Plant CLE peptides from two distinct functional classes synergistically induce division of vascular cells Ryan Whitforda,b, Ana Fernandeza,b, Ruth De Groodta,b, Esther Ortegaa,b,1, and Pierre Hilsona,b,2 aDepartment of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Technologiepark 927, 9052 Ghent, Belgium; and bDepartment of Molecular Genetics, Ghent University, 9052 Ghent, Belgium Communicated by Marc C. E. Van Montagu, Ghent University, Ghent, Belgium, September 20, 2008 (received for review October 17, 2007) The Clavata3 (CLV3)/endosperm surrounding region (CLE) signal- (LRR-RLKs) and CLV3-related genes are differentially regu- ing peptides are encoded in large plant gene families. CLV3 and the lated across secondary meristem tissues, suggesting the involve- other A-type CLE peptides promote cell differentiation in root and ment of a similar signaling system in vascular development as shoot apical meristems, whereas the B-type peptides (CLE41– well. CLE44) do not. Instead, CLE41 inhibits the differentiation of Zinnia Clavata3 (CLV3) represents the short-range mobile ligand of elegans tracheary elements. To test whether CLE genes might code the CLV1 receptor that together regulates differentiation (13) for antagonistic or synergistic functions, peptides from both types and belongs to a family of predicted plant proteins, named were combined through overexpression within or application onto CLV3/endosperm surrounding region (CLE) (10, 14–16). All Arabidopsis thaliana seedlings. The CLE41 peptide (CLE41p) pro- CLE proteins share common characteristics: They are small moted proliferation of vascular cells, although delaying differen- (Ͻ15 KDa), have a putative N-terminal secretion signal, and tiation into phloem and xylem cell lineages. Application of CLE41p possess a conserved 14-aa CLE domain at or near their C or overexpression of CLE41 did not suppress the terminal differ- terminus. Synthetic CLE domain-derived peptides can alter stem entiation of the root and shoot apices triggered by A-type CLE cell fate and cellular differentiation within primary shoot and peptides. However, in combination, A-type peptides enhanced all root meristems (17). Furthermore, in situ mass spectrometry of the phenotypes associated with CLE41 gain-of-function, leading revealed that the bioactive domain derived from the CLV3 to massive proliferation of vascular cells. This proliferation relied preprotein is a modified dodecapeptide, overlapping with the on auxin signaling because it was enhanced by exogenous appli- CLE domain (18). Direct evidence for the involvement of CLE PLANT BIOLOGY cation of a synthetic auxin, decreased by an auxin polar transport peptides in secondary meristems comes from the discovery of inhibitor, and abolished by a mutation in the Monopteros auxin another dodecapeptide, corresponding to the CLE domain of response factor. These findings highlight that vascular patterning CLE41, and identified in mesophyll cell cultures of Zinnia is a process controlled in time and space by different CLE peptides elegans as a tracheary element differentiation inhibitory factor in conjunction with hormonal signaling. (TDIF) (19). We describe the use of both synthetic CLE-derived peptides cambium ͉ hypocotyl ͉ RAM ͉ SAM ͉ TDIF and CLE overexpression lines to classify CLE genes into 2 separate classes based on their ability to promote terminal n higher plants, postembryonic organogenesis is mediated by differentiation of the primary shoot and root meristems in Imeristems. These specialized structures provide a reservoir of Arabidopsis. The synergistic interaction of these 2 classes of undifferentiated stem cells as well as a limited population of peptides to suppress differentiation and promote auxin- proliferating cells, often referred to as transit-amplifying (TA) mediated cell proliferation within the secondary meristem (vas- cells that are fated for differentiation (1). To date, molecular cular cambium) demonstrates that specific CLE genes have dual research has focused on the Arabidopsis primary meristems of functionality and cell-type-specific responsiveness. root and shoot apices, but more recent studies have sought to dissect molecular programs underpinning the control of second- Results ary meristems, including the vascular cambium (2, 3) that is a Root Growth Assays Define Two Functional Classes of CLE-Derived circumferential stem cell niche including the fusiform initials Peptides. To investigate the respective role of CLE genes in root from which secondary xylem and phloem originate (2). During development, Arabidopsis seedlings were grown on medium the course of differentiation, fusiform initial daughters take on supplemented with one of 22 synthetic peptides encoded by the a TA state to increase the population of xylem and phloem corresponding CLE domain. Measured root-growth rates de- mother cells. Positioning and size of stem cell and TA cell fined two classes: 18 peptides arrested growth and 4 (CLE41p, populations, at least in root and shoot meristems, are controlled CLE42p, CLE43p, and CLE44p) did not. They are designated in a noncell-autonomous manner (1, 4, 5). This noncell-autonomous control of stem cell size and posi- A-type and B-type peptides, respectively [supporting informa- tioning has best been described in Arabidopsis primary meris- tion (SI) Fig. S1A and Table S1]. These quantitative results tems where a stable pool of undifferentiated stem cells are confirmed independent studies (11, 19, 20) with the caveat that maintained by a feedback loop mechanism involving the Clavata the synthetic peptides CLE1 to CLE7 do not arrest root apical (CLV) signaling pathway and the Wuschel (WUS) homeodomain transcription factor (6, 7). Similar regulatory mechanisms may be Author contributions: R.W., A.F., and P.H. designed research; R.W., A.F., R.D.G., and E.O. at work within root meristems, where the transcription factor performed research; R.W., A.F., R.D.G., E.O., and P.H. analyzed data; and R.W. and P.H. WUS-related-homeobox 5 (WOX5) controls stem cell mainte- wrote the paper. nance (8) and CLV3 and related genes can influence root The authors declare no conflict of interest. patterning (9–11), although to date the regulation of WOX5 by 1Present address: Centro de Investigacio´n del Ca´ncer, Universidad de Salamanca, Consejo a CLV-like pathway has not been demonstrated. Transcript Superior de Investigaciones Cientificas, E-37007 Salamanca, Spain. profiling of the vascular cambium, either across the cambial zone 2To whom correspondence should be addressed. E-mail: [email protected]. of aspen (Populus tremula) (2, 12) or in secondary tissues of the This article contains supporting information online at www.pnas.org/cgi/content/full/ Arabidopsis root-hypocotyl (3), showed that genes coding for 0809395105/DCSupplemental. several CLV-like leucine-rich repeat receptor-like kinases © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809395105 PNAS ͉ November 25, 2008 ͉ vol. 105 ͉ no. 47 ͉ 18625–18630 Downloaded by guest on September 23, 2021 meristem (RAM) growth at 1 ␮M (19, 20), but do at 10 ␮M (compare Fig. S1 A and B). To confirm genetically the results obtained with exogenous application of synthetic CLE peptides, we characterized avail- able transgenic plants that overexpress representative A- and B-type genes, CLE6OE and CLE41OE, respectively, under the control of the CaMV 35S promoter. In brief, CLE6OE T1 plants exhibited a short-root phenotype and microscopic analysis re- vealed that the activity of the RAM ceased gradually over time, similarly to its arrest observed after A-type peptide treatment. The shoot apical meristem (SAM) of CLE6OE seedlings also ceased activity gradually. In contrast, CLE41OE T1 plants had normal roots, but grew as compact dwarf plants and produced numerous small leaves, although the size and structure of their primary SAM seemed unaffected. Our results agree with sepa- rate studies of closely related CLE genes [(10, 21–23); for additional details, see SI Text]. Structure of Primary Meristems Treated with CLE Peptides. To better understand how the CLE peptides triggered root-growth arrest, representatives of either classes were applied to Arabidopsis transcriptional marker lines reporting mitotic divisions [CYCB1,1pro:GUS; (24)] and auxin response [DR5pro:GUS; (25)]. Compared with control plants (without added peptide), the primary root basal meristem of CLE41p-treated seedlings had identical structure and GUS pattern in both lines (Fig. S2 A, B, G, H, L, and M). In contrast, CYCB1,1pro:GUS seedlings treated with CLE6p, CLV3p, or CLE19p (all A-type peptides) had very low GUS levels in the shorter meristem, indicating that the gradual cessation of root growth correlated with the inhibition of cell division within the RAM (Fig. S2 C–E). CLE6p-treated roots also had a disorganized cellular structure with unusually large cells and differentiated vascular strands close to the meristem center (SI Text; Fig. S2N), in agreement with the previously observed terminal differentiation of the RAM in- duced by CLV3p, CLE19p, and CLE40p (11). None of the tested CLE-peptide treatments altered the position of the GUS max- imum in DR5pro:GUS root tips, although GUS activity was lowered after A-type peptide treatments, suggesting that RAM arrest may be associated with decreased auxin response (Fig. S2 I–K). To determine whether the initial distinction between A- and B-type CLE peptide activity is also reflected in other organs, we Fig. 1. Combined