Rice LHS1/OsMADS1 controls floret meristem specification by coordinated regulation of transcription factors and hormone signaling pathways

Abstract

SEPALLATA (SEP) MADS box transcription factors mediate floral development in association with other regulators. Mutants in five rice (Oryza sativa) SEP genes suggest both redundant and unique functions in panicle branching and floret development. leafy hull sterile1/OsMADS1, from a grass-specific subgroup of LOFSEP genes, is required for specifying a single floret on the spikelet meristem and for floret organ development, but its downstream mechanisms are unknown. Here, key pathways and directly modulated targets of OsMADS1 were deduced from expression analysis after its knockdown and induction in developing florets and by studying its chromatin occupancy at downstream genes. The negative regulation of OsMADS34, another LOFSEP gene, and activation of OsMADS55, a SHORT VEGETATIVE PHASE-like floret meristem identity gene, show its role in facilitating the spikelet-to-floret meristem transition. Direct regulation of other transcription factor genes like OsHB4 (a class III homeodomain Leu zipper member), OsBLH1 (a BEL1-like homeodomain member), OsKANADI2, OsKANADI4, and OsETTIN2 show its role in meristem maintenance, determinacy, and lateral organ development. We found that the OsMADS1 targets OsETTIN1 and OsETTIN2 redundantly ensure carpel differentiation. The multiple effects of OsMADS1 in promoting auxin transport, signaling, and auxin-dependent expression and its direct repression of three cytokinin A-type response regulators show its role in balancing meristem growth, lateral organ differentiation, and determinacy. Overall, we show that OsMADS1 integrates transcriptional and signaling pathways to promote rice floret specification and development.

Figure 1.

Global profile of genes and pathways deregulated in OsMADS1 knockdown panicles. A and B, Functional categorization of deregulated transcriptome (3-fold or more; P < 0.05) in developing panicles of OsMADS1-RNAi knockdown lines as compared with the wild type. Down-regulated transcripts are classified in A and up-regulated transcripts in B. Categorization was based on their predicted functional domains as annotated in various databases. C and D, RT-qPCR analyses of transcript levels for representative transcription factor genes down-regulated (C) and up-regulated (D). The fold change, with se bars, is calculated for the normalized transcript level in OsMADS1 knockdown versus wild-type panicles. RT-qPCR data are compared with data from microarray analysis.

Figure 2.

The OsMADS1-ΔGR fusion protein is functional on induction in transgenic rice. A, Transfer DNA segment in the construct PUbi:OsMADS1-ΔGR; P35S:OsMADS1amiR. The 35S promoter drives transcription of an amiRNA targeting the 3′ UTR of endogenous OsMADS1 transcripts. The maize ubiquitin cis-elements drive the expression of OsMADS1 translational fusion with the partial rat glucocorticoid receptor (ΔGR) domain. LB, Left border; RB, right border. B, A wild-type floret. C to E, Floret phenotypes in PUbi:OsMADS1-ΔGR; P35S:OsMADS1amiR transgenics grown in noninductive mock conditions. F to K, Scanning electron microscopy of epidermal cellular features in glumes, lemma, and palea in wild-type and mock-treated PUbi:OsMADS1-ΔGR; P35S:OsMADS1amiR lines. L and M, Florets of PUbi:OsMADS1-ΔGR; P35S:OsMADS1amiR plants grown in inductive conditions (dexamethasone treated). All floret organ phenotypes are rescued. N, Cellular features of rescued palea shown in L. O, Conversion of empty glumes to a lemma/palea-like organ in M, an overexpression phenotype seen in occasional spikelets. gl, Empty glumele; il, internal lemma; le, lemma; pa, palea. Bars = 1 mm in B to E and L to M and 100 µm in F to K and N to O. P, Western blot with nuclear extracts from leaves of PUbi:OsMADS1-ΔGR; P35S:OsMADS1amiR lines to detect OsMADS1-ΔGR protein before induction (lane 1) and after dexamethasone induction (lane 2).

Figure 3.

Direct transcriptional regulation, by OsMADS1, of transcription factor genes with meristem functions. A, Quantitation of normalized OsMADS34, OsMADS55, OsHB4, and OsKANADI2 transcript levels in wild-type (WT) and PUbi:OsMADS1-ΔGR; P35S:OsMADS1amiR panicles by RT-qPCR. Plants of both genotypes were mock treated or were given dexamethasone, cycloheximide, or a combination of both chemicals. B to D, Spatial localization of OsMADS55 transcripts in wild-type florets. FM with initiating lemma and palea primordia (B), floret with differentiating inner organs (C), and a nearly mature floret (D) are shown. ca, Carpel; fm, FM; le, lemma; lo, lodicule; pa, palea; st, stamen. Bars = 50 μm.

Figure 4.

Regulation of the auxin signaling pathway by OsMADS1. A, Fold change determined by RT-qPCR in the normalized expression of OsPIN1, OsARF-GAP, four ARFs (OsETTIN1, OsETTIN2, OsARF9, and OsARF18), and two auxin-responsive genes (OsGH3.4 and OsIAA9) in OsMADS1 knockdown florets. The fold change detected by microarray analysis is also shown. B, Expression levels for OsPIN1, OsARF-GAP, and OsETTIN2 in the panicles of wild-type (WT) and PUbi:OsMADS1-ΔGR; P35S:OsMADS1amiR plants treated individually with dexamethasone and cycloheximide and also in combination. The effects of these treatments were compared with that of wild-type plants. C to H, Spatial distribution of OsETTIN2 (C–E) and OsARF-GAP (F–H) transcripts in developing wild-type florets. C and F, Young FMs before organ initiation. D and G, FMs with differentiating floret organs. E and H, Florets with mature organs. Meristems and organs are labeled as in Figure 3. Bars = 50 µm in C, D, F, and G and 100 µm in E and H. I to L, Functional characterization of OsETTIN1 and OsETTIN2. Carpels are shown in the wild type (I), OsETTIN1-RNAi (J), OsETTIN2-RNAi (K), and OsETTIN1+OsETTIN2-RNAi (L). M to O, Abaxial and adaxial views of the style region above the ovary. The wild-type (M and N), OsETTIN1-RNAi (O), OsETTIN2-RNAi (P and Q), and OsETTIN1+OsETTIN2-RNAi (R) are shown. Bars = 200 µm in I to L and 50 µm in M to R.

Figure 5.

Negative regulation of components in cytokinin signaling by OsMADS1. A, Fold increase in the normalized transcript levels for two cytokinin biosynthetic genes (LOG and AK061341), four A-type response regulators (OsRR1, OsRR2, OsRR4, and OsRR9), and two B-type response regulators (OsRR16 and OsRR18) in OsMADS1 knockdown panicles versus wild-type panicles. The data from RT-qPCR are compared with those from microarray analysis. B, Transcript levels for OsRR1 and OsRR9 in wild-type (WT) and PUbi:OsMADS1-ΔGR; P35S:OsMADS1amiR plants treated with dexamethasone and cycloheximide individually and those given both chemicals compared with levels in mock-treated plants.

Figure 6.

Spatial distribution of OsRR1 and OsRR9 transcripts in developing wild-type florets. A to C, Expression pattern for OsRR1. D to F, Expression pattern for OsRR9. A and D, FMs with emerging lemma/palea primordia. B and E, Florets at the early stages of inner floret organ development. C and F, Florets with nearly mature organs. Organs are labeled as in Figure 3. Bars = 50 µm.

Figure 7.

ChIP assay for OsMADS1 occupancy at genomic loci of target genes. Schematic diagrams show exons (light gray box), introns (thin black line), 5′ UTRs (dark gray box), 3′ UTRs (diagonally striped box), and upstream sequences (thick black line) for each locus analyzed. A short bar numbered 1 indicates the position of the DNA fragment with CArG elements that was taken for PCRs shown in the top panel for each locus. The region numbered 2 in the schematic shows the position of the PCR amplicon for a CArG-less nonspecific control DNA segment shown in the bottom panel for each locus. A to E, ChIP analysis for selected target genes encoding meristem regulatory transcription factors. PCR amplicons are shown for OsMADS34 (A), OsMADS55 (B), OsHB4 (C), OsKANADI2 (D), and BLH1 (E). In each case, the Input lane is a control PCR with sheared chromatin, the No Ab lane is PCR on chromatin mock precipitated without antibodies, and the IP lane is PCR on immunoprecipitated chromatin. F to I, Enrichment of OsMADS1 at cis-elements of genes in the auxin pathway: OsPIN1 (F), OsARF-GAP (G), OsETTIN2 (H), and OsARF9 (I). J to L, OsMADS1 occupancy on cytokinin A-type response regulators OsRR1 (J), OsRR4 (K), and OsRR9 (L). M to O, Quantitative representation of the fold enrichment for PCR amplicons from ChIP as compared with the no-antibody control in each case. The average enrichment is shown for two biological replicates, each analyzed in triplicate reactions.

Figure 8.

Model depicting the likely mechanism of OsMADS1 action in regulating meristem specification, determinacy, and organ differentiation through different stages of floret development. Gray dots depict the spatial domains of OsMADS1 expression in a young spikelet with an incipient FM, in a young floret at the early stages of lateral organ patterning, and in a differentiated floret. OsMADS1 and its partners in various complexes directly repress OsMADS34 to promote the transition from spikelet to FM. It activates other meristem regulators and genes in the auxin signaling pathway. These events could regulate early events in meristem specification, its floral fate, and lateral primordia differentiation. OsMADS1-containing complexes are also negative regulators of the cytokinin pathway that can contribute to meristem determinacy. These pathways and the cross talk between them in developing florets culminate in a determinate meristem, with its typically differentiated organs.