The dicer-like1 homolog fuzzy tassel is required for the regulation of meristem determinacy in the inflorescence and vegetative growth in maize

Abstract

Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize (Zea mays) inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize fuzzy tassel (fzt) mutant has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and discovered that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. miRNAs are small noncoding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are moderately reduced in fzt plants and a few miRNAs are dramatically reduced. Some aspects of the fzt phenotype can be explained by reduced levels of known miRNAs, including miRNAs that influence meristem determinacy, phase change, and leaf polarity. miRNAs responsible for other aspects of the fzt phenotype are unknown and likely to be those miRNAs most severely reduced in fzt mutants. The fzt mutation provides a tool to link specific miRNAs and targets to discrete phenotypes and developmental roles.

Figure 1.

fzt Mutants Have Severe Vegetative and Inflorescence Defects. (A) Normal sibling (left) next to an fzt mutant (right). fzt mutants are much shorter than normal siblings. (B) Normal tassel. (C) A normal tassel spikelet containing two florets. Glumes have been removed to expose the florets. (D) fzt mutant tassel. Spikelets lack recognizable glumes. (E) fzt tassel spikelet containing extra florets. fzt florets contain abnormal stamens that do not shed pollen and other abnormal floral organs. (F) Normal ear. (G) Dissected ear spikelet from a normal ear contains a single floret. (H) fzt ear contains few silks and abnormal bracts and is sterile. (I) fzt ear spikelet contains extra florets, abnormal floral organs, and undeveloped stamens. White arrowheads indicate florets. Bars = 2 cm.

Figure 2.

fzt Contains a Mutation in dcl1. (A) Mapping data for fzt. (B) Genomic region of dcl1 with mutant alleles indicated. Black triangles indicate insertion sites of Mutator transposon insertions (dcl1-mum1-4). Orange boxes indicate protein-coding exons, and gray boxes indicate 3′ untranslated regions. (C) Schematic of the DCL1 protein with conserved domains indicated. The predicted effects of the mutant alleles on the DCL1 protein are indicated. a.a., amino acids.

Figure 3.

dcl1-fzt Inflorescences Make Abnormal Meristems. (A) to (H) Scanning electron micrographs of normal ([A] to [D]) and dcl1-fzt mutant ([E] to [H]) inflorescences. A young normal ear (A) is compared with a young dcl1-fzt mutant ear (E). dcl1-fzt IMs are flattened and broader than normal, indicating mild fasciation. dcl1-fzt SPMs are enlarged and not initiated in ordered rows. An older normal ear (B) is compared with a dcl1-fzt older ear (F). Normal tassels ([C] and [D]) are compared with dcl1-fzt mutant tassels ([G] and [H]). The white arrowhead in (H) indicates fasciated BMs. (I) Normal spikelet pair contains two SMs. Each SM is subtended by a glume. (J) fzt mutant spikelet “pair” contains extra SMs, and not all SMs are subtended by glumes. (K) Normal spikelet pair during floral development. Each spikelet consists of an upper FM and a lower FM. Floral organs are initiated in a stereotypical, ordered manner. (L) Older fzt mutant spikelet pair. Spikelets initiate extra FMs. Floral development is abnormal, and floral organs are not initiated properly. An indeterminate branch-like meristem persists (black arrow). Black asterisks indicate SMs, and white asterisks indicate FMs/developing florets. Bars in (A) to (H) = 0.5 mm; bars in (I) to (L) = 100 μm.

Figure 4.

dcl1-fzt Is Required for Normal Leaf Cell Differentiation. Macrohairs (arrows) are present on the adaxial surface of a normal leaf blade (A) but absent from the abaxial surface (C). dcl1-fzt [Mo17] contains fewer macrohairs on the adaxial surface (B), but macrohairs are present on the abaxial surface (D). Vascular polarity is also perturbed in dcl1-fzt [Mo17] mutants ([E] and [F]). A normal vascular bundle is shown in (E). Xylem cells (red asterisks) are positioned adaxially relative to the phloem cells (P). In dcl1-fzt [Mo17] mutants (F), the xylem cells are disorganized and extend farther toward the abaxial pole than normal. Bars in (A) and (B) = 1 mm; bars in (C) and (D) = 400 μm; bars in (E) and (F) = 50 μm.

Figure 5.

miRNA Levels Are Reduced in dcl1-fzt Mutants. (A) Comparison of individual miRNA levels in dcl1-fzt and A619 seedlings. (B) Comparison of individual miRNA levels in dcl1-fzt and normal sibling tassel primordia. Differentially expressed miRNAs (P < 0.05) are indicated by black bars, and nonstatistically significant miRNAs are indicated by gray bars. miRNAs are listed in order of ascending P values. miRNAs decreased in both tissues are boldface and underlined. miRNAs that represent the miRNA* from the miRNA duplex are indicated with asterisks. Data shown are a summary of three biological replicates.

Figure 6.

Analysis of Predicted miRNA Targets in Tassel Primordia. (A) MA plot showing all predicted miRNA targets (mF ≤ 7) for miRNAs decreased in dcl1-fzt tassel primordia. Red dots indicate miRNA targets differentially expressed in dcl1-fzt mutants (P < 0.05 and FDR < 0.05), and black dots indicate miRNA targets that are not differentially expressed. miRNA targets are not broadly increased in dcl1-fzt mutants. CPM, counts per million; FC, fold change. (B) MA plot showing predicted miRNA targets with mF ≤ 4. Dot color indicates statistical significance as in (A). Of the differentially expressed targets, the majority (12 of 14) are increased in dcl1-fzt tassel primordia. (C) qRT-PCR validation of RNA-seq analysis for select miRNA targets and pri-miRNA transcripts. Black bars indicate fold change calculated from qRT-PCR analysis; gray bars indicate fold change calculated from RNA-seq analysis; gray cross-hatched bars indicate transcripts that do not meet the statistical threshold for differential expression in RNA-seq experiments. qRT-PCR data are the result of three biological and three technical replicates. Error bars indicate se.