Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells

Abstract

Plant shoot development depends on the perpetuation of a group of undifferentiated cells in the shoot apical meristem (SAM). In the Petunia mutant hairy meristem (ham), shoot meristems differentiate postembryonically as continuations of the subtending stem. HAM encodes a putative transcription factor of the GRAS family, which acts non-cell-autonomously from L3-derived tissue of lateral organ primordia and stem provasculature. HAM acts in parallel with TERMINATOR (PhWUSCHEL) and is required for continued cellular response to TERMINATOR and SHOOTMERISTEMLESS (PhSTM). This reveals a novel mechanism by which signals from differentiating tissues extrinsically control stem cell fate in the shoot apex.

Figure 1

Phenotype of ham mutants. (A) Wild-type W138 Petunia. (B) Wild-type W138 flower with the internal whorls of organs. Five stamens (yellow arrow) surround two fused carpels (green arrow). (C) ham-B4281 plant, terminated during vegetative rosette growth. (D) ham mutant flower lacking two stamens and both carpels. (E) Wild-type vegetative apex. p3, p2, and p1 = leaf primordia in order of decreasing age; m = meristem. (F) Vegetative ham apex shortly after termination. Primordium initiation has ceased. The meristem displays ectopic trichomes (arrows). (G) As in F, 2 wk after termination, the central dome has increased in size and is covered with trichomes. (H) Wild-type inflorescence apex. fm = floral meristem, im = inflorescence meristem, br = bract, se = sepal. (I) ham inflorescence apex with ectopic trichomes. br = last initiated bracts. (J) ham floral meristem, showing termination after initiation of three stamens (st). In place of carpels, a flat apex is visible with a small outgrowth (arrow). Bars, 100 μm.

Figure 2

Histology of ham apices. (A) Wild-type vegetative meristem. The arrow indicates a periclinal division in the L2 layer of an initiating leaf primordium. (B) In situ localization of PhSTM transcript in a wild-type vegetative apex. The signal (blue) is excluded from the leaf primordia. (C) ham vegetative apex showing a cessation of organ initiation and periclinal division in the central zone (arrow). (D) In situ localization of PhSTM transcript in a ham apex shortly after termination. (E) Transverse section of developing stem, just below a wild-type meristem (section schematized, inset). e = epidermis, c = cortex, v = vasculature, p = pith. (F) Older ham apex in longitudinal section showing a layered structure of differentiated tissue. e, c, v, p as in E. Arrow = trichome. Bars, 50 μm.

Figure 3

Cloning and structure of HAM and TER. (A) Protein sequence alignment of the C-terminal portion of HAM with SHR and GAI. Absolutely conserved positions are red, and conserved residues are grey (>90%), yellow (>80%), or green (>70%), based on alignments of 12 GRAS sequences as in the cladogram of C. Atypical residues at conserved positions are not colored. VHIID, PFYRE, and SAW are domains as defined in Pysh et al. (1999). Triangle = dTph1 insertion in ham-B4281. (B) ham excision alleles. WT = wild-type sequence flanking the insertion in ham-B4281, wt = footprints restoring HAM function, mut = mutant footprint allele hamFT-7. (C) Tree produced by neighbor joining (ClustalG software) showing the similarities of 12 GRAS sequences. Numbers indicate bootstrap frequencies of each branchpoint in the cladogram. GenBank accession nos. AtGAI (CAA75492), AtSHR (NP195480), AtPAT1 (AAF73237), AtRGA (CAA75493), LeLS (AAD05242), AtSCL6 (NP191926), AtSCR (AAB06318), AtSCL15 (NP191622), ZmD8 (AAL10319), TeGRM (CAB51555), OsGAI (BAA90749), and PhHAM (AF481952). (D) Full protein alignment of TER (GenBank accession no. AF481951) and WUS (GenBank accession no. CAA09986). Conservation is given on the basis of these two orthologs only. Red residues indicate positions in the homeobox, and grey residues denote blocks of conspicuous colinearity. (E) As in B but for ter-B1382. (F) Wild-type Petunia inflorescence producing two bracts (br) and a flower per node. (G) HAM cosuppressor showing a node without bracts and flower (arrow). (H) In situ hybridization of HAM RNA on wild-type (upper) and cosuppressed (lower) floral meristems. Wild type shows a signal in the initiating petal primordia. The cosuppressor lacks this signal. Bar, 50 μm.

Figure 4

Expression pattern of HAM. (A) In situ localization of HAM transcript in a near median (top right inset) longitudinal section through a vegetative apex. Signal is in the developing primordia (blue arrow) and at the presumptive position of a newly initiating primordium (red arrow). (B) As in A, with a section located more peripherally (top right inset). The signal is seen in a developing primordium (red arrow), as well as in a ring-shaped pattern that corresponds to the developing stem vasculature (blue arrow). (C) As in A, but in a transverse section. The position of the section is indicated in the top right inset. The signal is observed in the inner ground tissues of the primordia (red arrow) and is weaker in the main vascular bundle of older primordia (grey arrow). The blue arrow indicates HAM expression in a ring-shaped pattern that merges with primordia P1 and P0 and corresponds to provascular tissue of the differentiating stem. P6, P5, and so forth indicate the consecutive order of primordium initiation with decreasing age. (D) As in C, but at a position just below the meristem (indicated in top right inset). HAM expression is seen as a ring that corresponds to the provasculature of the stem. (E) HAM localization during development of the floral meristem, as exemplified for initiating petal primordia. Expression is observed in inner cell layers at the site of petal initiation (red arrows) and in subtending provascular tissue of the developing pedicel (blue arrow). (F) Schematic representation of the HAM expression pattern as exemplified for a wild-type vegetative meristem. Bar, 50 μm.

Figure 5

Relations between ham and ter. (A) Wild-type Petunia during vegetative rosette growth. (B) Wild-type shoot apical meristem (SAM) with the first two true leaf primordia. Cotyledons have been removed. (C) In situ localization of TER (PhWUS) transcripts in a wild-type vegetative apex. (D) ter-B1382 seedling. Growth has ceased after production of the first two true leaves. (E) ter seedling apex after initiation of the two first leaves. A flat, disorganized structure replaces the SAM. (F) ter apex with an ectopic meristem (arrow, stop-and-go growth). (G) ter ham double mutant seedling. An additional leaf, compared with D, occurred with a low frequency in ter single mutants as well. (H) ter ham double mutant seedling apex. Initiation of ectopic leaves is observed (stop-and-go). (I) ter ham double mutant apex on an older plant. The SAM displays a trichome covered surface characteristic for ham single mutants. (J) In situ localization of PhWUS transcripts in a ham mutant apex shortly after termination. The signal is essentially normal. (K) In situ localization of PhWUS transcripts in a later ham mutant apex. Expression occurs in a disorganized pattern. (L) As in K. PhWUS expression in the main apex has disappeared. In the axillary position, expression is disorganized and deeply internal. Bars: B, 25 μm; C,E,J, 50 μm; F,I, 200 μm; H,K,L, 100 μm.