Two small regulatory RNAs establish opposing fates of a developmental axis

Abstract

Small RNAs are important regulators of gene expression. In maize, adaxial/abaxial (dorsoventral) leaf polarity is established by an abaxial gradient of microRNA166 (miR166), which spatially restricts the expression domain of class III homeodomain leucine zipper (HD-ZIPIII) transcription factors that specify adaxial/upper fate. Here, we show that leafbladeless1 encodes a key component in the trans-acting small interfering RNA (ta-siRNA) biogenesis pathway that acts on the adaxial side of developing leaves and demarcates the domains of hd-zipIII and miR166 accumulation. Our findings indicate that tasiR-ARF, a ta-siRNA, and miR166 establish opposing domains along the adaxial-abaxial axis, thus revealing a novel mechanism of pattern formation.

Figure 1.

leafbladeless1 (lbl1) establishes leaf polarity by demarcating the domains of hd-zipIII and miR166 expression. (A) lbl1-rgd1 seedling with thread-like abaxialized leaves. (B) Longitudinal section through a wild-type apex shows rld2 expression in the SAM, vasculature, and adaxially in leaf primordia (arrowheads). (C) In lbl1-rgd1, meristematic, and adaxial (arrowheads) rld2 expression is reduced. (D) Precursor levels for mir166i, mir166h, and mir166c are increased in lbl1-rgd1 compared with wild type, whereas expression of mir166a is reduced. (E) In wild type, miR166 is expressed below and on the abaxial side of the incipient leaf (arrow). (F) In lbl1-rgd1, miR166 is expressed at the base of the SAM and uniformly throughout the incipient (arrow) and P1 primordia. Arrowheads in E and F mark the base of the incipient leaf.

Figure 2.

lbl1 encodes a homolog of SUPPRESSOR-OF-GENESILENCING3 (SGS3). (A) Diagrammatic representation of the lbl1 gene. Boxes represent exons, and the zinc-finger, XS, and coil-coiled domains are highlighted in blue, red, and green, respectively. F and R represent primers used for RT–PCR analysis in C. Mutations in four mutant lbl1 alleles are indicated. (C) Cysteine; (H) histidine; (Y) tyrosine. (B) Alignment of the maize LBL1 and Arabidopsis SGS3 proteins, with red indicating identity and black indicating similarity. The zinc-finger, XS, and coil-coiled domains are underlined, and asterisks mark the amino acids mutated in lbl1-rgd1 and lbl1-372. (C) lbl1 transcript levels are reduced in the mutants compared with wild type. Loading and −RT controls are shown.

Figure 3.

lbl1 encodes an essential component of the ta-siRNA pathway. (A) Diagram of the tas3a transcript. The 21-nt intervals phased relative to the miR390 cleavage site are shown as black brackets and the red brackets indicate the positions of tasiR-ARF. An alignment of tasiR-ARF sequences from maize, rice, and Arabidopsis is shown below, and the green arrow indicates complementarity to the tasiR-ARF LNA probe. Note that tas3d also has complementarity to this probe. (B) RT–PCR showing the relative expression levels of mir390, the tas3 genes, and arf3a in wild-type and lbl1-rgd1 apices. Loading and −RT controls are shown. (C) Small RNA Northern showing that tasiR-ARF accumulates in wild-type, not in lbl1-rgd1 apices. (D) 5′ RACE analysis shows a single prominent arf3a cleavage product (arrowhead) only in wild type. RT–PCR of ubiquitin served as loading control. (E) Diagram of the arf3a transcript with tasiR-ARF complementary sites A and B. The 5′ ends of the tasiR-ARF cleavage products map to complementary site B.

Figure 4.

The ta-siRNA pathway acts on the adaxial side of developing leaf primordia and restricts expression of abaxial determinants. (A) Longitudinal section through a wild-type apex showing lbl1 expression in the meristem tip and adaxially in leaf primordia. The yellow arrowheads mark the position of the incipient or P0 primordium, and the black arrowhead indicates expression at the P3 margin. (B–E) In situ hybridization with the tasiR-ARF complementary LNA-modified DNA probe in longitudinal sections through wild-type (B–D) and lbl1-rgd1 (E) apices. (B) In wild type, tasiR-ARF accumulates predominantly on the adaxial side of developing leaves. Arrowheads highlight adaxial expression in P2 and P3 primordia. (C) A close-up of a distinct incipient leaf shows adaxial tasiR-ARF expression (arrow). (D) A close-up of a P1 primordium also shows adaxial accumulation of tasiR-ARF. (E) Some hybridization to the tasiR-ARF complementary LNA probe is observed in lbl1-rgd1, perhaps reflecting hybridization to tas3 precursor transcripts. (F, left panels) lbl1-rgd1 apices before and after laser capture microdissection. The incipient leaf is marked by yellow arrowheads. (Right panel) RT–PCR analysis on LCM samples of the SAM or P1 plus P2 leaf primordia showing the effect of lbl1-rgd1 on expression of arf3a, mir166a, mir166c, and mir166i. The SAM-specific homeobox gene rough sheath1 (rs1) was analyzed as LCM control (Schneeberger et al. 1995). Loading and −RT controls are also shown.