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The WUSCHEL and SHOOTMERISTEMLESS genes fulfil complementary roles in Arabidopsis shoot meristem regulation

Michael Lenhard1, Gerd Jürgens2 and Thomas Laux1,*

1 Institut für Biologie III, Universität Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany
2 Universität Tübingen, ZMBP – Entwicklungsgenetik, Auf der Morgenstelle 1, D-72076 Tübingen, Germany



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  		Fig. 1. Ectopic STM expression suppresses cell differentiation. (A) Light micrograph of a non-transgenic stm5 mutant seedling 8 days after germination. Cotyledon petioles are fused and no leaves have been formed. (B) Light micrograph of an stm5 mutant seedling expressing CLV1::STM 8 days after germination. The first pair of leaves formed by the SAM is visible (arrow). The bases of the cotyledon petioles are fused as in the seedling shown in A. We used the CLV1 promoter, which is active in the centre of the embryonic shoot meristem primordium from heart-stage onward, and whose initial activation does not require STM function (Long and Barton, 1998Go), since no STM promoter has been described that mimics the endogenous mRNA expression pattern. (C,D) Micrographs of DAPI-stained seedlings. (C) stm5 mutant seedling 5 days after germination. No meristematic cells are visible inside the fused cotyledon petioles (arrow). (D) CLV1::STM-expressing stm5 mutant seedling 5 days after germination. A meristematic region is evident from the bright signal from cytoplasmically dense cells inside the fused petioles (arrow). (E,F) Scanning electron micrographs. (E) Wild-type seedling 10 days after germination. c, cotyledon; l, leaf. (F) ANT::STM-expressing seedling with a strong phenotype 21 days after germination. The petioles of the cotyledons (cp) are broader than in wild type (compare with E). Leaves (l, arrow) are not expanded and are rolled up at their margins. h, hypocotyl. (G) Light micrograph of a mature second rosette leaf of a wild-type plant. (H) Light micrograph of a mature second rosette leaf of an ANT::STM-expressing plant with a weak phenotype. The petiole (asterisk) is broader than wild type and lateral outgrowths have developed into leaf-like structures (arrow). (I-L) Cross-sections of plastic-embedded leaf material from seedling 12 days after germination, stained with Toluidine Blue. (I) Petiole of the first rosette leaf of a wild-type plant. A vascular bundle (arrow) with differentiated cells lacking cytoplasm is surrounded by large, vacuolated cells. (J) Basal part of the first rosette leaf of an ANT::STM-expressing seedling. The cells in place of the vascular strand (arrow) are cytoplasmically dense and the cells throughout the petiole are less expanded than in G. (K) The lamina of the first rosette leaf of a wild-type plant. Note the high degree of vacuolation and the large intercellular spaces (asterisk). (L) The lamina of the first rosette leaf of an ANT::STM-expressing seedling. Cells throughout the leaf are smaller than in I and contain more cytoplasm, indicating that differentiation is suppressed. Scale bars are 500 µm in C-H, 100 µm in I-L.

 


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  		Fig. 2. Marker gene expression in ANT::STM plants. (A-H) Light micrographs of GUS-stained, cleared seedlings. (A) CycB1;1::CDBGUS expression in wild type. Staining is restricted to the SAM region and young leaves (arrowhead), but is absent from the expanded first pair of rosette leaves (arrow). (B) CycB1;1::CDBGUS; ANT::STM-expressing seedling of the same age as the one in A. Staining is seen throughout the first pair of rosette leaves (arrow). (C) CycB1;1::CDBGUS; ANT::STM seedling with intermediate phenotype. Ectopic GUS staining is observed in the lateral outgrowths of the leaves (arrows). (D) ANT::STM; ANT::GUS-expressing seedling. The transgenes are strongly expressed in the vasculature of the cotyledons (c), leaf primordia (arrowhead) and in older leaves with stronger staining at the tips (arrow), as well as in their lateral outgrowths (not visible). (E) KNAT1::GUS expression in wild type. Staining is restricted to the SAM region and hypocotyl, yet is absent from leaves. (F) KNAT1::GUS; ANT::STM-expressing seedling. Ectopic GUS staining is seen in the vasculature of the cotyledons (c) and in strongly affected leaves (arrow). (G) KNAT2::GUS expression in wild type. Staining is restricted to the SAM region and is absent from cotyledons (c) and leaves (arrow). (H) KNAT2::GUS; ANT::STM-expressing seedling. Ectopic GUS staining is observed in the vasculature of the cotyledons (c) and in leaves (arrow). (I,J) In situ hybridization with a CLV3 antisense riboprobe. In both wild-type (I) and ANT::STM-expressing (J) seedlings, CLV3 mRNA is exclusively detected in the stem cells in the three outermost layers of the SAM. Scale bars are 1 mm in A-H, 100 µm in I,J.

 


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  		Fig. 3. Marker gene expression in 35S::WUS-GR-expressing plants. (A) 35S::WUS-GR-expressing seedlings (lower left) show the same phenotype with inhibition of cotyledon expansion, root growth and greening as 35S::WUS; 35S::GUS-expressing seedlings (upper left) when germinated on dexamethasone containing medium, but not on control medium (lower right). (B) Longitudinal section through a GUS-stained CLV3::NLSGUS-expressing plant. Staining is restricted to the stem cells of the SAM, mirroring the CLV3 mRNA expression pattern (compare with Fig. 2I). (C-J) Light micrographs of GUS-stained and cleared seedlings. Seedlings in C,E,G,I were treated with mock solution for 2 days, while seedlings in D,F,H,J were induced with 5 µM dexamethasone for the same time. (C,D) After dexamethasone treatment of 35S::WUS-GR; CLV3::NLSGUS seedlings (D), strong ectopic GUS expression is observed in cotyledons (c), leaves (l) and hypocotyl (h), mainly associated with vascular strands, while expression is restricted to the stem cells of the SAM in uninduced seedlings (arrowhead, C). (E,F) No difference in the GUS staining pattern is observed between dexamethasone induced (F) and uninduced (E) 35S::WUS-GR; KNAT1::GUS-expressing seedlings. (G,H) No difference in the GUS staining pattern is observed between dexamethasone induced (H) and uninduced (G) 35S::WUS-GR; KNAT2::GUS-expressing seedlings, even though the first morphological effects of ectopic WUS activity on young leaves – reduced expansion of the lamina and upright position – are already visible (arrowhead). (I,J) Occasional ectopically staining cells are visible along the vasculature of the first pair of rosette leaves in dexamethasone-treated 35S::WUS-GR; CycB1;1::CDBGUS-expressing seedlings (arrowhead in J), which were never observed in mock-treated seedlings of the same genotype (arrowhead in I). Scale bars are 5 mm in A, 100 µm in B and 500 µm in C-J.

 


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  		Fig. 4. Independent functions of WUS and STM. Light micrographs of live seedlings (A,E-H) and GUS-stained, cleared seedlings (B-D,I-L). (A,B) ANT::WUS; ANT::GUS-expressing wild-type seedlings 12 days (A) and 10 days (B) after germination. An enlarged SAM has developed in place of leaves (A) which strongly expresses the transgenes (B). (C-E) ANT::WUS; ANT::GUS-expressing stm5 mutant seedlings 10 days (C) and 18 days (D,E) after germination. Transgene expression has only been initiated in a few cells (arrow) inside the fused cotyledon petioles in the seedling in C from which a mass of small meristematic cells develops subsequently (D,E arrow). In E, the fused cotyledon petioles have been cut open for clarity. (F) Non-transgenic stm5 mutant seedling 18 days after germination. Several leaves have been formed and have ruptured the fused wall of the cotyledon petioles. (G) ANT::STM-expressing wild-type seedling. Leaves are reduced to finger-like, lobed structures (arrow) and the petioles of the cotyledons (c) are broadened. (H) ANT::STM-expressing wus1 mutant seedling. Leaves (arrow) and cotyledon petioles (c) are affected as in G. (I,J) ANT::STM; ANT::GUS-expressing wild-type (I) and wus1 mutant (J) seedlings. In both cases, strong GUS staining is visible in the vascular strands of the cotyledon petioles (arrowheads) and in young leaf primordia (arrows) at the shoot meristem. (K,L) WUS::NLSGUS- (K) and ANT::STM; WUS::NLSGUS- (L) expressing seedlings. In both cases, GUS staining is restricted to a small central cell group in the shoot apical meristem (arrowheads). The additional smaller region of staining in K is an axillary meristem. (M) Longitudinal section through a GUS-stained WUS::NLSGUS-expressing seedling. GUS activity is detected specifically in a small central cell group of the SAM, reflecting the WUS mRNA expression pattern (Mayer et al., 1998Go). Scale bars are 1 mm in A-L, 100 µm in M.

 


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  		Fig. 5. Synergistic effects of coexpression of WUS and STM. (A-D) Scanning electron micrographs of seedlings 14 days after germination. (A) ANT::WUS-expressing seedling. An enlarged SAM has formed in place of leaves. The cotyledon petioles (cp) are unaffected and separated from the meristematic cells by a sharp boundary (arrow). h, hypocotyl. (B) ANT::STM-expressing seedling. Cotyledon petioles (cp) are broadened, but do not show meristem-like cells. (C,D) ANT::WUS; ANT::STM coexpressing seedlings. No cotyledon petioles have been formed and fields of small, meristematic cells (arrows) extend into the lamina of cotyledons (c). (E,F) Histological sections of plastic embedded material stained with Toluidine Blue. (E) Longitudinal section through the apex of an ANT::WUS-expressing seedling 8 days after germination. Note the massively overproliferated shoot meristem with small, cytoplasmically dense cells (arrow). (F) Longitudinal section through the apex of an ANT::WUS; ANT::STM-expressing seedling 8 days after germination. The regions of small meristematic cells are expanded into the cotyledons (arrows). The spots of darker stained cells are an artefact of processing. (G,H) In situ hybridization using a CLV3 antisense riboprobe. (G) In ANT::WUS-expressing seedlings, CLV3 mRNA is detected in the outermost cell layers of the enlarged shoot meristem (black arrow), but not in cells of the cotyledon petioles (white arrow). (H) By contrast, ANT::WUS; ANT::STM coexpressing seedlings show CLV3 expression both in the enlarged shoot meristem (black arrow) and in the meristematic regions on the cotyledons (white arrow). Scale bars are 500 µm in A-C, 200 µm in D and 100 µm in E-H.

 


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  		Fig. 6. The loss-of-function phenotypes of wus and stm mutants cannot be rescued by transgenic expression of the respective other gene. Light micrographs of GUS stained cleared embryos or seedlings (A-D,G,J-L,O,P) and of live seedlings (E,F,H,M,N). (A,B) The CLV1::STM transgene is strongly expressed in the SAM primordia (arrows) of wild-type (A) and wus1 mutant (B) embryos as indicated by staining for the activity of a linked CLV1::GUS reporter. Note the flat apex of the wus1 mutant embryo compared to the convex meristem in the wild type, suggesting that the former has terminated. (C) CLV1::STM; CLV1::GUS expression is detected in the SAM of 7-day old wild-type seedlings by GUS staining. (D) CLV1::STM; CLV1::GUS-expressing wus1 mutant seedlings 10 days after germination show strong GUS staining at the shoot apex. (E,F) The meristems in CLV1::STM; CLV1::GUS-expressing wus1 mutant seedlings (F) terminate indistinguishably from the meristems in non-transgenic wus1 mutants (E) (arrows). (G,H) In CLV1::WUS; CLV1::GUS-expressing wild-type seedlings 7 days after germination strong GUS staining is detected at the apex (G) which causes the development of an enlarged meristem (H, arrow). (I) In situ hybridization using a WUS antisense riboprobe on CLV1::WUS-expressing seedlings confirms transgene expression specifically in the centre of the enlarged shoot meristem, yet not on the flanks (arrow) where organs are initiated. (J,K) In CLV1::WUS; CLV1::GUS-expressing stm5 mutant seedlings the first GUS-staining cells are detected 7 days after germination inside the fused cotyledon petioles (arrow in J) which give rise to adventitious meristems (K, compare with M,N). (L-N) While non-transgenic stm5 mutants 12 days after germination show no sign of a SAM inside the fused cotyledon petioles (L), CLV1::WUS-expressing stm5 mutant seedlings (M,N) of the same age contain a conspicuous meristematic structure (arrows) that is surrounded by small leaf primordia (arrowhead in N). (O) In CLV1::WUS; CLV1::GUS-expressing wild-type plants 25 days after germination, the meristem is massively enlarged (arrow). (P) CLV1::WUS; CLV1::GUS-expressing stm5 mutant plants of the same age show only small meristematic regions that express the GUS reporter gene (arrow). In addition, leaves are small and sometimes fused as in non-transgenic stm5 mutant plants. Scale bars are 50 µm in A,B, 1 mm in C-H,J-P, and 100 µm in I.

 


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  		Fig. 7. Marker gene expression in CLV1::WUS-expressing wild-type and stm5 mutant plants. Longitudinal sections hybridized in situ with CLV3 (A,B), KNAT1 (C,D,F,G), KNAT2 (H-J) antisense and KNAT1 sense (E) riboprobes. CLV3 and KNAT2 sense riboprobes did not produce any staining (not shown). (A) In CLV1::WUS-expressing wild-type plants 14 days after germination, cells in the three outermost layers of the meristem show strong CLV3 expression. (B) CLV1::WUS-expressing stm5 mutant plants 14 days after germination exhibit CLV3 expression in a band at the top of the induced structure inside the fused cotyledon petioles. The same result was obtained when analyzing 10 day old seedlings (not shown). (C) In non-transgenic wild-type seedlings, KNAT1 expression is detected at the base and periphery of the SAM and close to the base of young leaf primordia (black arrow), but is absent from the central zone of the SAM (white arrow). In addition, expression is detected in cells close to the vasculature (arrowhead). (D) In CLV1::WUS-expressing wild-type plants 10 days after germination, KNAT1 expression is detected at the periphery of the enlarged meristem (black arrows) and adjacent to the vasculature (arrowhead). Although weak, this staining was consistent throughout serial sections. The central region of the meristem (white arrow) shows only weak background staining that is also found in leaves (asterisk) and in sections hybridized with a KNAT1 sense probe (compare with E). (E) Hybridization with a KNAT1 sense riboprobe produces only weak non-specific staining. (F,G) While no KNAT1 mRNA can be detected in the induced structures of 10 day old CLV1::WUS-expressing stm5 mutant seedlings (F), plants of the same genotype at 14 days after germination (G) exhibit clear KNAT1 expression at the base (arrow) and in patches on the flanks of the induced structures (arrowhead). However, no expression is seen close to the vasculature in either seedling. (H) KNAT2 mRNA can be detected in the periphery of the enlarged meristem of CLV1::WUS-expressing wild-type plants 14 days after germination (black arrows), while only weak and even staining is visible in the centre of the meristem (white arrow) and in leaves (asterisk) which most likely represents non-specific background staining. (I,J) In 10-day old CLV1::WUS-expressing stm5 mutant seedlings (I), no KNAT2 expression can be detected, which is however seen in seedlings of the same genotype 14 days after germination (J) on the flanks (arrow) and at the base (arrowhead) of the induced structure. The asterisk in I indicates a fragment of the vasculature which appears darker because of its secondary cell wall. Scale bars in A-J are 100 µm.

 

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© The Company of Biologists Ltd 2002