the root meristemless1 cadmium sensitive2 gene defines … · mutation does not affect axial and...
TRANSCRIPT
The Plant Cell, Vol. 12, 97–109, January 2000, www.plantcell.org © 2000 American Society of Plant Physiologists
The
ROOT MERISTEMLESS1
/
CADMIUM SENSITIVE2
Gene Defines a Glutathione-Dependent Pathway Involved in Initiation and Maintenance of Cell Division during Postembryonic Root Development
Teva Vernoux,
a,1
Robert C. Wilson,
b,1,2
Kevin A. Seeley,
b,3
Jean-Philippe Reichheld,
a
Sandra Muroy,
b
Spencer Brown,
c
Spencer C. Maughan,
d
Christopher S. Cobbett,
d
Marc Van Montagu,
a
Dirk Inzé,
a
Mike J. May,
a,4
and Zinmay R. Sung
b,5
a
Universiteit Gent, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium
b
Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, California 94720
c
Institut des Sciences du Végétal-CNRS, Service de Cytométrie, avenue de la Terrasse, 91198 Gif-sur-Yvette, France
d
Department of Genetics, University of Melbourne, Parkville 3052, Australia
Activation of cell division in the root apical meristem after germination is essential for postembryonic root develop-ment. Arabidopsis plants homozygous for a mutation in the
ROOT MERISTEMLESS1
(
RML1
) gene are unable to estab-lish an active postembryonic meristem in the root apex. This mutation abolishes cell division in the root but not in theshoot. We report the molecular cloning of the
RML1
gene, which encodes the first enzyme of glutathione (GSH) biosyn-thesis,
g
-glutamylcysteine synthetase, and which is allelic to
CADMIUM SENSITIVE2
. The phenotype of the
rml1
mu-tant, which was also evident in the roots of wild-type Arabidopsis and tobacco treated with an inhibitor of GSHbiosynthesis, could be relieved by applying GSH to
rml1
seedlings. By using a synchronized tobacco cell suspensionculture, we showed that the G
1
-to-S phase transition requires an adequate level of GSH. These observations suggestthe existence of a GSH-dependent developmental pathway essential for initiation and maintenance of cell division dur-ing postembryonic root development.
INTRODUCTION
One of the most remarkable developmental features ofhigher plants is their capacity to generate new organsthroughout their life cycle. Postembryonic development arisesessentially from groups of highly organized, mitotically ac-tive cells called meristems. Meristems generate cells thatwill enter specific differentiation programs while simulta-neously maintaining a population of proliferating, undifferen-tiated cells. A fundamental aspect of meristems is that theyallow the supply of cells to be related to environmental con-straints or demands, thereby permitting the fine-tuning ofplant growth and development to the prevailing environmen-tal conditions. The primary root and shoot meristems, which
are located at the apex of the root and shoot, are estab-lished during embryogenesis (Laux and Jürgens, 1997), andthey initiate postembryonic development after germination.
The Arabidopsis root presents a remarkably regular andsimple radial structure in which single layers of defined cellnumber comprising the epidermis, cortex, endodermis, andpericycle surround the vascular tissues (Dolan et al., 1993).The cellular organization of the Arabidopsis root is first es-tablished during embryogenesis by specific divisions withinthe ground tissues (Scheres et al., 1994). After germination,the internal structure of the Arabidopsis root is maintainedby nearly invariant patterns of cell division when grown un-der controlled conditions (Scheres et al., 1994). The simplic-ity of the Arabidopsis root and the amenability of thisorganism to genetic and molecular analysis have made it anexcellent system in which to dissect the mechanisms con-trolling plant cell growth and differentiation.
Addressing the question of how cell division is controlledduring developmental processes is fundamental to under-standing plant development. Inherent in this question is thecontribution of the cell cycle to plant development and mor-phogenesis. Indeed, direct insights into the role of the cell
1
These authors contributed equally to this work.
2
Current address: Department of Agriculture and Natural Sciences,Hedmark College, N-2322, Ridabu, Norway.
3
Current address: Gelman Sciences, P.O. Box 3850, Ann Arbor, MI48106.
4
Current address: Plant Genetic System N.V., Jozef Plateaustraat22, B-9000, Gent, Belgium.
5
To whom correspondence should be addressed. E-mail [email protected]; fax 510-642-4995.
98 The Plant Cell
cycle in plant development have been obtained by modulat-ing the expression of genes whose products are involved inthe core cell cycle machinery. For example, roots of Arabi-dopsis overexpressing a mitotic cyclin showed an increasein cell number and a significant increase in root size, sug-gesting that cyclins could be a limiting factor during growth(Doerner et al., 1996). However, no alteration in the organi-zation of the root was observed. A normally shaped rootalso was generated from a smaller than normal number ofcells in tobacco plants expressing dominant-negative formsof the cell cycle–dependent Cdc2 kinase (Hemerly et al.,1995). Organ shape thus appears to be relatively insensitiveto variations in cell number. Thus, cell division may contrib-ute to growth in response to a higher level of regulation thatcontrols meristem organization. Indeed, laser ablation ex-periments have demonstrated that positional signaling fromsurrounding cells is an essential aspect of cell identity andmeristem organization in the Arabidopsis root (Van den Berget al., 1995, 1997). However, the mechanisms controllingcell division during development remain largely unknown.
Genetic studies using Arabidopsis roots provide a power-ful means to address the function of genes that regulate celldivision during root growth and development, and numerousArabidopsis mutants affected in root development are avail-able. Some of these exhibit specific alterations in meristemorganization and cell division patterns (reviewed in Schereset al., 1996; Schiefelbein et al., 1997). A mutation in the
ROOT MERISTEMLESS1
(
RML1
) gene, however, does notaffect embryonic development but results instead in plantswith an extremely short mature root composed of the samenumber of cells and cell files as the embryonic root (Chenget al., 1995). The
rml1
mutation does not affect axial and ra-dial patterns of root cell organization, suggesting that the
RML1
gene does not play an obvious role in cell fate specifi-cation. However,
rml1
mutants fail to initiate cell divisionwhen germinated and, as a result, are unable to establishand maintain an active, undifferentiated meristematic zonein the root. Lateral and adventitious roots from callus of
rml1
mutants have similar defects (Cheng et al., 1995). In contrastto the highly defective cell division phenotype of
rml1
roots,cell division occurs in the apical shoot meristem, producinga small shoot with vegetative and sexual organs. Thus, the
RML1
gene is required primarily for the regulation of cell di-vision in root apical meristems (Cheng et al., 1995).
Here, we describe the cloning and sequence of the
RML1
gene. Its coding sequence and functional analysis revealedthat surprisingly,
RML1
is allelic to
CADMIUM SENSITIVE2
(
CAD2
) (Cobbett et al., 1998), encoding the previously de-scribed first enzyme of glutathione (GSH;
g
-glutamylcys-teinyl glycine) biosynthesis,
g
-glutamylcysteine synthetase(
g
-GCS; EC 6.3.2.2; May and Leaver, 1994); as a result alltissues of
rml1
mutants are devoid of GSH. GSH is a ubiqui-tous tripeptide involved in cellular redox homeostasis thathas been shown to be present in high concentrations in allplant tissues (May et al., 1998). We present evidence thatGSH deficiency leads to a cell division block during the G
1
phase. Furthermore, our results demonstrate that GSH isnecessary for the initiation as well as maintenance of cell di-vision. The role of GSH-dependent cell division in plantroots is discussed.
RESULTS
RML1
Is Allelic to
CAD2
, the Structural Gene for
g
-GCS
To study the molecular function of the RML1 protein, wedevised a strategy to isolate the
RML1
gene by using a re-striction fragment length polymorphism (RFLP)–based chro-mosome walk technique, as shown in Figure 1.
RML1
wasmapped earlier to the middle of chromosome 4 betweenRFLP markers pCITd104 and
AGAMOUS
(
AG
) (Cheng et al.,1995). A yeast artificial chromosome (YAC) contig previouslyhad been established between pCITd104 and
AG
on chro-mosome 4 (Schmidt et al., 1995), and RFLP probes derivedfrom the ends of YAC clones in this region were used to an-alyze a segregating population of 605 plants. After havingmapped
RML1
between marker mi422 and the left end ofmarker CIC7H1 (CIC7H1LE), we deduced that the YACclone CIC5A4 should span the gene (Figures 1A and 1B).Total DNA from the yeast strain harboring CIC5A4 was usedto construct a cosmid library enriched for
RML1
sequencesin pCLD04541, a T-DNA–transferable vector that stably con-fers kanamycin resistance to transformed plants (Bent et al.,1994).
An overlapping cosmid contig was established by com-paring patterns generated from restriction digestions andcross-hybridizations between cosmids and by segregationanalyses (Figure 1C and data not shown). Six overlappingcosmids, of which four detected no recombinants in thesegregating population, were mobilized into Agrobacteriumfor transformation of Arabidopsis plants that were heterozy-gous for the
rml1-1
allele. Two cosmids, 20A7 and 26A8,which overlap by
z
10 kb, produced 86 transgenic T
1
plantsthat were hemizygous for the T-DNA and heterozygous forthe
rml1
allele. These plants yielded only kanamycin-resis-tant T
2
seedlings, which were all phenotypically wild type;thus, the 5- to 10-kb sequence shared between cosmids20A7 and 26A8 was sufficient for complementation andtherefore contained the
RML1
gene.A cDNA library was screened with the sequence shared
between the complementing cosmids 20A7 and 26A8, andtwo cDNA clones were isolated. Both clones showed se-quence identity with a previously published Arabidopsis se-quence. The gene itself encodes the first enzyme of GSHbiosynthesis,
g
-GCS (May and Leaver, 1994). The cadmium-sensitive Arabidopsis mutant
cad2-1
recently was alsoshown to be defective in the
g
-GCS gene (Cobbett et al.,1998); however,
cad2-1
cannot be morphologically distin-guished from wild-type Arabidopsis. The roots of
cad2-1
seedlings developed normally unless challenged by 0.5 to 1
GSH-Dependent Control of Root Cell Division 99
m
M Cd, in which case they turned brown (Howden et al.,1995).
rml1
mutants died when germinated on medium con-taining 1
m
M Cd, indicating an extreme sensitivity to Cd(data not shown).
To test for allelism, the homozygous
cad2-1
mutant wascrossed with
rml1
heterozygotes. Given the normal root de-velopment in
cad2-1
, we expected that if it was allelic with
rml1
, the
cad2-1
allele would complement the Rml1 stuntedroot, but not the Cd-sensitive phenotype, in
cad2-1
/
rml1
plants. All plants in the F
1
progeny grew normally, indicatingthat the Rml1 root phenotype was complemented. F
1
plantswere also tested for Cd sensitivity on medium containing 0.5
m
M CdSO
4
along with wild-type and
cad2-1
controls. Ap-proximately 50% of the progeny were Cd sensitive (
rml1-1
,
Figure 1. Positional Cloning of the RML1 Gene.
(A) Genetic map of the RML1 region of chromosome 4 of Arabidopsis. The genetic map is represented by the open-ended solid line. The num-bers over double-headed arrows indicate the distances between RFLP markers expressed as absolute numbers of recombinants in the F2 pop-ulation. The regions between the left line break and marker g3883 and between the right line break and marker KG-32 are not drawn to scale.(B) YAC (and bacterial artificial chromosome) contig. Clones anchored to the molecular markers are connected by dashed lines to the geneticmap in (A). Solid circles denote right ends of clones; solid squares, left ends; and open squares, ends not aligned. YAC clones yUP20C1 andCIC7A5 are most likely chimeric clones (open circles connected to dashed lines). The region of CIC7A5 left of the line break is not drawn toscale.(C) Cosmid walk from RFLP marker mi422 to RML1. All clones were constructed in the T-DNA–transformable cosmid vector pCLD04541 (Bentet al., 1994) from genomic DNA isolated from the yeast strain harboring YAC clone CIC5A4, except for the bacterial artificial chromosome cloneTAMU12H8, which served to bridge the gap between clones MB6A and 30E6, and cosmid clone BICB1, isolated from an Arabidopsis genomiclibrary kindly provided by K. Mayer (Institute of Plant Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland). Symbols are thesame as given in (B), and the box indicates the region for which no recombinants were detected using RFLP markers derived from the cosmidclones. The z10-kb overlapping sequence between complementing cosmids 20A7 and 26A8 was used to isolate cDNA clones encodingg-GCS.(D) Identification of the rml1 mutation. Sequence of the g-GCS cDNA clone spanning nucleotides 820 to 890 and the encoded amino acid se-quence spanning residues 236 to 259. The 6-bp deletion (824 to 829) mutation of the cad2-1 allele is underlined; this mutation yields a two–amino acid deletion (asterisks) and a one–amino acid substitution in the encoded protein (boxed). The single base pair (G to A) substitution mu-tation of rml1 is indicated by the letter A in boldface (dots indicate nucleotides identical to the wild-type [WT] sequence); this mutation gives riseto an aspartate to asparagine substitution in the encoded protein at residue 258 (boxed). The cysteine residue at position 251 is thought to bepart of the active site of g-GCS.
100 The Plant Cell
31 resistant to 39 sensitive,
k
2
5
0.3, P
.
0.5;
rml1-2
, 77 re-sistant to 77 sensitive,
k
2
5
0, P
.
0.9), indicating that theCd-sensitive phenotype was not complemented. Collec-tively, these data indicate that
cad2-1
and
rml1
are allelic;thus, the
rml1
plants are mutated in the
g
-GCS gene.The primers used to identify the
cad2-1
mutation (Cobbettet al., 1998) were used to sequence the two
rml1
alleles,
rml1-1
and
rml1-2
(Cheng et al., 1995). Surprisingly, both al-leles harbored the same single G-to-A substitution mutationthat encodes an asparagine residue at position 258 of theRML1 protein rather than an aspartate residue (Figure 1D),indicating that
rml1-1
and
rml1-2
are in fact the same allele.Therefore, throughout this article, we refer to
rml1-1 andrml1-2 as the rml1 mutant and allele. The rml1 mutation is 60bp from the cad2-1 mutation, which marks a 6-bp deletionresulting in the deletion of two amino acids at positions 237and 238 and a valine-to-leucine substitution at position 239in the encoded g-GCS protein (Figure 1D; Cobbett et al.,1998).
The rml1 Mutant Is Deficient in g-GCS Activity and as a Result Is Almost Devoid of GSH
To elucidate how the rml1 mutation affects protein function,we measured g-GCS activity in rml1 and wild-type plants.As shown in Table 1, g-GCS activity was undetectable in ex-tracts of the rml1 mutant. Wild-type seedlings exhibitedg-GCS activity comparable with that observed previously(Cobbett et al., 1998). The activity of the second enzyme ofGSH biosynthesis, GSH synthetase (GSHS), also was mea-sured, and this activity essentially was not affected in rml1mutants (Table 1). Quantification of the intracellular concen-tration of GSH was performed by using an HPLC assay(Cobbett et al., 1998). When compared with those of thewild type, extracts of rml1 mutants contained only 2.7% ofthe extractable GSH (Table 1). g-Glutamylcysteine (g-GC),the product of g-GCS, was undetectable. In contrast, theamount of cysteine, one of the precursors of g-GC, was ap-proximately threefold higher than that in the extracts fromwild-type plants. Importantly, confocal scanning laser mi-croscopy analysis of whole rml1 seedlings labeled with the
GSH-specific dye monochlorobimane (Sánchez-Fernándezet al., 1997) revealed that the distribution of GSH in all or-gans was uniformally low when compared with the distribu-tion seen in wild-type seedlings (data not shown). Thus, therml1 mutation results in a reduction in the activity of g-GCSin all cells to below the level of detection by using HPLC.The mutation leads to an extreme reduction in the intracellu-lar pool of GSH and to a substantial increase in one of theprecursors, cysteine.
Absence of Cell Division in the rml1 Postembryonic Root Results from the Depletion of Endogenous GSH
To investigate whether the root development phenotype ofthe rml1 mutant was directly linked to the impairment inGSH biosynthesis or to a pleiotropic effect of the mutation,we supplemented the growth medium with the product ofg-GCS activity, g-GC, or its downstream anabolite GSH, todetermine whether any of these substances could rescueroot growth in the rml1 mutants. As described by Cheng etal. (1995), rml1 shoot tips can produce leaves, but cells inthe root tip did not divide after germination, with the primaryand secondary roots achieving a length of only z1 mm (Fig-ure 2A). Importantly, rml1 could not be rescued when germi-nated on a medium containing glutamate and cysteine, thesubstrates of g-GCS (data not shown). In contrast, when ei-ther 250 mM g-GC or GSH was added to the growth mediumbefore germination, the rml1 mutants developed roots up to8 to 9 mm in length by 14 days after germination (Figures 2Band 2C). Because homozygous rml1 mutants exhibit re-duced fertility, heterozygous parental strains segregatingone-fourth rml1 plants were routinely used for these experi-ments.
At concentrations of 250 mM g-GC or GSH, rml1 mutantsin the segregating population could still be distinguishedfrom wild-type and heterozygous plants. At higher g-GC orGSH concentrations, the distribution of root lengths of seg-regating rml1 populations was indistinguishable from that ofthe wild type, whereas on Murashige-Skoog (MS) medium(Murashige and Skoog, 1962), the root lengths of the rml1segregating population showed the expected 3:1 wild type
Table 1. Biochemical Characterization of Wild-Type and rml1 plants
Lines Cysteinea (nmol/g DW)b g-GCa (nmol/g DW) GSHa (mmol/g DW)g-GCSa
(nmol/min/mg Protein)GSHSa
(nmol/min/mg Protein)
Wild type 109 6 10 (100) 60 6 6 (100) 3.02 6 0.35 (100) 0.80 6 0.02 (100) 0.98 6 0.05 (100)rml1 323 6 19 (296) NDc 0.08 6 0.01 (2.7) ND 0.88 6 0.04 (90)
a The biochemical determination of thiols and of g-GCS and GSHS activities was conducted with extract from 2-week-old liquid-grown plantssubjected to HPLC as described in Methods. Numbers within parentheses indicate the percentage compared with the wild-type value.b DW, dry weight of tissue.c ND, undetectable.
GSH-Dependent Control of Root Cell Division 101
plus heterozygote to rml1 mutant ratio (Figure 2E). In con-trast, we could not rescue rml1 mutants with the oxidizedform of GSH, GSSG, with ascorbate, another important anti-oxidant, or with DTT, a synthetic thiol (data not shown).Thus, exogenous GSH (and more precisely its reduced form)alleviated the block imposed on cell division by depletingthe cellular GSH pool. Although growth is determined bothby cell division and elongation, we do not know the exactrole of GSH in processes regulating the elongation of rootcells.
To determine whether GSH is required continuously dur-ing root growth, we grew rml1 mutants for 3 days on GSH-containing media and then transferred them to media lack-ing GSH. As shown in Table 2, we found that in the absenceof exogenous GSH, root growth decreased rapidly andstopped totally after 2 days, whereas roots remaining onGSH-containing media grew 10 to 11 mm each day. There-fore, exogenous GSH was necessary not only for initiatingbut also for maintaining rml1 root growth.
To confirm further that GSH depletion was directly re-sponsible for the absence of postembryonic growth in therml1 root, we germinated wild-type Arabidopsis on mediacontaining 2.5 mM L-buthionine-(S,R)-sulfoximine (BSO).BSO is a nontoxic and highly specific inhibitor of the first en-zyme of GSH biosynthesis, g-GCS, and its application re-sults in the depletion of cellular GSH (Griffith and Meister,1979; May and Leaver, 1993). As shown in the middle row ofFigure 2D, wild-type seedlings exhibited growth similar torml1 mutants (top row) by 7 days after germination on me-dium containing BSO. By comparison, wild-type seedlingsgrown in the absence of BSO developed long roots (Figure2D, bottom row), confirming the direct link between the phe-notype of the rml1 mutant and impairment in GSH biosyn-thesis. Thus, the depletion of intracellular GSH, eitherphysiologically or genetically, inhibits postembryonic rootgrowth in Arabidopsis. The addition of BSO to the growthmedium of Arabidopsis seedlings inhibited root apical mer-istematic activity but allowed shoot meristematic activity tooccur after germination. Like the control plants, 3-week-oldrml1 mutants (Cheng et al., 1995) and BSO-treated wild-type plants produced six or seven leaves, although theleaves were somewhat shorter than those of plants grown inthe absence of BSO (S. Muroy and Z.R. Sung, unpublisheddata).
Figure 2. rml1 Rescue and Phenocopy.
(A) rml1 seedlings at 14 days after germination (DAG) grown on two-fifths MS medium. Note the short (z1 mm) roots.(B) rml1 seedlings at 14 DAG grown on two-fifths MS medium sup-plemented with 250 mM g-GC.(C) rml1 seedlings at 14 DAG grown on two-fifths MS medium sup-plemented with 250 mM GSH.(D) Top, rml1 seedlings at 7 DAG grown on two-fifths MS medium;middle, wild-type seedlings at 7 DAG grown on two-fifths MS me-dium supplemented with 2.5 mM BSO, a specific inhibitor of g-GCS;bottom, wild-type seedlings at 7 DAG grown on two-fifths MS me-dium. Note the similarity in root growth between the rml1 mutantsand the wild-type plants grown on BSO. Wild-type plants developrelatively long roots at the same age when grown on BSO-free me-dium.
(E) Histograms show the results of a representative experiment inwhich wild-type and segregating rml1 populations (containing rml1/rml1:RML1/rml1:RML1/RML1 seeds in a 1:2:1 ratio) were germi-nated on MS medium or medium supplemented with 500 mM g-GCor 750 mM GSH. The plants were grown vertically for 6 days beforethe root lengths were measured.Bars in (A) to (D) 5 1 mm.
102 The Plant Cell
DNA Content of Cells in the Root Tip of rml1Arabidopsis Mutants
The cloning and functional analyses of the rml1 mutant sug-gested a specific role for the antioxidant GSH in the controlof root growth in the postembryonic root of Arabidopsis. Be-cause root growth results from increases in cell number andcell length, and cell division arrest is the most dramatic phe-notype in rml1 roots (Cheng et al., 1995), we investigatedthe possibility that GSH regulates the cell division cycle inthe root apex. To elucidate the effect on cell division of thedepletion of cellular GSH that occurs as a result of the rml1mutation, nuclear DNA content of the tissue of rml1 mutantswas analyzed by using flow cytometry, as shown in Figure 3.The ploidy distributions for nuclei isolated from the shoot tipwere identical in 3-day-old rml1 and wild-type Arabidopsisplants (Figures 3A and 3B) and similar to that previously ob-served (Galbraith et al., 1991), confirming that cell division isnot affected in rml1 shoots (Cheng et al., 1995). In the roottip of both rml1 and wild-type 3-day-old seedlings, differentextents of polyploidy were observed (Figures 3C and 3D).The presence of polyploid cells in Arabidopsis roots did notallow us to identify a specific defect in the cell cycle arisingfrom the rml1 mutation. However, we observed significantdifferences in the distribution of the nuclei in the root. Forexample, the 8C peak was predominant in rml1 plants,whereas the 4C peak was predominant in wild-type plants(Figures 3C and D). Moreover, the 32C population was un-detectable in rml1 root tips (Figure 3C).
Using a Tobacco Cell Suspension to Understand the Phenotype of the rml1 Mutant
To study specifically the effect of a GSH depletion on cellcycle progression, we sought to exploit a cell suspensionsystem that could be synchronized. Because an Arabidopsiscell suspension that can be synchronized is currently un-available, we used a highly specialized tobacco BY-2 cellsuspension that has been widely adopted for cell cyclestudies in plants (Nagata et al., 1992).
We first germinated tobacco plants on a medium contain-ing 2.5 mM BSO. As observed with Arabidopsis plants, de-pletion of intracellular GSH in tobacco seedlings completelyabolished root growth (Figures 4A and 4B), whereas shootmeristematic activity was essentially unaffected (Figure 4C).Therefore, depletion of intracellular GSH has similar effectson tobacco postembryonic root development, as seen withArabidopsis plants, suggesting a general mechanism inplants.
We then checked whether GSH depletion could affect celldivision in a BY-2 cell suspension culture. Exponentiallygrowing BY-2 cells were treated with 1 mM BSO. From aninitial value of 4.71 6 0.09 mg (g dry weight)21 of tissue, thecellular concentration of GSH progressively decreased to0.21 6 0.02 mg (g dry weight)21 after 12 hr and to 0.11 6
0.05 mg (g dry weight)21 at 24 hr, while remaining stable inan untreated culture (data not shown). The effect of thistreatment on cell cycle progression was studied by usingflow cytometric analysis (Figure 4D). After 12 hr of BSOtreatment, a pronounced depletion of the nuclei populationduring S phase was observed; after 24 hr, .95% of the nu-clei from BSO-treated cells were in G1 phase compared with60% in the control (Figure 4D). The almost total depletion ofthe G2 population after 24 hr of BSO treatment indicates thatthe cells remaining in G2 at 12 hr most probably representcells that have not completed their cycle rather than cells ar-rested in G2 phase as a result of depletion of intracellular
Table 2. Daily Root Growth of rml1 Plants
Treatmenta First Day (mm)b Second Day (mm) Third Day (mm)
1 GSH 11.3 6 1.7 10.8 6 1.7 11 6 1.42 GSH 3.5 6 0.6 1.1 6 0.2 0
a Two-day-old rml1 seedlings were grown for 4 days on a mediumsupplemented with 250 mM GSH before being transferred onto amedium supplemented with 250 mM GSH (1 GSH) or lacking GSH(2 GSH).b Numbers represent the mean of 12 roots 6SD.
Figure 3. Cellular DNA Content in rml1 and Wild-Type Plants.
DNA contents were analyzed by using flow cytometric analysis. Thedifferent ploidy levels are indicated.(A) and (C) Extracts from rml1 shoots and root tips, respectively.(B) and (D) Extracts from wild-type shoots and root tips, respec-tively.
GSH-Dependent Control of Root Cell Division 103
GSH. The cellular concentration of GSH was essentially thesame at 12 and 24 hr. The effects of supplementing BSOwith GSH yielded the same results when compared with thecontrol plants (data not shown), demonstrating that the ef-fects of BSO on cell cycle progression are directly linked todepletion of endogenous GSH. These data suggest that themechanisms acting to block the cell cycle in roots whenGSH is depleted also exist in tobacco BY-2 cells. Moreover,the G1-to-S transition appears to be a target for GSH-dependent control of progression through the cell cycle.
G1-to-S Phase Transition in Tobacco Cell Suspension Requires an Adequate Concentration ofIntracellular GSH
Two possibilities might explain the G1 block induced by de-pletion of the intracellular GSH pool: either de novo synthe-sis of GSH is required at the G1-to-S transition, or moresimply, there is a specific requirement for an adequate cellu-lar concentration of GSH. To distinguish formally betweenthese two possibilities, we analyzed the effects of an in-crease or a depletion of intracellular GSH on entry into Sphase in highly synchronized BY-2 cells. The cells weretreated either with exogenous GSH during the G1 phase orwith different concentrations of BSO before entry into G1 toensure a sufficient depletion of cellular GSH (see Methods).The effect of these treatments on cell cycle progression wasfollowed by flow cytometric analysis, measurement of DNAsynthesis, and the determination of the cellular concentra-tion of GSH, as shown in Figure 5. Synchronized cell sus-pensions that had not been exposed to these treatmentswere used to establish the timing of cell cycle events. Attime 0, all of the cells were in the beginning of G1 phase(data not shown). Three hours later, cells were at the G1-to-Stransition; at 6 hr, cells were in mid-S phase; at 8 hr, theywere in late S phase; and at 12 hr, they were in G2-to-M (Fig-ure 5A). The cellular concentration of GSH increased slightlyfrom 0 to 1 hr, remained stable until 6 hr, and began to in-crease only after 8 hr (Figure 5C).
The relatively stable GSH levels during the first 6 hr arguesagainst the hypothesis that de novo synthesis of GSH is re-quired for accomplishment of the G1-to-S transition. Fur-thermore, the rapid increase in the intracellular GSHconcentration triggered by applying GSH (100 mM or 1 mM)to the cells had no effect on the timing or duration of cell cy-cle phases, as indicated by measurement of DNA synthesis(Figure 5B) and cytometric analysis (Figure 5A). In contrast,application of BSO (100 mM or 1 mM) to the synchronizedBY-2 cells resulted in a reduction in the intracellular GSHconcentration (Figure 5C), DNA synthesis (Figure 5B), andthe proportion of cells in the G2 phase (Figure 5A). It shouldbe noted that some cells appear to be insensitive to BSOtreatment and are not blocked in the G1 phase when treatedwith 100 mM or 1 mM BSO (Figures 5A and 5B). Such cellsprogress normally through the G2 phase and into mitosis, as
Figure 4. Using a Tobacco Cell Suspension to Determine the Phe-notype of the rml1 Mutants.
(A) SR1 tobacco seedlings at 2 days after germination (DAG) grownon medium supplemented with 2.5 mM BSO.(B) SR1 tobacco seedlings at 2 DAG grown on medium lacking BSO.(C) Detail of the shoot apex of a 2-DAG SR1 tobacco seedling ger-minated and grown in the presence of 2.5 mM BSO and visualizedon a Reichert Polyvar microscope (Leica, Heerbrugg, Switzerland)using Nomarski differential interference contrast optics. Note the de-veloping leaf primordia (arrow).(D) Tobacco BY-2 cell suspensions were treated with water (Con-trol), 1 mM BSO, or as a further control to test the reversibility of theeffects of BSO, 1 mM BSO and 100 mM GSH. The effects of deple-tion of intracellular GSH on cell cycle progression were followed byusing flow cytometry. Depletion of S-phase cells is evident at 12 hr(solid arrowhead) and of G2-phase cells at 24 hr (open arrowhead).Similar results were obtained in three independent experiments.Bars in (A) and (B) 5 1 mm; bar in (C) 5 100 mm.
104 The Plant Cell
measured using the mitotic index (Figure 5 and data notshown), indicating that there is no secondary block duringG2 phase. Therefore, we conclude that exit from the G 1
phase and entry into S phase require a minimum level ofGSH (Figures 5A and 5B).
Downregulation of Cell Cycle Genes at the G1-to-S Phase Transition in Tobacco Cell Suspensions
To study the mechanisms underlying the G1 block inducedby depletion of GSH, we investigated the expression ofgenes encoding two mitotic cyclins, CycA1.1 and CycA3.2,and histone H4. Figure 6 shows that in untreated synchro-nized cell suspensions, CycA3.2 mRNA started to accumu-late at the G1-to-S phase transition at 3 hr, with kineticssimilar to H4 mRNA, whereas CycA1.1 mRNA started to ac-cumulate at mid-S phase at 6 to 8 hr (Figure 6). These varia-tions in the transcript amounts for the two cyclins are similarto that previously observed (Reichheld et al., 1996) and sug-gest a possible role for CycA3.2 in the G1-to-S phase transi-
tion and for CycA1.1 during S phase (Figure 6; Reichheld etal., 1996).
To assess directly the impact of changes in the intracellu-lar concentration of GSH on the cell cycle machinery, we in-vestigated whether the temporal pattern of cell cycle geneexpression described above was modified by the addition ofGSH or BSO. In agreement with the flow cytometric andDNA synthesis analyses (Figures 5A and 5B), exogenousGSH at 100 mM or 1 mM did not modify the expression ofthe cell cycle genes analyzed (Figure 6 and data not shown).However, depletion of GSH by BSO treatment markedlymodified the amplitude but not the timing of cell cycle geneexpression (Figure 6). Moreover, the effect of BSO treatmenton cell cycle gene expression was dose dependent (Figure6), suggesting that the intracellular concentration of GSH di-rectly influences cell cycle gene expression, either throughdirect molecular interaction or by interfering with signaltransduction.
We also analyzed the temporal pattern of parB geneexpression. parB encodes a tobacco glutathione–S trans-ferase (GST) that has been shown to be induced when to-bacco cells enter the cell cycle (Takahashi and Nagata,1992). Therefore, analysis of parB expression is of interest inthis context because the gene encodes a protein implicatedin the metabolism of GSH and is also linked to progressionthrough the cell cycle. In control cells, the steady state levelof parB mRNA showed marked variations during the cell cy-cle. parB gene expression is maximal at 6 hr (mid-S phase)(Figure 6), supporting the proposition that the product of thisgene may interact functionally with the cell cycle. Exoge-nous GSH stimulated an increase in the amplitude and theduration of parB transcript accumulation (Figure 6). BSOtreatment blocked parB mRNA expression after 3 hr, al-though treatments of 1 mM BSO led to visibly lower steadystate levels of parB mRNA than did treatments of 100 mM(Figure 6). BSO treatment did not modify the expression ofother genes whose functions are not related to the cell cyclebut related to aspects of cellular redox status (ascorbateperoxidase and Mn superoxide dismutase; data not shown).Depletion of the intracellular concentration of GSH on geneexpression thus appears to be specific to cell cycle gene ex-pression rather than to indiscriminate perturbations of tran-scription.
DISCUSSION
RML1 Encodes the Arabidopsis g-GCS
We have cloned the Arabidopsis RML1 gene based on itsmap position. Sequence analysis indicates that it encodes apreviously described metabolic enzyme, g-GCS, which isthe first enzyme of GSH biosynthesis (May and Leaver,1994; Figure 1). In Arabidopsis, g-GCS is encoded by a sin-gle nuclear gene (May and Leaver, 1994). Biosynthesis of
Figure 5. Entry into S Phase Is Regulated by the Cellular Concen-tration of GSH in Synchronized Tobacco BY-2 Cell Suspensions.
Synchronized tobacco BY-2 cells were treated with exogenous GSH(0.1 mM) at the beginning of G1, defined as time 0 of the experiment,or with BSO (0.1 or 1 mM) 2 hr before entry into G1 phase. Controlcells were treated with water at time 0.(A) Flow cytometric analysis of the cell cycle. Similar results wereobtained in three independent experiments.(B) DNA synthesis (3H-thymidine incorporation) at 6 hr, at which timeit reaches its maximum (as previously determined). Data are themeans 6SD from three replicates. C, control; prot, protein.(C) Total GSH content in control cells (solid circles), cells treatedwith 0.1 mM GSH (solid squares), 0.1 mM BSO (open squares), and1 mM BSO (open circles). Data are the means 6SD from three repli-cates. gdw, grams dry weight of tissue.
GSH-Dependent Control of Root Cell Division 105
GSH in all organisms studied to date occurs in two steps. Inthe first step, which is catalyzed by g-GCS, the presumedrate-limiting enzyme in GSH biosynthesis (Cobbett et al.,1998), g-GC is synthesized from L-glutamate and L-cysteine.The second step, which is catalyzed by GSHS, directlyyields GSH and is not affected in the rml1 mutants (Table 1).
The location of the mutation in the rml1 mutants was ana-lyzed in two allelic variants of rml1. These are rml1-1 andrml1-2 (Cheng et al., 1995) that were shown to represent thesame substitution at position 258 of the derived amino acidsequence and thus represent the same allele. The mutationis located close to a highly conserved cysteine residue atposition 251 in Arabidopsis, which is believed to be part ofthe active site (Lueder and Phillips, 1996). This region is theonly sequence that is closely related in the g-GCS of Arabi-dopsis and that of other organisms (May et al., 1998). Theabsence of detectable g-GCS activity in rml1 plants stronglysupports the proposition that this cysteine residue plays amajor role in the enzymatic activity of the Arabidopsisg-GCS. In agreement with this hypothesis, another mutationin the same gene, cad2-1, is more distant from the essentialcysteine residue, and g-GCS activity is reduced only to 40%in these mutant plants (Cobbett et al., 1998). The rml1 muta-tion probably modifies the accessibility of the cysteine resi-due or its reactivity.
Root Phenotype of rml1 Is Specifically Related to GSH
As indicated by the extremely low cellular concentration ofGSH and the complete rescue by exogenous GSH (Table 1and Figure 2), the developmental phenotype observed inrml1 mutants is related to the impairment in GSH biosynthe-sis. Due to the presence of a strong nucleophilic thiol group
on the cysteine residue, GSH is a powerful reductant and isone of the most important low molecular weight antioxidantsin plants and other organisms. It is present in high concen-trations and has proposed roles in the storage and transportof reduced sulfur, in the synthesis of proteins and nucleic acids,and as a modulator of enzyme activity (May et al., 1998).
Exogenous ascorbate could not rescue the rml1 mutants,despite partially overlapping functions between ascorbateand GSH (May et al., 1998). Similarly, the rml1 mutantscould not be rescued by a synthetic thiol, DTT. Thus, the ef-fects on root development observed in rml1 mutants arehighly dependent on the intracellular concentration of GSHand not simply on cellular antioxidant capacity. Moreover,shoot growth in rml1 suggests that the developmental ef-fects of the rml1 mutation are not simply due to a metabolicdefect, resulting from the impairment of general biochemicalprocesses requiring GSH. Therefore, the rml1 mutation re-veals that GSH fulfills more specific developmental func-tions, which until now have remained obscure despiteintense interest in this cellular antioxidant (see below).
Surprisingly, the cad2-1 mutation in g-GCS does not re-sult in a perturbation of cell division (Howden et al., 1995).However, in this mutant, the intracellular concentration ofGSH is 15 to 30% of that in the wild type (Cobbett et al.,1998), whereas it is only 2.7% in rml1 (Table 1). In the ab-sence of further mutations, we can thus assume that thephysiological differences between these two mutants are di-rectly linked to differences in the cellular concentration ofGSH. A threshold concentration must therefore exist, belowwhich developmental effects are observed in the root. Theexistence of this threshold suggests that in wild-type Arabi-dopsis, GSH is in large excess or that some compensatorymechanisms exist that become insufficient when the intra-cellular GSH concentration is too low, reinforcing the idea
Figure 6. Downregulation of Cell Cycle Genes by Depletion of Intracellular GSH Concentration in Synchronized Tobacco BY-2 Cells.
RNA gel blot hybridization analysis of histone H4 (H4), cyclin A1.1 (CycA1.1), cyclin A3.2 (CycA3.2), and parB (encoding GST) transcripts in syn-chronized tobacco BY-2 cells. The cells were treated with exogenous GSH (0.1 mM) at the beginning of G1 phase (defined as time 0 of the ex-periment) or with BSO (0.1 or 1 mM) 2 hr before entry into G1 phase. Control cells were treated with water at time 0. Loading was monitored byethidium bromide staining of the membrane (rRNA). Similar results were obtained in three different experiments.
106 The Plant Cell
that rml1 mutants are affected in a developmental processspecifically requiring GSH.
A GSH-Dependent Developmental Pathway Controls Cell Division in the Postembryonic Root
A few genes that affect cell division in shoot meristems havebeen cloned, such as SHOOT MERISTEMLESS (STM) andCLAVATA1 and 3 (CLV1 and CLV3), that could be involvedin signal transduction pathways (Meyerowitz, 1997). Thespecific effect of the rml1 mutation on cell division in thepostembryonic root has suggested that RML1 could besuch a protein (Cheng et al., 1995). The identity of RML1 asg-GCS, which is a metabolic enzyme, is thus rather surpris-ing. However, we have shown that in two different plant sys-tems, Arabidopsis and tobacco, depletion of intracellularGSH, which is the direct effect of the rml1 mutation, com-pletely inhibits cell division in the root. From the phenotypeof rml1 Arabidopsis plants and from the specific cell cycleblock induced in tobacco cell suspension by depletion of in-tracellular GSH (Figures 4C and 5), the rml1 mutation has aclear effect on cell cycle progression; thus, an adequate in-tracellular concentration of GSH is required for normal pro-gression through the cell cycle.
rml1 seedlings can produce leaves and flowers (Cheng etal., 1995), suggesting that progression through the cell cycleis not seriously affected in the shoot apical meristem. Thisindicates that the rml1 mutation (or depletion of intracellularGSH) does not affect division of all plant cells. A possible in-direct effect of GSH on root cell cycle may be through mod-ulation of upstream signal transduction processes. Becausethe oxidized form of GSH, GSSG, could not rescue the rml1mutants, the effects of GSH on root development are mostprobably derived from the reducing capacity of the thiol. In-deed, redox mechanisms function in the regulation of trans-duction pathways in animals. A number of transcriptionfactors have been reported that show redox-dependentchanges in their ability to bind DNA (Abate et al., 1990;Toledano and Leonard, 1991; Mihm et al., 1995). Moreover,activation of the animal cell cycle in response to growth fac-tors appears to be modulated by the intracellular GSH con-centration (Liang et al., 1989; Suthantiran et al., 1990),suggesting the existence of GSH-dependent transductionpathways in mammalian cells.
The absence of cell cycle activity in the postembryonicroots of rml1 mutants suggests the existence of a similarGSH-dependent transduction pathway that regulates thecell cycle in the postembryonic root and may indicate thatthis is a general mechanism in all organisms. GSH biosyn-thesis appears necessary to activate and maintain cell divi-sion in the postembryonic root. Such a GSH-dependentpathway would then play a role not only in activation butalso in maintenance of cell cycle activity in the root after ger-mination. Alternatively, GSH may not directly affect the re-dox state of target developmental regulators. Some GSTs
have been shown to be regulated during the cell cycle (e.g.,parB; Nagata et al., 1992; this study). GSH-dependent mod-ulation of the cell cycle activity during postembryonic rootdevelopment could involve specific GSH conjugates througha non-redox mechanism, although implication of GSTs indevelopmental processes clearly awaits experimental dem-onstration.
The fact that the rml1 mutation affects primarily postem-bryonic cell division in the root suggests that the embryo re-ceives GSH from the maternal tissue; in the postembryonicshoot, putative GSH-dependent factors, if present, are regu-lated by other redox-active molecules. However, we cannotexclude that differences in sensitivity to GSH between theroot and the shoot may account for the root specificity of therml1 mutation. Clearly, at this stage, the isolation of othermutants defective in the RML1 gene, and notably of a mu-tant synthesizing no GSH (if not lethal), is an indispensablestep to define precisely the function of GSH in root develop-ment.
GSH-Dependent Control of the G1-to-S Phase Transition
Quantification of the nuclear DNA content of cells in the rml1root tip did not allow us to identify a block in cell cycle–spe-cific gene expression due to the presence of endoredupli-cated cells (Figure 3). Given the absence of DNA synthesisafter germination (Cheng et al., 1995), this situation must re-flect embryonic events that are influenced by maternallysupplied GSH. However, because drug-mediated depletionof GSH in tobacco cell suspensions blocked entry into Sphase, the physiological impact of the rml1 lesion is likelyalso to be manifested as a G1 block in postembryonic roots.The depletion of intracellular GSH in tobacco cell suspen-sion downregulated two different A-type cyclins that are pu-tatively involved in the G1-to-S phase transition and inprogression into S phase (Reichheld et al., 1996; Figure 6).Cyclins have been shown to be potential targets for growthcontrol in plants (Doerner et al., 1996), and the downregula-tion of these two cyclins therefore could be partially respon-sible for the specific G1 block induced by depletion ofintracellular GSH rather than being simply the result of thecell cycle arrest or of a diminution of the population of cy-cling cells.
Cyclin-dependent kinase inhibitor (CKI) activation alsocould account for the G1 block induced by depletion of intra-cellular GSH. p21 is a CKI that was shown to be involved inmammalian cells in the negative regulation of the G1-to-Stransition (Elledge et al., 1996). Using the GSH-depletingdrug diethyl maleate, Russo et al. (1995) observed a dose-dependent induction of the p21 protein in mammalian cells.This induction was concomitant with a G1 block. Therefore,depletion of intracellular GSH could block root cells in the G1
phase in a similar fashion. The description of the first CKI inplants (Wang et al., 1997) and the availability of mutants,such as rml1, coupled with the amenability of the BY-2 cell
GSH-Dependent Control of Root Cell Division 107
suspension, clearly offer tools to dissect the molecularmechanisms underlying the G1 block induced by depletionof intracellular GSH.
METHODS
Plant Growth Conditions, Strains, and Treatments
Unless stated otherwise, Arabidopsis thaliana ecotype Columbiaplants (root meristemless1 [rml1] and the wild type) were grown asdescribed in Cheng et al. (1995). Supplementation of the growth me-dium with g-glutamylcysteine (g-GC), glutathione (GSH), the GSHoxidized form (GSSG), L-buthionine-(S,R)-sulfoximine (BSO), DTT,ascorbate, and glutamate and cysteine (Sigma) at various concentra-tions was performed as follows. Appropriate amounts of filter-steril-ized aqueous stock solutions containing these substances wereadded to sterile Petri dishes, and molten two-fifths Murashige Skoog(MS) medium (Murashige and Skoog, 1962) that had been cooled to488C was added while swirling the plates. After plating surface-ster-ilized seeds, the plates were stored in the dark at 48C for 4 days be-fore placing them in nearly vertical racks (z158 angle) at 218C undershort-day (8 hr of light/16 hr of darkness) conditions. Seedlings werephotographed with a single-lens reflex camera (model 8000S; NikonCorporation, Tokyo, Japan).
Nicotiana tabacum cv Petit Havana (SR1) seeds were grown in thepresence and absence of BSO as described above. After plating sur-face-sterilized seeds, the plates were placed at 248C under long-day(16 hr of light/8 hr of darkness) conditions and photographed with a35-mm camera (Olympus Optical Company, Tokyo, Japan).
Tobacco BY-2 Cell Suspension and Synchronization
A tobacco Bright Yellow 2 (BY-2) cell culture was maintained as de-scribed by Nagata et al. (1992). For experiments with exponentiallygrowing BY-2 cells, 3-day-old suspensions were used. For experi-ments with synchronized cells, synchronization was performed asdescribed by Reichheld et al. (1996). Briefly, sequential treatmentwith 3 mg mL21 aphidicolin (an inhibitor of DNA synthesis; Sigma) andthen with 1.54 mg mL21 propyzamide (an antitubulin drug; SumitomoChemical Company, Tokyo, Japan) was used to study the G1-to-Sphase transition specifically. For treatment with exogenous GSH orBSO at various concentrations at the G1-to-S phase transition, GSHwas added at the indicated concentration 2 hr after the removal ofpropyzamide (this later point being time 0 of the experiment); BSOwas added directly after the removal of the propyzamide. DNA syn-thesis was measured as described by Reichheld et al. (1996).
Positional Cloning and Sequencing of RML1
Generation of the segregating F2 mapping population and restrictionfragment length polymorphism (RFLP) mapping were performed aspreviously described (Cheng et al., 1995). Isolation of yeast artificialchromosome (YAC) ends and subsequent detection of ecotype-spe-cific RFLPs were as described in Schmidt et al. (1995). To constructthe T-DNA transformation-ready cosmid library, total DNA isolatedfrom the yeast strain harboring the RML1-spanning YAC cloneCIC5A4 was partially digested with MboI and size-fractionated over a
sucrose gradient to obtain 20- to 25-kb fragments. These fragmentswere then ligated to BamHI-digested pCLD04541 vector DNA andtransfected into Escherichia coli.
Approximately 3000 recombinant clones were obtained, and thesewere cultured in 96-well microtiter plates, lifted onto nylon mem-branes (Hybond; Amersham) for screening, and then stored as glyc-erol stocks at 2808C. End fragments from the cosmids of interestwere identified by electrophoresing DNA restricted with SacI, ClaI,and both enzymes. Each left a 30- to 40-bp polylinker tag on the in-sert end fragments; the resulting gels were transferred to nylon mem-branes and hybridized with each of two radiolabeled PvuII-BamHIfragments derived from the polylinker of pCLD04541.
We defined cosmid end fragments flanking the ClaI site as left-endfragments and those flanking the SacI site as right-end fragments.Cosmids were mobilized into Agrobacterium tumefaciens by elec-troporation according to the manufacturer’s instructions (Bio-Rad).Heterozygous rml1 plants were transformed with Agrobacterium bythe vacuum infiltration procedure (Bechtold et al., 1993); the resultingT1 seed was harvested from individual infiltrated plants and selectedon two-fifths MS medium containing kanamycin at 50 mg mL21. As acontrol, transformation was also performed with Agrobacterium har-boring an empty pCLD04541 plasmid. The kanamycin-resistant T1
seedlings from plants also segregating rml1 were selfed, and the re-sulting T2 seedlings were scored for both kanamycin resistance andthe Rml1 phenotype. Insert DNA from the two complementing cosmids,20A7 and 26A8, were radiolabeled and used to screen z300,000l-Prl2 cDNA clones (stock No. CD4-7; Arabidopsis Biological Re-source Center, Columbus, OH) according to established protocols(Sambrook et al., 1989). Two cDNA clones hybridizing to both cos-mids were analyzed by restriction digestion and partial sequencing.
Genomic DNA was sequenced as described previously (Cobbettet al., 1998). The GenBank accession number for RML1/CAD2 isAF068299.
Thiol Determination and Enzyme Activities
Total GSH content of BY-2 cells was determined using the recyclingenzymatic assay, as described by May and Leaver (1993). g-GC,cysteine, and total GSH, g-GCS, and GSH synthetase (GSHS) activ-ities in rml1 plants were measured, as described by Cobbett et al.(1998), from liquid-grown Arabidopsis tissues (May and Leaver,1993). Protein was determined according to the method of Lowry,with modifications by Peterson (1977).
Flow Cytometry
For flow cytometric analysis, protoplasts of tobacco BY-2 cells wereprepared for 1 hr with 2% cellulase Onozuka R10 and 0.1% pecto-lyase (Kikkoman Company, Tokyo, Japan). The cells were incubatedat 378C, washed, and lysed in Galbraith’s buffer (Galbraith et al.,1983), filtered in 1% formaldehyde through 10-mm nylon mesh,treated with RNase A, and stained with propidium iodide (50 mgmL21). Cytometric analysis was performed using 104 nuclei on anEPICS flow cytometer (Beckman Coulter, Roissy, France). For flowcytometric analysis of nuclear DNA content in roots, 10 to 30 roottips were chopped with a razor blade in Galbraith’s extraction buffer(Galbraith et al., 1983) and analyzed as described by Gendreau et al.(1998). For all results discussed in the text, two populations were es-timated as significantly different if their deviation exceeded 5%.
108 The Plant Cell
RNA Analysis
Total RNA was extracted using Trizol according to the manufac-turer’s instructions (Boehringer Mannheim). RNA gel blot analysiswas performed essentially as described by Reichheld et al. (1996),and radioactively labeled fragments corresponding to the codingregion of H4A748 (histone 4 cDNA clone A748) from Arabidopsis(Chabouté et al., 1987), parB (Takahashi and Nagata, 1992), andCycA1.1 and CycA3.2 (Reichheld et al., 1996) from tobacco wereused as probes.
ACKNOWLEDGMENTS
We thank Beatrice Satiat-Jeunemaitre, Jan Traas, and Mary AliceYund for comments on the work and critical reading of the manu-script. The cosmid vector pCLD04541 was kindly provided by BrianStaskawicz, YAC clones by Caroline Dean, and parB cDNA byToshiyuki Nagata. This work was supported by a National ScienceFoundation grant (No. IBN-9513522) to Z.R.S. and by grants from theBelgian Program on Interuniversity Poles of Attraction (Prime Minis-ter’s Office, Science Policy Programming and Grant No. 38) and theVlaams Actieprogramma Biotechnologie (No. ETC 002) to M.V.M.T.V. is recipient of Rhône-Poulenc Agrochimie and the Institut de laRecherche Agronomique for predoctoral training grants; R.C.W. re-ceived a U.S. Department of Agriculture postdoctoral grant (No. 95-37305-2299); J.-P.R. received a grant from the European Commis-sion, Research Training Project (No. ERBFMBI-CT96-1274); M.J.M.is a recipient of the European Molecular Biology Organization for apostdoctoral fellowship; and C.S.C. was supported by a grant fromthe Australian Research Council.
Received August 10, 1999; accepted November 10, 1999.
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DOI 10.1105/tpc.12.1.97 2000;12;97-109Plant Cell
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DevelopmentPathway Involved in Initiation and Maintenance of Cell Division during Postembryonic Root
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