The Plant Journal (2002) 29(1), 73±85
TUMOROUS SHOOT DEVELOPMENT (TSD) genes are required for co-ordinated plant shoot development Markus Frank1, Anne Guivarc'h2, Eva KrupkovaÂ1,², Irina Lorenz-Meyer1, Dominique Chriqui2 and Thomas SchmuÈlling1,*,² 1 UniversitaÈt TuÈbingen, ZMBP/Allgemeine Genetik, Auf der Morgenstelle 28, D-72076 TuÈbingen, Germany, and 2 Universite Pierre & Marie Curie, Laboratoire CEMV, Bat. N2, 4, place Jussieu, F-75252 Paris Cedex 05, France Received 30 July 2001; revised 24 September 2001; accepted 5 October 2001. * For correspondence (fax +49 30 83854345; e-mail
[email protected]). ² Current addresss: Free University of Berlin, Institute of Biology, Applied Genetics, Albrecht-Thaer-Weg 6, 14195, Berlin, Germany.
Summary This report describes the identi®cation of novel plant genes that are required to ensure co-ordinated post-embryonic development. After germination the tumorous shoot development mutants of Arabidopsis thaliana develop disorganized tumorous tissue instead of organized leaves and stems. This results in green callus-like structures, which are capable of unlimited growth in vitro on hormone-free medium. The tsd mutants are recessive and belong to three complementation groups (tsd1, tsd2, tsd3). The genes were mapped to the bottom of chromosomes 5 and 1, and the top of chromosome 3, respectively. Histological analyses showed that the tsd mutants have different developmental defects. The shoot apical meristem of tsd1 formed only rudimentary leaves and was characterized by a degenerating L1 cell layer. tsd2 mutants had reduced cell adhesion and altered cell division planes in the L2 and L3 cell layers. The tumorous tissue of tsd3 mutants originated from the base of the leaf. Cytokinin levels that are inhibitory to the growth of wild-type seedlings bring about an enhanced growth response in all the tsd mutants. The steady state transcript levels of the histidine kinase CKI1 gene and the KNAT1 and STM homeobox genes were increased in tsd mutants, while mRNA levels of cell cycle genes were not altered. We hypothesize that the TSD gene products negatively regulate cytokinin-dependent meristematic activity during vegetative development of Arabidopsis. Keywords: Arabidopsis, cell proliferation, cytokinin, tumor, shoot, meristem.
Introduction In ¯owering plants, morphogenesis depends upon the tight control of pattern formation, the number of cell divisions, and upon the control of cell growth. The fundamental body plan is determined during embryogenesis when shoot and root apical meristems (SAM, RAM) are established as the terminal elements of the apicalbasal axis (Steeves and Sussex, 1989). During postembryonic development most of the mitotic activity is restricted to these primary meristems, which maintain themselves and initiate the formation of organs. Some groups of specialised cells retain mitotic competence and can divide later, for example the meristemoids, which form the stomatal complexes. Very little is known about how local patterns of cell divison are generated and maintained (Meyerowitz, 1997). The SAM has been described as being organized into three distinct zones: the central zone (CZ) with slowly ã 2002 Blackwell Science Ltd
dividing cells that ensure the renewal of stem cells; a surrounding peripheral zone (PZ); and an underlying rib zone (RZ) (Kerstetter and Hake, 1997; Steeves and Sussex, 1989). The SAM initiates leaves and axillary buds from the actively dividing PZ (NougareÁde and Rembur, 1978), and the pith from the RZ. Another level of organisation of the SAM is division into superimposed clonal cell layers: a super®cial layer L1 and a sub-surface layer L2 form the tunica, divide mainly anticlinically and give rise to the epidermis and sub-epidermal tissues, respectively. Cells of the deeper layer L3 divide more randomly and form the inner parts of leaves and stems, including the pith and vascular strands. Genetic approaches have identi®ed several genes that participate in the establishment and maintenance of the meristem and the regular formation of new cells and organs. Known co-ordinating factors of cell proliferation and differentiation in the SAM are transmem73
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Table 1. Genetic analysis of the tsd mutants
Cross
Generation
Number of progeny
Progeny with tsd phenotype
Segregation ratio
c2
TSD1, TSD1 X TSD1, tsd1 TSD1, tsd1 X TSD1, tsd1 TSD2, TSD2 X TSD2, tsd2 TSD2, tsd2 X TSD2, tsd2 TSD3, TSD3 X TSD3, tsd3 TSD3, tsd3 X TSD3, tsd3
F1 F2 F1 F2 F1 F2
50 466 50 477 50 650
0 122 0 106 0 167
1 : 2.8 1 : 3.5 1 : 2.9
0.35Ëa P > 0.05 1.96a, P > 0.05 0.16a, P > 0.05
a 2
c calculated for an expected 3 : 1, wild type:mutant ratio
brane receptor proteins (e.g. CLV1; Clark et al., 1997) and homeodomain-containing transcription factors (e.g. STM, Long et al., 1996; KNAT1, Lincoln et al., 1994; WUS, Mayer et al., 1998). The gene products interact in regulatory loops to regulate the balance between continued division and cellular speci®cation (Bowman and Eshed, 2000; Fletcher and Meyerowitz 2000; Lenhard and Laux, 1999; Brand et al., 2001). Their expression is con®ned to distinct domains in the SAM (Lincoln et al., 1994; Long et al., 1996; Nishimura et al., 1999; Schoof et al., 2000). Communication between these domains is essential for co-ordination of cell division and growth activities (Fletcher et al., 1999; Lucas et al., 1995). Plant cell division, growth and differentiation are also greatly dependent upon phytohormones. Cytokinins, one class of phytohormone, were originally discovered because of their ability to promote, in concert with auxins, plant cell division (Miller et al., 1955; Frank and SchmuÈlling, 1999). Cytokinins are causally involved in inducing and maintaining growth of disorganized plant tissue, that is plant tumors (Morris, 1995). In plant tissue culture they are widely used because of their ability to induce shoot formation, indicating a role for this hormone beyond the cycling of cells (Skoog and Miller, 1957). Endogenous cytokinin overproduction in transgenic plants causes pleiotropic phenotypic alterations including cytokinin-auxotrophic growth of calli in vitro (SchmuÈlling et al., 1999). Analysis of cytokinin-overproducing and cytokininde®cient plants has con®rmed a stimulatory role in the regulation of cell division activity in the SAM and young leaves (Rupp et al., 1999; Werner et al., 2001). Recent data indicate regulatory interaction between cytokinins and class I knox homeobox genes that are active in the SAM, such as KNAT1 and STM (Chuck et al., 1996; Frugis et al., 1999; Hewelt et al., 2000; Ori et al., 1999; Rupp et al., 1999; Sinha et al., 1993). A number of mutants that have a modi®ed cytokinin metabolism or an altered response to exogenously applied cytokinins have been isolated in Arabidopsis. Loss of function mutants of the cytokinin receptor CRE1 are unable
to respond to cytokinin in tissue culture and show cytokinin-insensitive root growth (Inoue et al., 2001). Other mutants with a reduced growth response to cytokinins are ckr1 (allelic to ein2; Cary et al., 1995; Su and Howell, 1992), cyr1 (Deikman and Ulrich, 1995) and stp1 (Baskin et al., 1995). The cin1 to cin5 mutants are disturbed in the cytokinin-dependent ethylene growth response (Vogel et al., 1998). A single cytokinin-overproducing mutant, amp1 (altered meristem program 1), has been isolated (Chaudhury et al., 1993). Overexpression of the sensor histidine kinase gene CKI1 induces cytokininindependent proliferation of callus and mimicks cytokinin effects in plants (Kakimoto, 1996). Mutations in the CKH1, CKH2 and PAS genes exhibit enhanced cell proliferation in response to exogenous cytokinin (Faure et al., 1998; Kubo and Kakimoto, 2000). In an attempt to isolate mutants that show cytokininindependent dedifferentiation and growth we have previously reported the identi®cation of tissue culture lines from mutated Arabidopsis seedlings that where able to grow in vitro as shooty callus in a cytokinin-autotrophic fashion (Frank et al., 2000). The phenotype was correlated with deregulated expression of CKI1 and class I knox homeobox genes. However, these lines were obtained from a batch screen and could not be characterised further due to infertility of the mutants. Here we describe the isolation of phenotypically similar Arabidopsis mutants, which were obtained from single mutagenized lines. These mutants have disorganized shoot tissue growth and an enhanced growth response towards cytokinins.
Results Isolation of the tsd mutants Surface-sterilized Arabidopsis seeds were germinated in vitro on hormone-free MS medium and screened after 2 and 4 weeks for individuals that showed callus-like growth. Progeny of individual M1 plants were screened in order to be able to recover heterozygous siblings of ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 73±85
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Table 2. Map positions of TSD loci
Mutation
Marker
Chromosome scored
Chromosomal location
Recombination
Genetic distance
tsd1 tsd2 tsd3
MQL5 nga111 nga172
84 100 100
chr. 5, lower arm chr. 1, lower arm chr. 3, upper arm
4 10 0
4.8 cM 10 cM 1 cM
Figure 1. Postembryonic development of tsd mutants on hormone-free medium. (a) Wild type seedling (left) and tsd1 5 days after germination. (b) tsd1 mutant 14 d.a.g., 21 d.a.g. (c) and 28 d.a.g. (d). (e) Phenotype of the tsd2 mutant 14 days and (f) 21 d.a.g. (g) Phenotype of the tsd3 mutant 14 d.a.g. and (h) 28 d.a.g.
infertile mutant seedlings. Five mutants were isolated among approximately 2500 EMS-mutagenized M2 families. One additional mutant was identi®ed in a population of En-1 transposon insertion lines (Wisman et al., 1998) but was found not to be tagged (data not shown). All mutants could be maintained inde®nitely in vitro as a green, poorly differentiated callus, ful®lling our screening criteria. Progeny obtained by self-fertilization of heterozygous M2 plants of the same families were rescreened to con®rm the phenotype and then used for backcrosses with wild type. Genetic analyses were performed to test the nature of the mutation, test for allelism and localize the mutated genes in the Arabidopsis genome. Segregation analysis of the mutant phenotype in F1 and F2 populations showed that all tsd phenotypes segregated as a single Mendelian recessive trait. The F1 of a backcross has a wild type phenotype and the F2 segregates in a ratio consistent with 3 : 1, wild-type:mutant (Table 1). Complementation and mapping analyses showed that the tsd loci belong to three complementation groups (tsd1, tsd2, tsd3). The tsd1 and tsd2 mutants complemented each other and each mutant mapped to a different chromosome. TSD1 was localized on the bottom of chromosome 5, TSD2 on the bottom of chromosome 1 and TSD3 on the top of chromosome 3 ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 73±85
(Table 2). Two independent mutations were identi®ed for each mutant. Further analyses were carried out with the alleles obtained in the Col-0 gL1 background (tsd1±1, tsd2±2, tsd3±1), which are named in the following tsd1, tsd2 and tsd3, respectively. Development of the tsd mutant seedlings All homozygote tsd mutant seedlings look alike during the ®rst days after germination. They are all smaller than wildtype, with shorter, thicker hypocotyls and dark green, ¯eshy cotyledons. Figure 1(a) exempli®es this phenotype for seedlings of tsd1. Five to seven days after germination differences between the individual mutants become more apparent. About 14 d.a.g. cotyledons of tsd1 mutants become necrotic. The shoot apex forms a callus-like structure with appending leaf-like organs, which appear to be arrested in development (Figure 1b±d). tsd1 seedlings form only a short deformed thick root, which grows poorly and stops growth completely several days after germination (data not shown). We noted only a late and relatively weak in¯uence of the mutation on embryonic development. No alterations were detected until the bent cotyledon stage. Compared with the post-embryonic
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Markus Frank et al. Figure 2. Histological analysis of the tsd1 mutant. (a) Longitudinal section through a wild type seedling ®ve d.a.g. (b) Enlargement of the SAM shown in (a). (c) Longitudinal section through the pre¯oral SAM of wild type 14 d.a.g. (d) Longitudinal section through the shoot of a tsd1 seedling 5 d.a.g. (e) Median section of SAM of tsd1 7 d.a.g. (f) Median section of the apical callus of tsd1 14 d.a.g. (g,h) Meristem-like and leaf-like structures formed in the apical callus of tsd1 after several weeks of subculture. Bar size, 50 mm.
developmental changes we detected only a late and relatively mild embryonic phenotype. Alterations were visible only in embryos after the bent cotyledon stage. Mature embryos showed a reduced organ elongation with shorter hypocotyls and more round-shaped and smaller cotyledons, which tended to stay fused (Frank, 1999). Young tsd2 seedlings show, in addition to the reduced stature, a large accumulation of anthocyanins in the apical part of the hypocotyl. The cotyledons remain fused and their surface appears more irregular than in tsd1 and wildtype. They necrotize at about 14 d.a.g. (Figure 1e). The callus-like tissue produced by the SAM develops numerous leaf-like structures, which elongate more than in tsd1 mutants (Figure 1f). The leaf tissue appears vitri®ed and falls apart easily as adhesion of cells is reduced. In contrast to tsd1, tsd2 mutants develop roots that grow similarly to wild-type (Figure 1e). Callus formation in the tsd3 mutants differs from tsd1 and tsd2. tsd3 mutant seedling are also characterized by a short hypocotyl and root (Figure 1g). Cotyledons are vitreous and soon after germination they show epinastic growth. About 7 d.a.g. the SAM starts to form leaf-like structures, which resemble leaves without petioles.
Around 4 weeks after germination these leaf-like organs form callus tissue at their base, which continues to produce further leaf-like structures (Figure 1h). The root of tsd3 stops growth after elongating several mm. A common characteristic of all tsd mutant calli is the ability to proliferate inde®nitely on hormone-free MS medium. In contrast, wild-type calli require exogenous auxin and cytokinin. All mutant calli grew more rapidly on hormone-free medium than wild-type calli on hormonecontaining medium (data not shown). Histological analysis of the tsd1 and tsd2 mutants In order to obtain more information about the origin and structure of the disorganized tissue that was formed by the shoot apex of the tsd mutants we analysed longitudinal sections through the SAM at different developmental stages. Wild-type seedlings form a dome-shaped SAM with anticlinal cell divisions in the L1 and L2 layers. The only periclinal divisions in L2 occur in the peripheral zones at the onset of leaf initiation (Figure 2a,b). About 14 d.a.g. the SAM develops a bulging pre¯oral meristem (Figure 2c). Figure. 2(d,e) shows that several days after germinã Blackwell Science Ltd, The Plant Journal, (2002), 29, 73±85
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Figure 3. Electron microscopic analysis of the shoot apical meristem. Longitudinal sections of wild type SAM (a,b) and the SAM of tsd1 (c,d). Note the higher electron density of the cytoplasm in tsd1, the degeneration of cells of the L1 layer and the presence of amyloplasts in meristematic cells. Magni®cation is 3600 3 (a,c) and 7200 3 (b,d), respectively. Seedlings were 7 days old.
ation the SAM of tsd1 mutant seedlings looks structurally distinct from wild-type SAM. The L1 appears to have a more irregular surface with numerous small cells. In the inner L3 layer, the appearance of relatively small cells clearly divided by newly developing cell walls indicates more and disorganized cell divison activity in this region (Figure 2e). At 7 d.a.g. the SAM of tsd1 has enlarged without forming leaves. Leaf primordia initiated at the SAM border stop developing and become incorporated into the growing apical callus. Two weeks after germination tsd1 mutants have formed an apical callus of app. 500±1000 mM diameter (Figure 2f). The callus is formed mainly from cells that originate from the anarchic L2 and L3 proliferation and is covered with a non-continuous cell layer. The L2 is no longer recognizable as a distinct cell layer. Groups of smaller, actively dividing cells can be distinguished in the outer part of the callus, while larger, vacuolated cells form the callus body (Figure 2f). Groups of smaller cells occasionally form ordered meristem- and leaf-like structures, which show structural de®ciencies and do not develop further (Figure 2g,h). Abundant starch granules can be identi®ed in cotyledons of tsd1, which are not present in wild-type cotyledons (data not shown). In addition, the structural disorganization in tsd1 is not limited to the shoot apex and appending tissues. The hypocotyl of tsd1 is thicker and cells are shorter than in wild-type (Figure 2d and data not shown). Furthermore, the vascular tissue is distorted and does not form continuous strands. Contrasting with the regular organization ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 73±85
in cell ®les observed in wild-type roots, the tsd1 root consists of only a few irregular and thickened cells (Figures 7, 8 and data not shown). Electron microscope pictures of wild-type SAM showed the classic, regular arrangement of tunica layers and a normal cellular infrastructure (Figure 3a,b). Cells were poorly vacuolated, contained juvenile chloroplasts and small intercellular spaces. In contrast, 5 d.a.g. tsd1 mutant seedlings showed distinct changes. Although they contain a dense accumulation of ribosomes, the SAM cells became stronger vacuolated and contain larger amyloplasts. They were also characterized by the presence of abundant dictyosomes along the cell walls. Electron dense material has accumulated at the cuticle, the plasmalemma, dictyosomes and the nuclear envelope (Figure 3c). Cell adhesion was partially disturbed as indicated by large intercellular spaces that have formed between L1 and L2. Plasmodesmata that are distinguishable between L1 and L1/L2 cells in wild-type were absent in tsd1 mutants. Cells of the L1 layer ± and occasionally of the L2 layer ± became necrotic, the cell layer was disorganized and eventually collapsed (Figure 3c,d and data not shown). Vacuolar accumulation of dense membrane material accompanied the necrotic evolution of these cells (Figure 3d, arrow). Nuclei were more ovoid or amiboid than spherical. In addition, 10 d.a.g. the cotyledons contain an abundance of storage molecules, namely large and abundant amyloplasts, very dense protein bodies and osmophilic lipid bodies, which are all absent in wild-type (data not shown).
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Markus Frank et al. Figure 4. Histological analysis of the tsd2 mutant. (a) Longitudinal sections through hypocotyl and SAM of tsd2 seven d.a.g. (b) Longitudinal section through the tsd2 SAM 10 days d.a.g. (c) Transverse sections through a wild type leaf and a leaf of tsd2 (d) 7 d.a.g.
Disruption of the L1 layer was con®rmed by the analysis of expression of the L1-speci®c ATML::GUS marker gene expression in the tsd1 background (Sessions et al., 1999). However, blue patchy staining on the surface of the tsd1 callus at later developmental stages indicated that the L1 was not completely destroyed. Cells with L1 identity were still existing and propagated or were newly formed (data not shown). Microscopic analysis of the SAM of tsd2 showed that the apex was ¯attened compared with the dome-shaped form of wild-type (Figure 4a). Only the L1 layer showed a regular structure. The L2 showed periclinal cell divisions and the L3 contained larger vacuolated cells. Typical of tsd2 is a disturbance of cell adhesion. In all tissues there are groups of rounded cells, which lack physical contact. This is particularly evident in leaves. Wild-type leaves have an orderly layered structure of differentiated cells (Figure 4c). In contrast, the leaves of tsd2 consist of poorly differentiated cells, which were not truly recognizable as epidermal, spongy or palisade cells (Figure 4d).
The tsd mutants respond to cytokinins with increased growth Because of the mutant's ability to grow without the addition of hormones we were interested in studying the hormonal content and the growth reaction in response to exogenous hormones. Analysis of the content of auxin and cytokinins revealed no signi®cant differences between mutant and wild-type callus or seedlings (M. Strnad and H. van Onckelen, pers. comm.). Minor differences were possibly due to the altered tissue composition of the
mutants and were not analysed further. Wild-type and mutant seedlings had the same qualitative response when germinated and grown on media containing auxin or gibberellic acid. Increasing auxin concentration inhibited growth and triggered root hair formation, while gibberellic acid stimulated hypocotyl elongation (data not shown). Figure 5 shows that growth of wild-type seedlings was inhibited by exogenous cytokinin. In contrast, the shoots of all tsd mutant seedlings reacted by hyperplasia (Figure 5a±c and data not shown).
The tsd mutants have an altered gene expression Previous analyses had shown that the tumorous shoot phenotype can be associated with the deregulation of the histidine kinase gene CKI1, meristem-specifying class I knox homeobox genes and/or cell cycle genes (Frank et al., 2000). In order to test whether the tsd mutants would ®t into the same operational concept and as a ®rst step to get insight into possible molecular mechanisms causing the mutant phenotype, we analysed steady state transcript levels of different genes in the tsd mutants and compared them with the levels in wild-type seedlings and callus. Figure 6(a) shows that the transcripts of CKI1 were barely detectable in the control tissues but that a strong signal was detected with RNA of tsd1 and tsd2. tsd3 showed a weaker enhancement of the CKI1 transcript. All three mutants displayed an increased transcript abundance of KNAT1 and STM (Figure 6b) while CycD2 and CycD3 transcripts were similar to wild-type (Figure 6c). The expression of some of these genes was further analysed by crossing tsd1 with transgenic Arabidopsis ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 73±85
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Figure 5. Growth of wild type (a), tsd1 (b) and tsd2 (c) on cytokinin-containing medium. On the left of each ®gure is a a seedling grown for 24 days on hormone free medium, on the right a seedling grown on medium containing 15 mM 2iP.
Figure 6. Northern blot analyses of tsd seedlings. (a) Steady state mRNA levels of the CKI1 gene. (b) Steady state mRNA levels of the KNAT1 and STM genes. (c) Steady state mRNA levels of CycD2 and CycD3 genes. Abbreviations: wt, s, wild type seedling d.a.g.; wt, c, wild type callus grown on medium containing 1 mg l±1 NAA and 0.1 mg l±1 iP. Control hybridization was carried out with a 25S rRNA probe.
harboring promoter-GUS fusion genes for the KNAT1 (Chuck et al., 1996), KNAT2 (Laufs et al., 1998; Pautot et al., 2001) or cycB1 promoter (ColoÂn-Carmona et al., 1999). In the latter construct the GUS enzyme carries a destruction box signalling proteolytic destruction at the end of M phase. Therefore, only cycling cells are marked (ColoÂnCarmona et al., 1999). Figure 7(a) shows that cell divisions in the shoot of wild-type are limited to the SAM and the bases of developing leaves. In the root, single cells of the ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 73±85
meristem were stained (Figure 7b). Cell divisions in the young seedlings (3 d.a.g) of the tsd1 mutants are also mainly con®ned to the SAM and RAM (Figure 7c). Five d.a.g. cell divisions in the root have ceased and are only seen in the shoot (Figure 7d). Later in development (14 d.a.g.) cell divisions are localized to distinct regions of the shoot callus, which possibly correspond to the clusters of smaller cells seen in Figure 2(f) (Figure 7e). KNAT1 expression in wild-type seedlings is detected in the SAM and the vasculature (Figure 8a; Ori et al., 2000). Staining of the SAM is detected early after germination in the tsd1 mutant background, while staining of the vasculature is weak and seen only rarely (Figure 8b). At later developmental stages staining in the tsd1 mutant background was entirely con®ned to the meristem and growing callus. Figure 8(c) shows that the SAM enlarges rapidly, which leads eventually to fasciation and the formation of numerous separate meristematic regions at the callus surface. The stained areas extend into the inner part of the callus (data not shown). Similar to KNAT1::GUS, expression of KNAT2::GUS, which marks the inner tissue of the SAM in wild-type (Pautot et al., 2001), is strongest in the SAM (Figure 8d). Cotyledons of tsd1 showed two stripes of KNAT2::GUS expression, which was absent in the wild-type (Figure 8e). KNAT2::GUS expression in the tsd1 callus primarily occupied a large area (Figure 8f), indicating that the inner region of the SAM might be extended.
Discussion We have isolated three mutants of Arabidopsis that show severe developmental defects. Common characteristics of all tsd mutants are: the formation of disorganized tumorlike shoot tissue; their ability to grow in vitro on hormone free medium; and a hypertrophic growth reaction to cytokinins. The tsd1 and tsd2 mutant calli are derived from the SAM, which shows aberrant cell division activity and has lost the ability to form proper lateral leaf organs. tsd3 mutants differ from the previous two as the formation of tumorous tissue is a later event and it originates not from the SAM but from the base of the leaf. Because of these and other differences it is likely that different
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Markus Frank et al. Figure 7. CycB1::GUS expression in wild type and the tsd1 mutant. Expression in the wild-type is found in the SAM, young leaves (a) and root tips (b). Expression in tsd1 3 d.a.g. (c), 14 d.a.g. (d) and 25 d.a.g. (e), respectively, is localized to de®ned regions of the apical callus. No expression is detected in roots later than 3 d.a.g.
Figure 8. KNAT1::GUS and KNAT2::GUS expression in wild type and the tsd1 mutant. (a) KNAT1::GUS expression in a wild type seedling 7 d.a.g. and in the tsd1 background 5 d.a.g. (b) and 21 d.a.g. (c). KNAT2::GUS expression in a wild type seedling 2 d.a.g. (d) and in the tsd1 mutant 5 d.a.g. (e) and 21 d.a.g. (f; the apical callus was split after staining to show the inner tissue).
molecular mechanisms are the causes of failure in normal development. The tsd mutants are related to other known mutants of Arabidopsis. They resemble most closely the pasticcino mutants (pas1, pas2, pas3), which respond to cytokinin
with hypertrophy of the shoot (Faure et al., 1998). The pas1 gene codes for an immunophilin, which has a role in cell divison (Vittorioso et al., 1998). However, the pas mutants show distinct developmental differences when compared with the tsd mutants, in particular with tsd1. For example ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 73±85
Novel Arabidopsis differentiation mutants root formation and elongation are less affected in pas mutants and formation of ®nger-like leaves indicates that the effects on leaf differentiation are less pronounced than in tsd mutants (Faure et al., 1998). PAS genes map to different chromosomal locations and pas1 does not complement tsd1 (Faure et al., 1998; data not shown). The ckh1 and ckh2 mutants in tissue culture are more sensitive to cytokinin than wild-type (Kubo and Kakimoto, 2000). However, the mutant plants form fertile shoots and the genes map to different chromosomal locations than TSD genes. tsd2 mutants share some features with cristal (cri1; Delarue et al., 1997), which has a vitreous appearance and disturbed cell adhesion in leaves. Vitri®cation can be induced in vitro by cytokinins (Leshem et al., 1988) and cri1 mutants have an enhanced cytokinin content (Santoni et al., 1997) but CRI1 does not map to the same chromosomal location as TSD2 (Delarue et al., 1997). tsd3 mutant seedlings resemble other mutants with an aberrant differentiation, in particular gurke (gk; Torres-Ruiz et al., 1996). Unlike this mutant tsd3 has lost cell division control leading to callus-like growth. TSD3 maps on chromosome 3, GK maps on chromosome 1 (Torres-Ruiz et al., 1996). The histological analysis of tsd1 and the analysis of gene expression give some clues, which lead to a preliminary interpretation of the phenotype. The main features of the SAM of tsd1 mutants are a degenerating L1 layer that sometimes affects L2 cells, an overall increase in cell division activity in L3 and L2 cell layers and the loss of clear cytological zones and the inability to develop leaves, although leaf primordia and rudimentary leaves are initiated. Communication between cell layers plays a crucial role in meristem organization. An example is the regulatory interaction between CLV and WUS, which have partly overlapping expression domains (Schoof et al., 2000). A non-cell autonomous activity has also been described for several other meristem identity genes, such as AG and ¯oricaula, the LFY homolog of Antirrhinum (Carpenter and Coen, 1995; Sieburth et al., 1998). It was shown in tomato that cells of the L3 layer control organ initiation and meristem size (Szymkowiak and Sussex, 1992, 1993). Transport of mRNAs and protein across layer boundaries has been reported (Lucas et al., 1995). It could be that disturbed communication between different zones of the SAM are a reason for the mutant phenotype. Overproliferation of the L3 cells in tsd1 seedlings might lead to lack of organization of the corpus and of procambium differentiation and ®nally cause mechanical tension in the tissue, eventually leading to disruption of the L1. An alternative possibility in the same context is that destruction of the L1 layer is a primary event. The L1 layer is possibly important for functionally organizing underlying cell layers. This idea is based upon the observation that during shoot meristem formation from disorganized callus the ®rst visual sign of the formation of a true ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 73±85
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meristem from meristematic centers is the appearance of a uniserrate layer with only anticlinal divisions (Nitsch and Lance-NougareÁde, 1967). The formation of the L1 layer is also a very early event during zygotic (Mans®eld and Briarty, 1991) and somatic embryogenesis (Norreel, 1973). In addition to an organizing role the L1 has a function as a mechanical barrier (Kutschera, 1992). Therefore, one possibility is that the lack of a functional L1 is the cause for uncontrolled cell divisions in L2 and L3 as well as the lack of regular leaf initiation. The reduced cell adhesion, which is visible in tsd1 and even more prominent in tsd2, could have a substantial role in the generation of the phenotype as well. This has been demonstrated for the pas mutants (Faure et al., 1998), gn/ emb30 (Shevell et al., 2000) and kor (Nicol et al., 1998; Zuo et al., 2000). Besides structural constraints, the biochemical and molecular composition of the extracytoplasmic component can in¯uence intercellular signalling as well by enhancing or attenuating the exchange of the signal molecules. The transcript abundance of several genes that are functionally linked to differentiation of the shoot meristem is enhanced in the tsd mutants. This is particularly intriguing in the case of CKI1, which was also found previously to be enhanced in shooty callus lines resembling tsd mutants (Frank et al., 2000). CKI1 encodes a histidine kinase of the two-component signalling pathway, which is related to the cytokinin receptor CRE1 (Inoue et al., 2001). Overexpression of CKI1 causes a phenotype that mimics several aspects of enhanced cytokinin activity, that is greening and cytokinin-independent proliferation of shooty callus (Kakimoto, 1996). CKI1 transcripts were not detected in wild type seedlings or callus and were strongly increased in tsd1 and tsd2 mutant tissue, and weakly increased in tsd3 mutants (Figure 6a). In fact, CKI1 expression is con®ned in wild-type to reproductive tissues (T. Kakimoto, pers. comm.). This indicates that the lack of repression of CKI1 is speci®cally linked to the mutations in TSD1 and TSD2. Therefore, one important function of TSD1 and TSD2 might be the negative regulation of CKI1 gene expression. However, overexpression of CKI1 can only explain part of the phenotype (cytokinin-independent propagation, shooty appearance) as CKI1 overexpressors are fertile plants (Kakimoto, 1996). It could be that negative regulation affects a larger set of genes, which share the expression domain with CKI1. Deregulated expression of a whole gene set could be incompatible with proper shoot development. For example, loss of function mutation in the chromatin remodeling factor gene PKL leads to pleiotropic effects including the activation of lipid biosynthesis and other embryonic features in the root and the suppression of various differentiation processes which are triggered by gibberellin (Ogas et al., 1999).
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tsd mutants also contained enhanced steady state mRNA levels of class1 knotted1-like homeobox (knox) genes of Arabidopsis, a feature that they share with the shooty callus lines (Frank et al., 2000). These knox genes are expressed in different domains of the shoot meristem, their expression disappears prior to leaf primordia formation (Kerstetter and Hake, 1997). STM, as well as the maize KNAT1 counterpart knotted1, have been shown to be essential for meristem maintenance (Barton and Poethig, 1993; Endrizzi et al., 1996; Vollbrecht et al., 2000). Deregulated expression of the class1 knox genes suggests that they suppress differentiation of meristematic cells (Chuck et al., 1996; Lincoln et al., 1994; Williams, 1998). Therefore, increase in expression of these genes might have a role in establishing the tsd phenotype and enhance cell growth if their expression is increased further by exogenous cytokinins (Rupp et al., 1999). This could be particularly relevant in the rib meristem, which has been reported to contain cytokinin-responsive cells (Mauseth, 1979). Interestingly, expression of WUS, a distantly related homeodomain protein that is required for speci®cation of stem cells (Mayer et al., 1998), in a broader domain in the SAM causes a phenotype that to some extent resembles tsd1. Meristems are fasciated, larger in size and several layers of densely staining cells overlay a corpus of large vacuolated cells (Schoof et al., 2000). However, at present it is not excluded that the enhanced steady state levels of the homeobox genes in tsd mutants is a consequence of the mutant's tissue morphology (resulting in an expanded expression domain) rather than its cause. Another important result of the transcript analysis is that the abundance of CycD2 and CycD3 transcripts is not altered in the mutant tissue (Figure 6). Difference in transcript levels of cyclin genes, in particular of CycD3, is often correlated with tumorous growth in animal cells (Hunter, 1997). Also in Arabidopsis, overexpression of CycD3 leads to uncoordinated growth of tissues (RiouKhamlichi et al., 1999). While our previous work has shown that disorganized shooty growth can be correlated with altered CycD3 gene transcript levels (Frank et al., 2000), this seems not to be the case in the tsd mutants. Taken together, TSD genes are required for coordinated development of Arabidopsis. The histological and molecular analyses have yielded some clues to interpret TSD gene function. These results indicate a role for TSD gene products in the structural organization of the plant body, possibly linked to functions of cytokinin. More speci®cally, it could be that TSD genes negatively regulate cytokinindependent processes in the SAM, which lead in the absence of additional cytokinin to disorganized growth and with additional exogenous cytokinin to an overproliferation of the responsive cells. Finally, only identi®cation of the mutated genes will elucidate the underlying
molecular changes. Map-based cloning of the mutated genes is in progress.
Experimental procedures Plant growth conditions and tissue culture Callus cultures of mutants were grown on hormone-free MS medium (3% sucrose, 9% agar) at 24°C and a 16-h light/8-h dark cycle. Calli were transferred weekly to fresh medium. Wild-type calli were induced by plating wild-type seeds on MS medium containing 1 mg l±1 NAA and 0.1 mg l±1 iP. Wild type calli formed 4 weeks after germination. They were sub-cultured weekly on MS medium containing 1 mg l±1 NAA and 0.1 mg l±1 iP. The growth reaction of seedlings to exogenous hormones was tested on MS medium supplemented with either auxin (0.1±15 mM NAA), gibberellic acid (1 mM ± 10 mM) or cytokinin (0.1±15 mM iP). Surface-sterilized seeds were sown and the growth reaction was recorded 3 weeks after germination. Soil-grown plants were cultivated in a growth chamber at 24°C and 16 h light.
Mutagenesis and screening procedure For mutagenesis, M1 seeds of Arabidopsis thaliana (L.) Heynh. accession Col-0 gL1 were soaked for 12±16 h in 0.25±0.3% v/v ethyl methane sulfonate, then washed with water (JuÈrgens et al., 1991). M2 seeds were harvested from approximately 2500 individually grown M1 plants and used for screening. Based on the frequency of embryo lethal mutations and albino and sur1 mutants, the expected average gene mutant frequency in this population was approximately 1 3 10±3. Additional screening was carried out upon approximately 600 M2 families of A. thaliana accession C24 (kindly provided by H. Hellmann) and lines carrying insertions of transposon En-1 (Wisman et al., 1998). For screening of mutants, seeds of individual M2 families were surface sterilized, 40 seeds per family were plated on MS medium, vernalized for 2 days at 4°C, transferred to standard growth conditions and screened for callus-like growth of seedlings 14 d.a.g. and 28 d.a.g. Seedlings of the same family that did not show dedifferentiation were transferred to soil in order to obtain M3 seeds from heterozygous progeny. Heterozygous M3 plants were backcrossed twice with wild-type. tsd1±1, tsd1±2, tsd2±1 and tsd3±2 were identi®ed in accession Col-0 gL1, tsd3±1 was identi®ed in accession C24, and tsd2±2 was identi®ed in the En-1 mutated population. The latter mutant was kindly provided by M.-T. Hauser.
Genetic mapping Homozygous mutants plants were infertile. Therefore, fertile heterozygote mutant plants were crossed with wild type plants of accession Landsberg erecta. The progeny of these crosses was self-fertilized and F2 progenies that segregated for the tsd mutant phenotype were used for mapping. DNA was extracted from individual green F2 calli according to Rogers and Bendich (1988). Recombination frequencies between mutant phenotype and simple sequence length polymorphism (SSLP) or cleaved ampli®ed polymorphic sequences (CAPS) were measured essentially according to established methods (Bell and Ecker, 1994; Konieczny and Ausubel, 1993). Genetic distances were calculated according to the Kosambi function (Koornneef and Stam, 1992). ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 73±85
Novel Arabidopsis differentiation mutants RNA blot analysis Total RNA was extracted from plant tissues according to Verwoerd et al. (1989). 50 mg RNA was separated in a denaturing 1.5% agarose-formaldehyde gel, transferred to nylon membranes (Amersham hybond N) and hybridized with radioactive-labelled DNA probes as described previously (Frank et al., 2000). The lowest stringency wash was performed in 0.2 3 SSC, 0.1% SDS at 65°C. As a control for loading, the blot was rehybridized with a 25S rDNA probe.
Microscopy Samples were ®xed during 4 hours at 4°C in 4% paraformaldehyde and 1% glutaraldehyde in PBS. After several washes in PBS, they were post®xed in 1% osmium tetraoxide for 2 hours. Samples were then washed with distilled water, dehydrated through an ethanol series, treated by propylene oxide and ®nally embedded in an epoxy resin (Durcupan, Fluka, Buchs, Switzerland). For light microscopy, semithin sections (3 mm) were placed on glass slides and stained with Paragon (0.37 g toluidine blue and 0.27 g basic fuchsine in 30% ethanol). For electron microscopy, ultra-thin sections (0.5 mm) were placed on copper grids coated with formvar and counterstained with uranyle acetate and lead citrate (Reynolds, 1963). Sections were observed under a Philips EM 201 electron microscope (Philips, Eindhoven, The Netherlands).
GUS staining Histochemical analysis of the GUS reporter enzyme was performed essentially according to Jefferson et al. (1987), modi®ed by Hemerly et al. (1993). Sample tissues were ®xed in 90% ice-cold acetone for 1 hour and incubated for 1±12 hours in reaction buffer. Endogenous pigments were destained with 70% ethanol and the GUS staining pattern recorded under a stereomicroscope (Olympus SZX9, Olympus, Hamburg, Germany) or a microscope (Zeiss Axioskop, Jena, Germany) equipped with a photographic device.
Acknowledgements We thank P. Doerner, J. Traas, S. Hake, A. Sessions and D. Weigel for providing seeds of cycB1::GUS, KNAT2::GUS, KNAT1::GUS and ATML1::GUS transgenic plants, respectively. We are indebted to M.T. Hauser and M. Rie¯er for providing a second mutant allele of tsd1 and tsd2, respectively. We thank H. Hellmann for providing the EMS mutant collection of accession C24. We are grateful to Catherine Scott-Taggart for proofreading. We acknowledge support of the Deutsche Forschungsgemeinschaft (SFB 446). E. Krupkova received a stipend of the DAAD. M. Frank was a stipend of the Studienstiftung des deutschen Volkes.
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