WAVY LEAF1, an ortholog of Arabidopsis HEN1, regulates shoot development by maintaining MicroRNA and trans-acting small interfering RNA accumulation in rice

Abstract

In rice (Oryza sativa), trans-acting small interfering RNA (ta-siRNA) is essential for shoot development, including shoot apical meristem (SAM) formation and leaf morphogenesis. The rice wavy leaf1 (waf1) mutant has been identified as an embryonic mutant resembling shoot organization1 (sho1) and sho2, homologs of a loss-of-function mutant of DICER-LIKE4 and a hypomorphic mutant of ARGONAUTE7, respectively, which both act in the ta-siRNA production pathway. About half of the waf1 mutants showed seedling lethality due to defects in SAM maintenance, but the rest survived to the reproductive phase and exhibited pleiotropic phenotypes in leaf morphology and floral development. Map-based cloning of WAF1 revealed that it encodes an RNA methyltransferase, a homolog of Arabidopsis (Arabidopsis thaliana) HUA ENHANCER1. The reduced accumulation of small RNAs in waf1 indicated that the stability of the small RNA was decreased. Despite the greatly reduced level of microRNAs and ta-siRNA, microarray and reverse transcription-polymerase chain reaction experiments revealed that the expression levels of their target genes were not always enhanced. A double mutant between sho and waf1 showed an enhanced SAM defect, suggesting that the amount and/or quality of ta-siRNA is crucial for SAM maintenance. Our results indicate that stabilization of small RNAs by WAF1 is indispensable for rice development, especially for SAM maintenance and leaf morphogenesis governed by the ta-siRNA pathway. In addition, the inconsistent relationship between the amount of small RNAs and the level of the target mRNA in waf1 suggest that there is a complex regulatory mechanism that modifies the effects of microRNA/ta-siRNA on the expression of the target gene.

Figure 1.

Phenotypes of waf1 and sho embryos. A, The wild type. B, waf1-1. C, waf1-2. D, sho1-1. E, sho2. The insets in A, B, and D show higher magnification views of the shoot apex of the wild type, waf1-1, and sho1-1, respectively. The yellow dotted line indicates the outline of the SAM. White and black arrowheads indicate embryonic shoot and radicle, respectively. 1, First leaf; 2, second leaf; 3, third leaf; c, coleoptile. Bar = 500 μm.

Figure 2.

Phenotypes of waf1 in the early vegetative phase. A to J, Wild-type and severe waf1 plants. A, Wild-type seedling at 5 DAG. B and C, waf1-1 and waf1-2 seedlings at 5 DAG, respectively. D and E, Elongated waf1-1 and waf1-2 seminal roots at 10 DAG, respectively. F and G, Wild-type and waf1-1 SAMs at 5 DAG, respectively. Arrows indicate SAMs. H to J, In situ expression pattern of OSH1 in wild-type (H) and waf1-1 (I and J) SAMs at 5 DAG. K to S, Wild-type and mild waf1 plants at 10 DAG. K, The wild type. L and M, waf1-1 and waf1-2, respectively. N to S, Leaf phenotypes in waf1. N, Wavy leaf. O, Bifurcation of the waf1-1 leaf blade at the tip. P, Bifurcation of the waf1-1 leaf blade from the base. Q, waf1-1 leaf showing a separation of the filamentous structure (arrow) from the leaf blade. R and S, Cross-sections of the leaf blade in the wild type and waf1-1, respectively. Arrowheads indicate abnormally enlarged cells. Bars = 1 cm (A–E), 50 μm (H–J), 5 cm (K–M), and 100 μm (R and S).

Figure 3.

Root phenotypes of waf1-1. A, Longitudinal section of the wild-type seminal root tip. B, Longitudinal section of the waf11-1 arrested seminal root tip. C, Root systems of the wild-type (left) and waf1-1 (right) plants. Crown roots do not elongate in waf1-1. D, Root characters in waf1. From left to right: number of crown roots counted at 10 DAG, length of seminal roots at 10 DAG, and number of lateral roots per 1 cm of seminal root. Error bars indicate sd. [See online article for color version of this figure.]

Figure 4.

Phenotypes of waf1 in the reproductive phase. A, Wild-type panicle. B, waf1-1 panicle. Arrowhead indicates an elongated bract, and arrows indicate awn-like structures protruded from the lemmas. C, Wild-type spikelet. Lateral halves of the palea and lemma are artificially removed to see inner floral organs. D, waf1-1 spikelet lacking palea. Lemma is reduced in size but protrudes an awn-like structure (arrow), and the stamens lack anthers (arrowheads). E, waf1-1 spikelet lacking palea and floral organs. F, Severe waf1-2 spikelet. A rod-like terminal structure (arrow) is formed. [See online article for color version of this figure.]

Figure 5.

Molecular characterization of the WAF1 gene. A, Map position and structure of WAF1. Exons in pitted and checked boxes indicate the positions of the dsRNA-binding domain and the methyltransferase domain, respectively. Locations of the two mutations are indicated. B, Deduced amino acid alignments of WAF1 and HEN1. Dotted and solid lines indicate the dsRNA-binding motif and the methyltransferase motif, respectively. C, RT-PCR analysis of WAF1. EM, Embryos at 5 DAP; RT, roots; ST, stems; IL, immature leaves; ML, mature leaves; IP, young panicle at the primary rachis branch differentiation stage.

Figure 6.

Analysis of small RNAs. A, Northern hybridization for seven small RNAs. Small RNA accumulations are greatly reduced or nondetectable in both waf1-1 and waf1-2. Arrowheads indicate larger smearing signals detected only in waf1. Ethidium bromide-stained gels corresponding to 5S rRNA are shown at the bottom. B, Modification of the 3′ end of miR156. Synthesized RNA oligonucleotide (left) does not show increased mobility after incubation without (−β) NaIO₄/β-elimination but gains mobility (arrow) after incubation with (+β) NaIO₄/β-elimination. nt, RNA without incubation. Wild-type miR156 (center) does not show increased mobility after incubation without (−β) and with (+β) NaIO₄/β-elimination. miR156 in waf1-1 (right) gains mobility (arrow) after incubation with NaIO₄/β-elimination, indicating that the 3′ end of miR156 in waf1-1 is unmodified. Ethidium bromide-stained gels corresponding to tRNA are shown at the bottom.

Figure 7.

Expression changes of miRNA/ta-siRNA putative targets in the seven gene families in waf1-1 revealed by the Affymetrix GeneChip rice genome array. Vertical bars indicate expression levels of waf1-1 relative to that of the wild type. Their Rice Annotation Project Database (RAP-DB) locus, target gene family, and miRNA/ta-siRNA are indicated. Error bars indicate sd. A RAP locus with an asterisk is not verified by the degradome data as a miRNA target (Li et al., 2010). [See online article for color version of this figure.]

Figure 8.

Phenotypes of double mutants between waf1 and sho. A, C, and E, waf1-2 sho1-1. B, D, and F, waf1-1 sho2. A and B, Germination of double mutant seeds. The shoot does not emerge, but the seminal root is elongated. C and D, Longitudinal sections of germinating double mutant embryos. A shoot-like structure is not formed. E and F, Double mutant seeds from which the seminal root started elongation but soon arrested (arrows). Bars = 500 μm. [See online article for color version of this figure.]