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Dependence of Stem Cell Fate in Arabidopsis on a Feedback Loop Regulated by CLV3 Activity

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Science  28 Jul 2000:
Vol. 289, Issue 5479, pp. 617-619
DOI: 10.1126/science.289.5479.617

Abstract

The fate of stem cells in plant meristems is governed by directional signaling systems that are regulated by negative feedback. In Arabidopsis thaliana, the CLAVATA(CLV) genes encode the essential components of a negative, stem cell–restricting pathway. We used transgenic plants overexpressing CLV3 to show that meristem cell accumulation and fate depends directly on the level of CLV3 activity and that CLV3 signaling occurs exclusively through a CLV1/CLV2 receptor kinase complex. We also demonstrate that the CLV pathway acts by repressing the activity of the transcription factor WUSCHEL, an element of the positive, stem cell–promoting pathway.

The shoot apical meristem can initiate organs and secondary meristems throughout the life of a plant. A few cells located in the central zone of the meristem act as pluripotent stem cells: They divide slowly, thereby displacing daughter cells outwards to the periphery where they eventually become incorporated into organ primordia and differentiate (1). The maintenance of a functional meristem requires coordination between the loss of stem cells from the meristem through differentiation and replacement of cells through division. In Arabidopsis, theCLAVATA (CLV1, CLV2, andCLV3) genes play a critical role in this process, since loss-of-function mutations in CLV1, CLV2, orCLV3 cause an accumulation of stem cells and a progressive enlargement of shoot and floral meristems (2–7). The CLV3 gene encodes a small and potentially extracellular protein that is expressed in the stem cells of the shoot and floral meristems (2). The otherCLV genes encode a leucine-rich repeat (LRR)–receptor protein kinase (CLV1) and a LRR-receptor–like protein (CLV2) that are assumed to form a membrane-bound receptor protein complex, together with other intracellular components (5, 6, 8). It has been proposed that the CLV3 protein is secreted from the outermost meristem cell layers and interacts with the CLV1/CLV2 receptor complex in deeper cell layers (2, 6) to restrict the size of the stem cell population. In clv1, clv2, or clv3 mutants, the expression domains of CLV1and CLV3 enlarge coordinately. A simple interpretation is that this coordinated expansion is controlled by a positive, stem cell promoting pathway, which in turn is negatively regulated by the stem cell restricting CLV pathway (2, 3). The stem cell promoting pathway would then also promote the expression of theCLV genes by causing enlargement of the cell populations in which they are expressed.

We separated these two pathways by expressing a CLV3cDNA from the constitutively active cauliflower mosaic virus35S promoter (CaMV35S) in transgenicArabidopsis plants (9). In 82% of the 599CaMV35S::CLV3 transgenic plants studied, the shoot meristem ceased initiating organs after emergence of the first leaves (Fig. 1, A and B). In some plants, the meristem resumed activity and initiated misshapen leaves at random positions, including the central zone that harbors the stem cells in wild-type plants (Fig. 1, D through F). Another 16% of the lines formed inflorescence meristems and produced flowers that failed to initiate the inner whorls with stamens and carpels (Fig. 1, G and I) (10). The opposite phenotype is observed inclv3 loss-of-function mutants, where stem cells accumulate in the center of shoot and floral meristems and additional organs or undifferentiated tissue are formed (Fig. 1H).

Figure 1

Constitutive expression of CLV3 in transgenic plants. (A) Three-week-old wild-type plant. (B) CaMV35S::CLV3 transgenic plant. The shoot meristem arrests after initiation of the first leaves (arrow). (C) A wus mutant plant of the same age. (D) Two-month-oldCaMV35S::CLV3 plant. The inflorescence meristem is flattened and initiates leaves and abnormal flowers (arrow). (E) A terminated inflorescence meristem. (F) Longitudinal section through a terminated meristem as shown in (E). Cells at the apex are large and vacuolated. A single leaf originates from the center. (G) Wild-type flower. (H) A clv3-2 mutant flower. Organ number is increased, particularly carpel number. (I) Flower of aCaMV35S::CLV3 plant. Stamens and carpels are missing. Bars (A, B, and C): 2 mm; (D): 2 cm; (E, G, H, and I): 0.5 mm; (F): 50 μm.

We hypothesized that the phenotypic differences between the two classes of CaMV35S::CLV3 transgenic plants might be caused by differences in the expression levels of theCaMV35S::CLV3 transgene. We therefore analyzed the levels of CLV3 mRNA by in situ hybridization (11) and found that transgenic lines showing an early meristem arrest expressed high levels of CLV3 mRNA, whereas lines that formed shoots with flowers showed weaker ectopicCLV3 expression (Fig. 2, A through C). This indicates that stem cell fate in the meristem center is controlled by the level of the CLV3 signal.

Figure 2

CLV3 expression levels in transgenic plants. (A) Bright-field image of aCaMV35S::CLV3 seedling. The shoot meristem has terminated (arrow) early in vegetative development, after the production of the first leaves. (B through D) In situ hybridization with a radiolabeled CLV3 antisense probe. (B) Bright-field/dark-field double exposure of the sameCaMV35S::CLV3 seedling. CLV3is expressed at high levels throughout the plant. (C) Inflorescence meristem of aCaMV35S::CLV3 plant. EndogenousCLV3 mRNA is present at the shoot apex, and a low level of ectopic CLV3 expression is detectable throughout the rest of the plant. (D) Inflorescence meristem of aCaMV35S::CLV3 clv1-4 plant.CLV3 is expressed at high levels throughout the plant, particularly in the outermost cell layers of the enlarged meristem. Bar, 50 μm.

We then tested whether CLV3 signaling requiresCLV1 and CLV2 by introducing theCaMV35S::CLV3 transgene intoclv1 or clv2 mutants. The resulting transgenic plants expressed CLV3 at high levels, but exhibited the typical clv mutant phenotype (Fig. 2D). Thus,CLV3 signaling requires functional CLV1 and CLV2, and the phenotypes observed in theCaMV35S::CLV3 transgenic plants are the result of enhanced CLV3 signaling through the CLV1/CLV2 receptor complex.

To increase the expression level of CLV3 under control of its own promoter, we increased the copy number of the CLV3gene in the genome. Plants containing four functional copies of theCLV3 gene (12) exhibited no alterations in shoot and floral meristem development, suggesting that increased gene dosage does not lead to enhanced CLV3 signaling. The observed compensation of the gene dosage could be explained if one indirect consequence of wild-type CLV3 signaling is the down-regulation of or failure to maintain transcription from theCLV3 promoter in the stem cells. To test whetherCLV3 expression itself is an indirect target of theCLV3 signaling pathway, we used the UNUSUAL FLORAL ORGANS (UFO) promoter to control CLV3expression. The UFO gene is expressed outside of the normalCLV3 expression domain in a group of cells underneath the central zone of the shoot apical meristem (13). AUFO::CLV3 transgene was introduced into wild-type Arabidopsis plants and clv3-8 mutants that still express CLV3 mRNA but do not make a functional CLV3 protein product (14). TheUFO::CLV3 transgenic plants obtained resembled the CaMV35S::CLV3 transgenic plants, indicating that CLV3 can act outside of its normal expression domain. Patterns of CLV3 expression during embryogenesis were analyzed by RNA in situ hybridization (Fig. 3). In theclv3-8 mutants, CLV3 RNA is expressed in the central zone of the meristem (Fig. 3A). In torpedo stageUFO::CLV3 transgenic embryos, CLV3 RNA was detected in cells underlying and surrounding the central region of the meristem in a pattern typical for expression from theUFO promoter (Fig. 3B). However, endogenous CLV3expression in the meristem center was barely detectable (Fig. 3B), indicating that CLV3 signaling from the UFOexpression domain is sufficient to cause a down-regulation ofCLV3 transcription in the central zone, perhaps by repressing cell division or initiating differentiation in the cells at the apex of the meristem.

Figure 3

Feedback regulation of CLV3. In situ hybridization with digoxigenin-labeled CLV3 antisense probe. (A) Torpedo stage clv3 embryo. CLV3 is expressed in the center of the meristem (arrow). H, hypocotyl; Co, cotyledon. (B) Torpedo stage embryo of aUFO::CLV3 plant. CLV3 is expressed in the UFO pattern (dashed arrow), and expression in the meristem center is reduced (arrow). Bars, 10 μm.

We conclude that targets of the CLV signal transduction pathway include factors that promote transcription of theCLV3 gene in the central zone of the meristem or that control central zone cell identity or division. CLV3signaling would act to repress the activity of these regulatory factors, which would themselves promote CLV3 expression or stem cell maintenance. The WUSCHEL (WUS) gene product, a homeodomain transcription factor (15), promotes stem cell formation and maintenance in the meristem, thereby acting antagonistically to the CLV genes. Phenotypically,wus mutants strongly resemble our transgenic plants that constitutively express CLV3 from the CaMV35Spromoter (compare Figs. 1B and 1C). In both cases, stem cells are not correctly specified during embryogenesis and shoot and floral meristems are not maintained. Genetic analysis revealed that wusmutations are almost fully epistatic to clv mutations (15, 16), indicating that WUS could be a target gene that is repressed by the CLV signaling pathway. Consistent with this, the WUS expression domain expands laterally and into the overlying cell layers in meristems ofclv mutants (Fig. 4, A and B) (16). In addition, we did not detect anyWUS RNA in the arrested apices ofCaMV35S::CLV3 transgenic plants by in situ hybridization, indicating that one consequence ofCLV signaling is a severe reduction in the levels ofWUS transcripts (Fig. 4C). However, because CLV3is still expressed at low levels in wus mutants (17), the stem cell–promoting pathway may involve additional factors that may overlap in function with WUS and be potential targets for regulation by the CLV pathway, such as POLTERGEIST (18).

Figure 4

CLV3-dependent regulation ofWUS mRNA expression. In situ hybridization withWUS digoxigenin-labeled antisense probe. (A) Longitudinal section through a wild-type shoot meristem. WUSis expressed in deep regions of the meristem. (B) In the enlarged meristem of a clv3 mutant, the WUSexpression domain is expanded. (C) Arrested meristem of aCaMV35S::CLV3 plant. WUS RNA is not detectable. Bars, 20 μm.

In wild-type Arabidopsis plants, the CLV3 signal is likely to be released from the stem cells at the apex of the meristem and to activate a CLV1/CLV2 receptor complex in underlying cells (2). Signaling through the CLV pathway limits WUS activity by restricting its expression to a narrow domain of cells in deeper layers of the meristem. Constitutive signaling through CLV3 enhances this negative pathway, causing down-regulation of WUS and complete loss of stem cells. When the negative pathway is disrupted in clvmutants, the WUS expression domain expands laterally and upward, resulting in, or resulting from, an accumulation of stem cells. Activity of the positive pathway promotes the expression ofCLV3 or maintenance of the CLV3 expression domain. This mutual regulation, involving positive and negative interactions, provides a feedback system for maintaining the delicate balance required for proliferation of stem cells to proceed at the right time and in the right place.

  • * Present address: U.S. Department of Agriculture Plant Gene Expression Center, University of California Berkeley, Department of Plant and Microbial Biology, 800 Buchanan Street, Albany, CA 94710, USA.

  • To whom correspondence should be addressed. E-mail: ruediger.simon{at}uni-koeln.de

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