A. M., Bitonti, M. B., Arrigoni, O., and Liso, R. (1990). The sizeof the quiescent centre in roots of Allium cepa L. grown with ascorbic acid.New Phytol. 114, 507-509.

Jackson, D., (1991) In situ hybridization in plants. In Molecular PlantPathology: A Practical Approach. (ed. D. J. Bowles, S. J. Gurr, and M.McPherson) Oxford: Oxford University Press.

Jensen, W. A. (1958). The nucleic acid and protein content of root tip cells ofVicia faba and Allium cepa. Exp. Cell Res. 14, 575-583.

Jensen, W. A. (1962). Botanical Histochemistry. San Francisco: W. H.Freeman Company.

Jensen, W. A. and Kavaljian, L. G. (1956). The cytochemical localization ofascorbic acid in root tip cells. J. Biophys. Biochem. Cytol. 2, 87-93.

Kerk, N. and Feldman, L. (1994). The quiescent center in roots of maize:initiation, maintenance and role in organization of the root apical meristem.Protoplasma 183, 100-106.

Kerk, N. M. (1990). Gene expression during meristem initiation andorganization. Ph. D. Thesis, Yale University.

Liso, R., Calabrese, G., Bitonti, M. B., and Arrigoni, O., (1984).Relationship between ascorbic acid and cell division. Exp. Cell Res. 150,314-320.

Liso, B. R., Innocenti, A. M., Bitonti, M. B. and Arrigoni, O. (1988).Ascorbic acid-induced progression of quiescent centre cells from G1 to Sphase. New Phytol. 110, 469-471.

Liu, C. M., Z. H. Xu, and Chua, N. H. (1993). Auxin polar transport isessential for the establishment of bilateral symmetry during early plantembryogenesis. The Plant Cell 5, 621-630.

Maniatis, T., Fritsch, E. F. and Sambrook J. (1989). Molecular Cloning: ALaboratory Manual. Second edition. Cold Spring Harbor, New York: ColdSpring Harbor Laboratory Press.

Newcomb, E. (1951). Effect of auxin on ascorbic oxidase activity in tobaccopith cells. Proc. Soc. Exp. Biol. Med. 76, 504-509.

Oberbacher, M. F. and Vines, H. M. (1963). Spectrophotometric assay ofascorbic acid oxidase. Nature 197, 1203-1204.

Ohkawa, J. Okada, N., Shinmyo, A. and Takano, M. (1989). Primarystructure of cucumber (Cucumis sativus) ascorbate oxidase deduced fromcDNA sequence: homology with blue copper proteins and tissue specificexpression. Proc. Natl. Acad. Sci. USA 86, 1239-1243.

Oparka, K. J., Duckett, C. M., Prior, D. A. M. and Fisher, D. B. (1994).Real-time imaging of phloem unloading in the root tip of Arabidopsis. ThePlant J. 6, 759-766.

Puissant, C. and Houdebine, L. (1990). An improvement of the single-stepmethod of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction. BioTechniques 8, 148-149.

Raghavan, V. (1990). Origin of the quiescent center in the root of Capsellabursa-pastoris (L.) Medik. Planta 181, 62-70.

Raghavan, V. and Olmedilla, A. (1989). Spatial patterns of histone mRNAexpression during grain development and germination in rice. Cell Differ.Dev. 27, 183-196.

Rondet, P. (1961). Repartition et signification des acides ribonucleiques aucours d’embryogene chez Myosurus minimus. L. C. R. Acad. Sci. 253, 1725-1727.

Rost, T. L. and Jones,T. J. (1988). Pea root regeneration after tip excisions atdifferent levels: polarity of new growth. Annal. of Bot. 61, 513-523.

Salamaatova, T. S. (1993). Histochemical investigation of indole-3-acetic acidlocalization in a distal part of aerial roots of Monstera deliciosa. VestinikSankt-Peterburgskogo universiteta. Seriia 3, Biologiia 2, 78-82.

Schiavone, M. and Cooke, T. (1987). Unusual patterns of embryogenesis inthe domesticated carrot: developmental effects of exogenous auxins andauxin transport inhibitors. Cell Diff. 21, 53-62.

Shi, L., Miller, I., and Moore, R. (1993). Immunocytochemical localization ofindole-3-acetic acid in primary roots of Zea mays. Plant, Cell Environ. 16,967-973.

Stange, L. (1988). Disorganization of the meristem in Riella helicophylla byinhibition of auxin action. In Physiology and Biochemistry of Auxins inPlants (ed. M. Kutacek, R. S. Bandurski, and J. Krekule). Prague: AcademiaPrah.

Steeves, T. A. and Sussex, I. M. (1989). Patterns in Plant Development.Cambridge: Cambridge University Press.

Torrey, J. G. (1972). On the initiation of organization in the root apex. InDynamics of Meristem Populations. (ed. M. W. Miller and C. C. Kuehnert).New York: Plenum Press.

(Accepted 16 May 1995)

a biochemical model for the initiation and maintenance of the … · key words: quiescent center,...

Documents