Functional diversification of CLAVATA3-related CLE proteins in meristem maintenance in rice

Abstract

Postembryonic development in plants depends on the activity of the shoot apical meristem (SAM) and root apical meristem (RAM). In Arabidopsis thaliana, CLAVATA signaling negatively regulates the size of the stem cell population in the SAM by repressing WUSCHEL. In other plants, however, studies of factors involved in stem cell maintenance are insufficient. Here, we report that two proteins closely related to CLAVATA3, FLORAL ORGAN NUMBER2 (FON2) and FON2-LIKE CLE PROTEIN1 (FCP1/Os CLE402), have functionally diversified to regulate the different types of meristem in rice (Oryza sativa). Unlike FON2, which regulates the maintenance of flower and inflorescence meristems, FCP1 appears to regulate the maintenance of the vegetative SAM and RAM. Constitutive expression of FCP1 results in consumption of the SAM in the vegetative phase, and application of an FCP1 CLE peptide in vitro disturbs root development by misspecification of cell fates in the RAM. FON1, a putative receptor of FON2, is likely to be unnecessary for these FCP1 functions. Furthermore, we identify a key amino acid residue that discriminates between the actions of FCP1 and FON2. Our results suggest that, although the basic framework of meristem maintenance is conserved in the angiosperms, the functions of the individual factors have diversified during evolution.

Figure 1.

Structural Characteristics of FCP1 and Expression Patterns of FCP1 and FCP2. (A) Alignment of amino acids in the putative active CLE peptide domain of related CLE proteins. Amino acid residues that differ from those in FCP1 are indicated in red. (B) Schematic representation of FCP1 and FON2. Arrowheads indicate the positions of the introns. CLE, CLE domain; SP, signal peptide. (C) RT-PCR–based expression analysis of FCP1 and FCP2. Similar results were obtained from two biological replicates. Lane 1, shoot apex; lane 2, leaf blade; lane 3, inflorescence at the stage of floral organ differentiation (3 to 8 mm in length); lane 4, inflorescence at the stage of floral organ development, including meiosis (3 to 5 cm in length); lane 5, root. Rice ACTIN1 was analyzed as a control.

Figure 2.

In Situ Localization of FCP1 and FCP2. (A) In situ localization of FCP1 transcripts in the vegetative SAM. (B) and (C) In situ localization of FCP1 transcripts in the IM. (D) In situ localization of FCP1 transcripts in the FM. (E) In situ localization of FCP1 transcripts in the root tip. (F) In situ analysis using a sense probe of FCP1 in the SAM as a negative control. (G) In situ localization of FCP2 transcripts in the vegetative SAM. (H) In situ localization of FCP2 transcripts in the FM. (I) In situ localization of FCP2 transcripts in the root tip. An antisense probe was used except for (F). le, lemma; lp, leaf primordia; pa, palea. Bars = 100 μm.

Figure 3.

Effects of Overexpression of FCP1, FON2, and Modified FON2. (A), (B), and (E) to (K) Transformed wild-type plants. (C) and (D) Transformed fon1-5 plants. (A) to (D) Shoot phenotypes of plants transformed with Actin:FCP1. (E) Vegetative SAM of a plant transformed with empty vector. (F) Vegetative SAM of a plant transformed with Actin:FCP1. (G) Shoot phenotype of a plant transformed with empty vector. (H) Shoot phenotype of a plant transformed with Actin:FON2. (I) Shoot of a plant expressing FON2/mCLE-2 showing a seedling-lethal phenotype. (J) Shoot of a plant expressing FON2/mCLE-2 showing a seedling-survival phenotype. (K) Shoot of a plant expressing FON2/mCLE-2-5-10 showing a seedling-lethal phenotype. (L) Effects of amino acid substitutions in the modified FON2 peptide on the survival rate of transgenic plants. Numerals in the name of the construct indicate the positions of the substituted amino acid residues in the CLE domain, which are shown at right (see Supplemental Figure 2A online). lp, leaf primordia. Bars = 5 mm in (A) to (D) and (G) to (K) and 100 μm in (E) and (F).

Figure 4.

Inhibitory Effects of in Vitro Application of CLE Peptides. (A) Effects of FCP1 and FON2 CLE peptides on the elongation of wild-type roots. (B) Effects of modified CLE peptides on the elongation of wild-type roots. Amino acid sequences in the CLE peptide used are shown in Supplemental Figure 2B online. (C) Effects of FCP1 and FON2 CLE peptides on the elongation of fon1 roots. (D) to (F) Phenotypes of roots mock-treated (D) or treated with 30 μM FCP1 (E) or FON2 (F) CLE peptide for 7 d. (D′) and (E′) Magnified views of (D) and (E), respectively. For (A) to (C), the length of the seminal root (n = 12) was measured at 7 d after peptide application. Bars = 100 μm.

Figure 5.

Spatial Expression Patterns of QHB, Os SCR, and HISTONE H4 in the RAM Treated with CLE Peptides. (A) to (D) In situ localization of QHB transcripts in the RAM mock-treated or treated with the indicated CLE peptides. (E) and (F) In situ localization of Os SCR transcripts in the RAM mock-treated (E) or treated with FCP1 CLE peptides (F). (G) to (I) In situ localization of HISTONE H4 transcripts in the RAM mock-treated (G) or treated with FCP1 CLE peptides ([H] and [I]). (A′) to (I′) Magnified views of (A) to (I), respectively. Seedlings were treated with 30 μM peptide for 2 or 7 d. Arrowheads indicate the position of the QC region. Bars = 100 μm.

Figure 6.

Model of Meristem Maintenance in Rice. This model proposes that rice has two CLV-like pathways that negatively regulate stem cell identity. FON2 and FCP1 may act as signaling molecules at their putative receptors, FON1 and Y (representing an unknown protein). The FON2–FON1 pathway is responsible for regulating the IM and FM, whereas the pathway including FCP1 may be involved in regulating the vegetative SAM. FON2 may not recognize the putative receptor Y, because no abnormalities in the vegetative SAM were induced by FON2 overexpression. Inactivity of FON1 in the vegetative SAM has been suggested by our previous work (Suzaki et al., 2006). The large Xs indicate that the gray pathway with this mark is not functional. The question mark indicates processes that are unclear at present and remain to be elucidated in the future. The stem cell–promoting factor corresponding to WUS in Arabidopsis has not been identified to date in rice.