The CLE genes encode small, secreted polypeptides characterized by a highly conserved 14 amino-acid region at their carboxyl termini called the CLE domain.1 To date 32 family members have been identified in Arabidopsis, yet only three have been assigned functions: CLV3, CLE40 and CLE41 have been implicated in stem cell homeostasis in shoot, root and vascular meristems, respectively.2–5 Overexpression studies indicated that CLE genes may regulate additional biological processes as diverse as root and shoot growth, phyllotaxis, apical dominance and leaf shape and size control.6,7 This hypothesis is consistent with our recent expression analysis of Arabidopsis A-type CLE genes,8 in which we found that all examined tissues expressed one or more CLE genes, in overlapping patterns. Each CLE promoter exhibited a highly distinct and specific activity profile, and many showed complex expression dynamics during vegetative and reproductive growth.

Consistent with their roles in meristem maintenance, CLV3 and CLE40 are expressed early in embryogenesis when meristem initiation and organization take place.3,5 Yet there are no other reports of CLE gene expression in Arabidopsis embryos, and therefore it is not known to what extent this family of small peptides regulates intercellular signaling events during embryogenesis. We addressed this question by analyzing the expression patterns of selected CLE promoters in mature embryos and compared them with those in 11-day-old seedlings. We chose nine CLE genes whose promoters are active in different tissues of the seedling.8 Transgenic dried seeds carrying a single CLE promoter sequence driving the expression of the uidA reporter gene were imbibed in water for four days, the embryos dissected out of their seed coats, and beta-glucuronidase (GUS) reporter assays performed.9 Stained embryos were cleared with chloral hydrate10 and visualized using a Zeiss Axiophot microscope.

Five of the CLE genes analyzed showed similar promoter expression patterns in mature embryos and in seedlings. In embryos, the CLE11, 13, 16 and 17 promoters drove GUS activity in specific patterns in the root. CLE11 and CLE13 promoter activity was detected in the root cap and root apical meristem (Fig. 1A and B), CLE16 promoter activity was observed in the root cap and above the root apical meristem (Fig. 1C), and CLE17 promoter activity was seen weakly in the root apical meristem (Fig. 1D). Each of these CLE genes exhibited a similar expression pattern in seedling roots.8 CLE17 was additionally expressed in the embryo shoot apex and at the cotyledon margins (Fig. 1D). Similarly, in seedlings CLE17 was expressed in the vegetative shoot apex, and at the margins of the cotyledons and fully expanded leaves.8 In embryos, CLE27 promoter activity was strong in the hypocotyl, as well as in the medial region of the cotyledons along the main vein (Fig. 1E). In seedlings, CLE27 was strongly expressed in the hypocotyl and exhibited patchy expression in both cotyledons and leaves.8 Our analysis reveals that the expression of these CLE genes is established early during development and remains constant at later stages, suggesting that they may perform the same function throughout the Arabidopsis life cycle.

Remarkably, the other four CLE promoters drove embryo expression patterns that were strongly divergent from what was observed in seedlings. We found that the CLE1 promoter was active in the embryo throughout the hypocotyl and in the central region of the cotyledons (Fig. 1F), but was observed in seedlings solely in the vasculature of fully differentiated roots and at the root tips.8 CLE12 promoter activity in embryos was observed throughout the hypocotyl and the cotyledons (Fig. 1G), whereas in seedlings it was detected weakly in the leaf vasculature and more strongly in the root vasculature.8 In contrast, the CLE18 and CLE25 promoters did not drive reporter activity in mature embryos (Fig. 1H and I), despite being broadly and strongly expressed in seedlings.8

These four CLE gene promoters show dynamic shifts in their activity between different developmental stages. From our data we infer that CLE1 activity in hypocotyls and cotyledons is required solely during embryogenesis, and that the gene then acquires a distinct function in post-embryonic root development. Similarly CLE12 appears to acquire a post-embryonic function in the root vasculature, and its broad activity in the embryonic leaves becomes restricted to the leaf vasculature following germination. Finally, the absence of CLE18 and CLE25 promoter activity in mature embryos suggests that they may be dispensable for embryo formation, and might either specifically regulate post-embryonic signaling events in certain tissues or be involved in mediating responses to environmental stimuli to which embryos are not subjected. Alternatively, they may be expressed earlier during embryogenesis and become repressed during seed dormancy.

Our spatio-temporal expression analysis of a small group of CLE genes in mature embryos and seedlings indicates that the majority of these signaling molecules exert their roles beginning early in development, potentially contributing to tissue patterning and organization. Yet whereas some appear to contribute to the same biological processes throughout the plant life cycle, others seem to function in different tissues at different developmental stages. In addition, each CLE promoter studied here is active in vegetative and/or reproductive tissues that are not present in embryos, such as trichomes (CLE16 and CLE17) and style (CLE1).8 This observation suggests that CLE genes are widely recruited to new tissue-specific signaling functions during the course of plant development.