Figure 1.
Gene Regulatory Network Controlling Early Flower Development
Genes involved in the establishment of floral meristem identity, floral patterning, or floral organ formation, and their regulatory interactions are shown. Certain floral regulators were not included in the diagram, because their positions in the gene regulatory network relative to the genes shown are currently not well understood. Individual genes are represented by horizontal lines with bent arrows and gene symbols (see Table S11 for full gene names). For each gene, upstream inputs and downstream targets are indicated. Activators are connected to their targets by arrows, repressors by blunted lines. Blue dots underneath gene symbols indicate that direct binding to these genes has been demonstrated by chromatin immunoprecipitation or by a combination of in vitro binding studies and in vivo binding site disruptions. Note their small number in the diagram, indicating the limited availability of binding data. White circles represent protein complexes. ASK1 and UFO are part of an ubiquitin ligase complex (“Ubiq. lig.”). SEU, LEU, and perhaps BLR (question marks indicate that a direct interaction of BLR to SEU and/or LEU has not yet been demonstrated) are part of a transcriptional co-repressor complex (“transcrip. corepress. compl.”) controlling AG expression. Protein interactions between MADS-box transcription factors [41] are not depicted (with the exception of AP3 and PI, which are thought to act as an oligate heterodimer [58]), to simplify the diagram.
Arrows for FT symbolize a long-range transport of FT mRNA from leaves to shoot apices [59]. Dashed lines indicate that gene products do not function as transcriptional regulators. A red arrow marks the position of AP1 in the gene regulatory network. Diagram was generated using BioTapestry [60] and is based on published data (see Table S11 for selected references).
Figure 2.
Floral Induction in ap1 cal Double Mutant Plants by AP1-GR Activation
While no phenotypic response was observed in mock-treated (mock) plants (A), the activation of AP1-GR in the inflorescences of ap1 cal plants by dexamethasone (dex) treatment led to a massive induction of floral primordia (B). Images were taken 6 d after a single dexamethasone treatment. (C–D) Fertile flowers of 35S:AP1-GR ap1 cal plants 4 wk after treatment with a mock solution (C) and 13 d after a single dexamethasone treatment (D). AP1-GR activation restores the organ identity defects of ap1 cal mutant flowers. (E–I) Scanning electron micrographs of floral buds of 35S:AP1-GR ap1 cal plants at different time points (as indicated) after a single dexamethasone treatment. In (H), an asterisk indicates the position of a sepal that was removed for a better visibility of the inner whorl organs. Scale bars: 20 μm in (E) to (H) and 100 μm in (I).
Figure 3.
Experimental Design and Results
(A) Calculation of expression ratios. RNA samples from tissues collected on two consecutive days were co-hybridized to microarrays. Thus, ratios r1 to r5 are direct experimental ratios that were calculated for each gene represented on the array using its normalized signal intensities (s) at the individual time points.
(B) Summary of gene expression changes observed in the experiment. The number of genes that were up- or down-regulated 1, 2, 3, 4, or 5 days after AP1-GR activation relative to the previous time point is indicated. Black bars represent up-regulated and gray bars down-regulated genes.
(C) Heat map representing Z-score normalized signal intensities of 1,653 genes showing significant expression changes in the experiment (as derived from ratios r1-r5). Yellow indicates high, and blue indicates low expression. Genes were clustered into five groups (A–E; indicated on the left) with predominant expression during certain stages of early flower development (indicated on the right). The approximate developmental stage of the floral buds at the different time points is indicated in (A) and (C). IM: inflorescence-like meristem.
Figure 4.
Microarray Results for Selected Floral Regulatory Genes
Log10-transformed signal intensities at the individual time points of the experiment are shown.
(A) Expression dynamics of previously identified AP1 response genes that regulate the initiation of flower formation (see Figure 1). The floral meristem identity gene LEAFY (LFY) is rapidly upregulated upon AP1-GR activation, whereas TERMINAL FLOWER1 (TFL1) and AGAMOUS-LIKE 24 (AGL24) are repressed. The slight reduction in expression of FRUITFULL (FUL) was not judged statistically significant in our analysis.
(B) Repression of AGAMOUS-LIKE42 (AGL42), SHORT VEGETATIVE PHASE (SVP), and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1).
(C) Expression dynamics of genes involved in specifying the identity of floral organs. AP3: APETALA3; PI: PISTILLATA; AG: AGAMOUS; SEP3: SEPALLATA3; AP2: APETALA2.
(D) Activation of genes involved in floral patterning (SUP: SUPERMAN) or organ primordia formation (RBE: RABBIT EARS; PRS: PRESSED FLOWERS).
(E) Induction of genes involved in carpel or stamen primordia development was detected towards the end of the time course experiment (SHP1: SHATTERPROOF1; SHP2: SHATTERPROOF2; CRC: CRABS CLAW; NZZ/SPL: NOZZLE/SPOROCYTELESS). Expression of SHP1 was not judged significantly changed in the experiment in contrast to that of its paralog SHP2. This result is in agreement with the reported induction of SHP2 at stage 6, one stage earlier than that of SHP1 [61,62].
(F) The meristem regulatory genes WUSCHEL (WUS) and CLAVATA3 (CLV3) were gradually downregulated during the course of the experiment. The increase in WUS expression on day 5 likely marks the onset of its expression in stamen primordia [16].
(G) Similar expression profiles (correlation coefficient of 0.84) were observed for FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3) in agreement with their largely identical expression patterns in developing flowers [29].
(H) Co-expression of JAGGED (JAG) and its paralog NUBBIN (NUB). Gene identifiers and references are listed in Table S1.
Figure 5.
Expression Patterns of Four Genes Encoding B3-Domain-Containing Proteins in Early-Stage Wild-Type Flowers
(A) Microarray results: log10-transformed signal intensities at the individual time points are shown for the genes tested.
(B–I) Results of in situ hybridizations. Expression patterns were analyzed in early-stage flowers of wild-type plants. Arrows point to regions of expression. Expression of At2g35310 was first detected throughout the center of young floral buds (B). At later stages, expression was confined to stamen and carpel primordia (C). Expression of At3g53310 was first detected at stage 4 throughout very young stamen primordia (D). At stage 6, expression was observed in stamen, as well as in carpel primordia (E). Expression of At5g57720 was first detected in stage 3 floral buds adjacent to the emerging sepal primordia (F). Its expression at later stages resembled that of At2g35310 (compare panels [G and C]). Weak expression of At3g46770 in stamen and carpel primordia was first observed in stage 7 floral buds (H). At later stages, expression was confined to the margins of the central septum of the gynoecium (I). (C, G, and I) show transverse sections; in all other panels, longitudinal sections are shown. Numbers indicate approximate floral stages.
Scale bars: 30 μm (B, D, and F); 50 μm (E); 100 μm in all others.
ca, carpel; se, sepal; st, stamen.
Figure 6.
Results of In Situ Hybridizations for Selected Genes
Expression patterns were analyzed in early-stage wild-type flowers. Arrows point to regions of expression.
(A and J) Log10-transformed signal intensities at the individual time points are shown for the genes tested.
(B) Expression of At3g04290, which encodes a lipase, was first detected at stage 4 in the epidermis of emerging sepals. At later stages, expression was also observed in the epidermis of stamens (C).
(D to G) Expression of At1g05480, which encodes a SNF2-domain containing protein, was detected in developing stamens and carpels.
(H) Expression of At5g22430, which encodes a protein of unknown function, was first detected in the tip of sepals around stage 6 of flower development.
(I) Expression of At3g26744, encoding the bHLH transcription factor INDUCER OF CBF EXPRESSION 1, was found in the inflorescence meristem and throughout developing flowers.
(K and L) Expression of At2g04570, encoding a lipase related to At3g04290 (see above), was first detected in stage 7 floral buds in the epidermis of sepals and, in contrast to At3g04290, appeared to be confined to sepals also at later stages of flower development (compare L and C).
(M–O) Expression of At5g65590, which encodes a Dof-type zinc-finger protein, was first observed in a small number of cells in stage 2 floral meristems (M). At stage 3 (N), expression was detected in incipient sepal primordia, and expression appeared to continue exclusively in developing sepals at later stages of development.
(P–R) Expression of At1g21460, encoding a nodulin MtN3 family protein, was found at early stage 3 in a few cells in the center of the floral apex (P). At late stage 3, the expression domain of this gene was significantly enlarged (Q). No signal was found in the upper cell layers of the meristem. At later stages, expression was detected in stamen primordia (R).
(S–U) Expression of At5g66940, which encodes a Dof-type zinc-finger containing protein, was observed in a small number of cells in very young floral buds (S), as well as in patchy pattern in young floral organ primordia (T). At later stages, its expression was confined to stamens and carpels (U).
(V) Expression of At1g12080, which encodes a protein of unknown function, was first detected in a region at the base of stamen primordia, which gives rise to the filament. (G) and (I) are transverse sections; in all others longitudinal sections are shown. Numbers indicate approximate floral stages.
Scale bars: 30 μm (B and M–Q); 100 μm (C, I, S, U, and V); 50 μm in all other panels.
ca, carpel; IM, inflorescence meristem; pe, petal; se, sepal; st, stamen.
Figure 7.
Co-Expression of Genes during Early Flower Development
Log10-transformed signal intensities at the individual time points are shown for selected genes.
(A) Expression profiles for GA4 (At1g15550) and a gene (At1g78440) encoding a GA2-oxidase.
(B) Expression profiles for four genes, encoding members of the PINFORMED (PIN) family of putative auxin efflux carriers.
(C) Expression profiles for seven genes, encoding closely related class II TCP-family transcription factors.
Figure 8.
Occurrence of Related Sequences in Groups of Co-Expressed Genes
The proportion of closely related sequences in each of the five clusters shown in Figure 3C is indicated by black bars. White and gray bars represent the proportion of closely related sequences in (equally sized) sets of sequences randomly chosen from the list of 1,653 differentially expressed genes and the Arabidopsis genome, respectively. Bars indicate the standard deviation of the calculations (see Materials and Methods for details). Note the strong enrichment of related sequences in clusters A, D, and E.