Distinct regulatory role for RFL, the rice LFY homolog, in determining flowering time and plant architecture

Abstract

Activity of axillary meristems dictates the architecture of both vegetative and reproductive parts of a plant. In Arabidopsis thaliana, a model eudicot species, the transcription factor LFY confers a floral fate to new meristems arising from the periphery of the reproductive shoot apex. Diverse orthologous LFY genes regulate vegetative-to-reproductive phase transition when expressed in Arabidopsis, a property not shared by RFL, the homolog in the agronomically important grass, rice. We have characterized RFL by knockdown of its expression and by its ectopic overexpression in transgenic rice. We find that reduction in RFL expression causes a dramatic delay in transition to flowering, with the extreme phenotype being no flowering. Conversely, RFL overexpression triggers precocious flowering. In these transgenics, the expression levels of known flowering time genes reveal RFL as a regulator of OsSOC1 (OsMADS50), an activator of flowering. Aside from facilitating a transition of the main growth axis to an inflorescence meristem, RFL expression status affects vegetative axillary meristems and therefore regulates tillering. The unique spatially and temporally regulated RFL expression during the development of vegetative axillary bud (tiller) primordia and inflorescence branch primordia is therefore required to produce tillers and panicle branches, respectively. Our data provide mechanistic insights into a unique role for RFL in determining the typical rice plant architecture by regulating distinct downstream pathways. These results offer a means to alter rice flowering time and plant architecture by manipulating RFL-mediated pathways.

Fig. 1.

Phenotypes of RFL(S) and dsRNAiRFL plants. (A) Schematic diagram of dsRNAiRFL transgene. The ubiquitin promoter transcribes hairpin loop RNAs for RFL exon 1 and exon 2 segments. (B) Morphology of a flowering wild-type plant (Left, red arrowhead) regenerated through tissue culture and a dwarf nonflowering dsRNAiRFL (Right) plant of same age. (C) Distribution of days to flowering in dsRNAiRFL T₀ plants. Flowering time (x axis) is plotted against phenotype (y axis). The statistical significance is P < 0.0001for all phenotypic groups. (D) Schematic diagram of Ubi::RFL transgene. (E) A RFL(S) plant (Left) with early panicle heading (Inset, red arrowhead with closeup), compared with a wild type of same age (Right). (F) Distribution of flowering time in RFL(S) T₁ plants showing strong, moderate, and weak phenotypes.

Fig. 2.

Expression status of flowering activators and a repressor in RFL(S) and dsRNAiRFL plants. Quantitative RT-PCR showing fold change, with respect to wild type, in expression for OsSOC1 and RFT1 in leaves and RCN2 in the culm of RFL(S) and dsRNAiRFL plants of various ages.

Fig. 3.

Panicle growth and branching in RFL knockdown plants. (A) Mature deseeded wild-type and dsRNAiRFL panicles displayed for rachis length and branching. The primary branches (arrowheads), secondary branches (arrows), and spikelet pedicels (solid dots) are marked at representative positions. (B) Fold change in expression of branching regulators in dsRNAiRFL panicles compared with wild-type panicles determined by quantitative RT-PCR. (C) Schematic diagram of Ubi::RFL(AS) transgene. (D) Progressive reduction in the panicle branching and no secondary branches in these plants (line numbers at the bottom of each panicle). (E) Normalized fold change in the expression of branching regulators in RFL(AS) panicles. (Scale bars: 1.0 cm.)

Fig. 4.

Tiller development in RFL knockdown plants. (A) Basal portion of a wild-type plant with tillers (red arrowheads). (B) RFL(AS) plant with few tillers. (C) Basal part of the tissue culture regenerated wild-type plant. (D) dsRNAiRFL plant with no side tillers. (E–G) Histochemical distribution of GUS activity in vegetative axillary meristems. Pink-orange fluorescence at sites of axillary/tiller bud initials shows reporter activity. Basal nodes (F and G, arrow) and internodes (E, arrow) of transgenics culms with RFL promoter::GUS fusions. (E and G Insets) Shoot apical meristem. (H–I) RFL mRNA localization in wild-type 23-day-old culms. (H and I) RNA expression at leaf axils (H, arrow) and in a young tiller bud (I, arrow). (J) Culm with a tiller bud probed with sense RNA. (Scale bars: E–H, 50 μm; I and J, 20 μm.) (K) Semiquantitative RT-PCR of RFL transcripts in 4-, 13-, and 23-day-old culms (Upper) and control UBQ5 transcripts (Lower).

Fig. 5.

Genome-wide expression analysis of genes regulated by RFL. (A) Comparison of fold change in expression levels in wild-type versus dsRNAiRFL panicles for nine representative transcripts chosen from microarray data (SI Table 3). Data from microarrays are compared with that from quantitative RT-PCR analysis. (B–I) In situ RNA hybridization of an RFL-regulated transcript, AK101504 (PIN3-like). Transcripts at leaf axils (B, green arrow) and in a young tiller bud (C, cyan arrow) are indicated. (D) Transcripts in the panicle apex (purple arrowhead) and initiating primary branch (red arrowhead). (E) Expression at the apical end of a primary branch (red arrowhead) and in emerging secondary branches (white arrowhead). (F) Uniform expression in a young spikelet meristem (yellow arrowhead). (G) Transcripts in the emerging lemma (black arrow), palea (red arrow), and carpel anlagen (blue arrowhead) of spikelets with differentiating organs. (H) Expression in the vascular strands of an emerging primary branch (pink arrow). (I) Panicle probed with sense AK101504 RNA. (Scale bars: B–D, F, and I, 20 μm; E and G, 50 μm; H, 10 μm.)