Positive autoregulation of a KNOX gene is essential for shoot apical meristem maintenance in rice

Abstract

Self-maintenance of the shoot apical meristem (SAM), from which aerial organs are formed throughout the life cycle, is crucial in plant development. Class I Knotted1-like homeobox (KNOX) genes restrict cell differentiation and play an indispensable role in maintaining the SAM. However, the mechanism that positively regulates their expression is unknown. Here, we show that expression of a rice (Oryza sativa) KNOX gene, Oryza sativa homeobox1 (OSH1), is positively regulated by direct autoregulation. Interestingly, loss-of-function mutants of OSH1 lose the SAM just after germination but can be rescued to grow until reproductive development when they are regenerated from callus. Double mutants of osh1 and d6, a loss-of-function mutant of OSH15, fail to establish the SAM both in embryogenesis and regeneration. Expression analyses in these mutants reveal that KNOX gene expression is positively regulated by the phytohormone cytokinin and by KNOX genes themselves. We demonstrate that OSH1 directly binds to five KNOX loci, including OSH1 and OSH15, through evolutionarily conserved cis-elements and that the positive autoregulation of OSH1 is indispensable for its own expression and SAM maintenance. Thus, the maintenance of the indeterminate state mediated by positive autoregulation of a KNOX gene is an indispensable mechanism of self-maintenance of the SAM.

Figure 1.

Loss-of-Function Mutant of OSH1 Does Not Maintain the SAM after Germination. (A) OSH1 structure and the mutation in osh1. Gray lines indicate introns and 5′ and 3′ regions. Boxes indicate exons. Conserved domains of OSH1 are labeled in color as shown below the gene structure. (B) Phenotypes of wild-type (wt) (left) and osh1 (right) seedlings at 10 DAG. Arrowheads indicate the position of each leaf. (C) RT-PCR analysis for OSH1 and Actin (loading control) in leaves and shoot apices of wild-type and osh1 plants at 10 DAG. This experiment was performed two times independently. (D) Immunoblotting analysis of OSH1 protein in the wild type and osh1. Total nuclear protein extracts from 5-mm young panicles, shoot apices, and young leaf primordia at 10 DAG were loaded in each lane. Coomassie blue staining of membrane after immunoblotting shown at a bottom indicates that samples were loaded equally. SA, shoot apex; YP, young panicle. (E) Shoot apex sections of the wild type and osh1 during germination. The SAM before germination is indicated with a black line. Bars = 2 cm in (D) and 50 μm in (E).

Figure 2.

OSH1 and OSH15 Are Redundantly Required for SAM Formation. (A) to (C) Sections of wild-type (wt) embryos at 4 DAP (A), 5 DAP (B), and 10 DAP (C). Black arrowheads indicate the position of the SAM. (D) to (F) Sections of osh1 d6 double mutant embryos at 4 DAP (D), 5 DAP (E), and 10 DAP (F). White arrowheads indicate the position where SAM formation should be observed in the wild type. (G) An osh1 d6 double mutant at 10 DAG (cf. to Figure 1D). (H) Top view of (G). Bars = 100 μm in (A) to (F) and 5 mm in (G) and (H).

Figure 3.

OSH1 and OSH15 Are Redundantly Required for SAM Formation and Maintenance during de Novo Shoot Regeneration from Callus. (A) Regenerated (left) and germinated (right) seedlings of osh1. White arrowheads indicate the position of each leaf. (B) and (C) Sections of shoot apices of the regenerated wild-type (B) and osh1 plants (C). Arrowheads indicate the SAM. (D) Panicles of regenerated plants of the wild type (wt) (left) and osh1 (right). (E) and (F) Spikelets formed on regenerated plants of the wild type (E) and osh1 (F). a, anther; p, pistil; asterisk, abnormal short anther of osh1. (G) and (H) Regenerating shoot from the wild type (G) and osh1 d6 (H). Arrows indicate leaf-like structures formed from osh1 d6 callus. Bars = 2 cm in (A), 50 μm in (B) and (C), 5 cm in (D), and 2 mm in (E) to (H).

Figure 4.

Expression Levels of KNOX Genes Were Reduced in osh Mutants. (A) Expression of KNOX genes in germinating embryos. Relative expression level was calculated as the ratio to the expression level at 2 DAG in the wild type (wt) for each gene. (B) Expression of KNOX genes in the SAM formation stage of the embryo at 5 DAP. Relative expression level was calculated as the ratio to the expression level in the wild type for each gene. (C) In situ hybridization of OSH6 and OSH71 in wild-type and osh1 d6 embryos at around the stage of SAM formation. Bars = 100 μm. (D) Expression of KNOX genes in callus before and 1 week after CK treatment. Relative expression level was calculated as the ratio to the expression level before CK treatment in the wild type for each gene. In (A) to (C), the expression level of ubiquitin (ubq) was used as an internal control for normalization. The data represent the average of three independent biological replicates. Error bars represent the sd. Asterisks indicate significant reduction at P < 0.05 compared with the expression level in the wild type.

Figure 5.

OSH1 Protein Binds to Five KNOX Loci in Vivo. ChIP assay using anti-OSH1 antibody followed by qPCR analysis of the OSH1 (A), OSH6 (B), OSH15 (C), OSH43 (D), and OSH71 (E) locus. Gene structures are indicated below the graphs. Primers used in qPCR were named after their position relative to the transcription start site of each gene and are shown as black bars on the gene structures. Magenta bars above and below the gene structure represent conserved TGAC sequences in the plus and minus strands, respectively. The percentage of enrichment represents the amount of immunoprecipitated DNA relative to input DNA. Each data point is the average of three independent biological replicates. Error bars represent the sd. (A) to (E) ChIP assay from 5-mm young panicles of the wild type. For a negative control, control rabbit IgG was used. (F) ChIP assay from vegetative tissues. For a negative control, osh1 shoot apex and wild-type (wt) young leaf primordia were used.

Figure 6.

OSH1 Directly Binds to Conserved cis-Elements on the OSH1 Locus in Vitro. (A) OSH1 binding sites (BS) on the OSH1 locus. Gray lines indicate introns and the 5′ and 3′ regions. Boxes indicate exons. Asterisks above and below the gene structure represent each BS, which contains a conserved TGAC sequence, in the plus and minus strands, respectively. (B) EMSA using GST-OSH1HD. For mutant probes (m1-m8), evolutionarily conserved TGAC sequences were substituted with TCTC. Arrowheads represent the positions of shifted probes. GST alone was used as negative control, and “comp.” represents the competition assay using wild-type (WT) cold probes.

Figure 7.

Positive Autoregulation of OSH1 Is Essential for Its Expression and SAM Maintenance. (A) Reporter gene structures. Gray lines indicate introns and 5′ and 3′ regions. White and green boxes indicate exons and GFP, respectively. In the case of GFP-mOSH1, OSH1 binding sequences were substituted from TGAC to TCTC. (B) GFP-OSH1 expression in the shoot apex. (C) GFP-mOSH1 expression in the shoot apex. (D) Regenerating shoots from osh1 d6 callus carrying GFP-OSH1 on regeneration medium. (E) osh1 d6 callus carrying GFP-mOSH1 on regeneration medium. For regeneration, transgenic osh1 d6 calli were cultured on regeneration medium for 4 to 5 weeks. (F) A rescued osh1 d6 plant carrying GFP-OSH1 grew until the reproductive phase. (G) d6 and wild-type (wt) plants during the reproductive phase. Bars = 100 μm in (B) and (C), 5 mm in (D) and (E), and 10 cm in (F) and (G).

Figure 8.

The Model of a KNOX Autoregulatory Loop for SAM Maintenance. In this model, OSH1 expression is positively regulated via autoregulation. OSH1 protein directly and positively regulates its own expression and other KNOX genes in the SAM. KNOX genes and CK mutually activate each other, creating another positive autoregulatory loop.