Dynamic and compensatory responses of Arabidopsis shoot and floral meristems to CLV3 signaling

Abstract

In Arabidopsis thaliana, the stem cell population of the shoot system is controlled by regulatory circuitry involving the WUSCHEL (WUS) and CLAVATA (CLV1-3) genes. WUS signals from the organizing center (OC) to promote stem cell fate at the meristem apex. Stem cells express the secreted peptide CLV3 that activates a signal transduction cascade to restrict WUS expression, thus providing a feedback mechanism. Stem cell homeostasis is proposed to be achieved by balancing these signals. We tested the dynamics of CLV3 signaling using an inducible gene expression system. We show here that increasing the CLV3 signal can very rapidly repress WUS expression during development, which in turn causes a fast reduction of CLV3 expression. We demonstrate that increased CLV3 signaling restricts meristem growth and promotes allocation of peripheral meristem cells into organ primordia. In addition, we extend the current model for stem cell control by showing that meristem homeostasis tolerates variation in CLV3 levels over a 10-fold range and that high-level CLV3 signaling can be partially compensated with time, indicating that the level of CLV3 expression communicates only limited information on stem cell number to the underlying OC cells.

Figure 1.

Morphological Effects of Increased CLV3 Expression. (A) The shoot meristem (arrow) of wild-type plants is unaffected by ethanol treatments 11 DAI and continues to initiate flower primordia. (B) At 11 DAI, the inflorescences of iCLV3 plants lack young flower meristems and the shoot meristem has arrested (arrow). (C) The iCLV3 transgenic lines carry the alcA:GUS reporter gene. GUS activity is found in the CZ of the inflorescence meristem (arrowhead) and floral meristems after induction. Bar = 25 μm. (D) and (E) iCLV3 transgenic plants after a single 6-h induction pulse. (D) The majority of iCLV3 plants grows flowers with a reduced number of carpels (arrowheads) and form pseudo-whorls (arrow) higher up. (E) Terminated inflorescences (arrow) are observed in 7% of pulse-induced iCLV3 plants. (F) Decapitation of wild-type plants phenocopies the arrested meristem phenotype (arrow; compare with [E]). (G) to (J) Continuous inductions for 12 d. (G) Close-up of a terminating shoot as in (H). (H) Inflorescence at 17 DAI. The meristem has terminated with a pseudo-whorl of normal flowers (arrow). Flowers further down on the inflorescence axis carry only one carpel (arrowheads) or a filamentous organ in the center. (I) Close-up of an arrested meristem as in (H). (J) Flower with only three stamens and no carpels.

Figure 2.

Quantification of CLV3 and WUS Expression after iCLV3 Induction. Total RNA was isolated from shoot tips of induced plants at the indicated time points, and CLV3 or WUS transcripts were quantified by quantitative RT-PCR. Two independent RNA preparations were analyzed for each time point. Median values were calculated from triplicate real-time PCR analysis, and standard errors are shown. Levels of CLV3 and WUS RNAs before induction (time point 0) were set to 1. The y axis represents X-fold induction in a logarithmic scale. Means are of three experimental repetitions, and bars indicate the standard error. (A) Changes in CLV3 and WUS expression after a single 6-h ethanol pulse. (B) Changes in CLV3 and WUS expression after continuous inductions. Oligonucleotide primers were used that allowed iCLV3 transgene (induced CLV3) and CLV3 gene (endogenous CLV3) expression to be distinguished.

Figure 3.

Effects of CLV3 Induction on Plant Development. (A) Pulsed induction. iCLV3 transgenic plants were induced for 6 h, and the morphology of the first 15 flowers was analyzed for individual induced plants and plotted according to their position on the inflorescence axis. Gray, normal flowers with two carpels; black, 0 or 1 carpel; white, aberrant positioning of flowers (i.e., more than three flowers are formed within 3 mm of stem axis). Only plants affected by the induction were plotted (51 of 86). (B) Continuous inductions. Columns represent individual iCLV3 plants analyzed after shoot meristem termination. Flowers along the inflorescence axis were scored for carpel number. Gray, normal flower with two carpels; black, flower with 0 or 1 carpel; white, filamentous organ. Individuals were ordered along the x axis by total number of flowers produced and by the position of the first affected flowers along the inflorescence axis.

Figure 4.

Analysis of Cell Behavior. Scanning electron micrographs were taken from meristem replicas every 24 h, and cells were color-coded according to their areal growth rates within this time period (relative size increase, units % h⁻¹). Cell divisions occurring within 24 h are indicated by number of black dots per cell. Asterisks identify the cell at which the geometric center of the SAM surface is located at the beginning of the series. Flower primordia are initiated in a clockwise pattern and are numbered from the youngest primordium (P0) observed in the sequence to the oldest. Their borders as assessed by surface curvatures are outlined in black. Bars = 30 μm. (A) Sequence of an untreated control meristem. (B) Sequence of an induced meristem. Replicas were taken at days 2 (B1), 3 (B2), and 4 (B3) after the start of induction.

Figure 5.

Size Reduction of the SAM Surface Area after CLV3 Induction. Surface areas (in μm²) were measured from scanning electron micrographs for four induced apices (dashed lines) at 0 to 6 DAI, and two control meristems (solid lines) were analyzed on consecutive days.

Figure 6.

Induction of CLV3 Expression in iCLV3 Transgenic Plants and Consequences for WUS Expression. RNA in situ hybridizations with CLV3 ([A] to [E]) or WUS ([G] to [L]) antisense RNA probes, and alcA:GUS activity staining of inflorescence apices (F). Color reactions for the detection of WUS RNA ([G] to [L]) and CLV3 (A) were incubated overnight; all others gave a strong signal after 6 h of incubation. Uninduced controls ([A] and [G]); 6 HAI ([B] and [H]); 1 DAI ([C] and [I]); 2 DAI ([D] and [J]); 4 DAI ([E] and [K]); 10 DAI (L). Note the reduction in shoot meristem size from (G) to (K) and that the SAM is missing in (L) (arrow). The 2 and 3 indicate stage 2 or 3 flower primordia, respectively. Bars = 600 μm for (L) and 100 μm for all other panels. (A) to (E) CLV3 is expressed in the CZ of shoot and floral meristems (arrowheads in [A]). Expression increases dramatically after the inductive treatment. Note that color reactions in (B) to (E) were stopped after 6 h. Inset in (B) shows stage 2 flower expressing CLV3. (F) Activation of the alcA:GUS reporter gene is revealed by a blue precipitate. Staining is detected in the SAM (asterisk) and floral meristems. (G) WUS RNA is found in the SAM and two floral meristems. (H) to (L) After CLV3 induction, WUS RNA disappears rapidly from the SAM. At 24 HAI (I), only faint or no expression is detected in stage 2 flower meristems (arrowheads). WUS is again expressed in flower meristems at 48 and 96 HAI (insets in [J] and [K]), although CLV3 is also highly expressed at this stage (cf. with [E], stage 2 flower meristem). WUS RNA is present in developing ovules (arrowhead in [L]).

Figure 7.

Activity of CLV3 Promoter Variants and Effects on Meristem Size. Activity of CLV3 promoter derivatives (V1 to V4) in transgenic plants was measured by a fluorometric GUS assay and is shown as percentage of the activity of a wild-type CLV3 promoter controlling GUS activity (pCLV3 = 100%). After replacement of the GUS gene with a CLV3 cDNA, the transgenes were introduced into clv3-2 mutants. The diameters of the shoot meristems at 10 DAG and carpel numbers were measured and are shown as percentages of wild-type levels (two carpels =100%). n.t., no transgene. Bars indicate standard error; for GUS assays, n > 50 seedlings; for carpel number, n = 50; for meristem size, n ≥ 12.