Dual effects of miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana

Abstract

Leaves of flowering plants are produced from the shoot apical meristem at regular intervals, with the time that elapses between the formation of two successive leaf primordia defining the plastochron. We have identified two genetic axes affecting plastochron length in Arabidopsis thaliana. One involves microRNA156 (miR156), which targets a series of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. In situ hybridization studies and misexpression experiments demonstrate that miR156 is a quantitative, rather than spatial, modulator of SPL expression in leaf primordia and that SPL activity nonautonomously inhibits initiation of new leaves at the shoot apical meristem. The second axis is exemplified by a redundantly acting pair of cytochrome P450 genes, CYP78A5/KLUH and CYP78A7, which are likely orthologs of PLASTOCHRON1 of rice (Oryza sativa). Inactivation of CYP78A5, which is expressed at the periphery of the shoot apical meristem, accelerates the leaf initiation rate, whereas cyp78a5 cyp78a7 double mutants often die as embryos with supernumerary cotyledon primordia. The effects of both miR156-targeted SPL genes and CYP78A5 on organ size are correlated with changes in plastochron length, suggesting a potential compensatory mechanism that links the rate at which leaves are produced to final leaf size.

Figure 1.

Modulation of Plastochron Length by SPL Genes. (A) Diagram of SPL9 and SPL15 transcribed regions, with thin lines indicating introns. Arrowheads mark T-DNA insertion sites. (B) Rosettes of 30-d-old short-day-grown plants. (C) Appearance of visible leaves in wild-type, Pro35S:MIR156f, and spl9 spl15 plants grown in short days (n ≥ 10). Bars indicate sd.

Figure 2.

Effects of MIR156f Misexpression. (A) Thirty-day-old short-day-grown transgenic plants expressing MIR156f from different promoters, with expression domains indicated by blue color in the accompanying illustrations of shoot apices (see Supplemental Figure 3 online for expression data). (B) Leaf initiation rates of transgenic plants, calculated from short-day-grown T1 individuals (n ≥ 10; see Supplemental Table 1 online). Bars indicate sd. Asterisks indicate a significant difference from the wild type (Student's t test with Bonferroni correction, P < 0.04).

Figure 3.

In Situ Hybridizations Showing Expression Patterns of SPL9 and miR156. (A) to (H) Expression of SPL9. (A) and (B) Wild-type vegetative (A) and reproductive shoot apex (B). (C) Pro35S:MIR156f vegetative shoot apex. (D) to (F) Wild-type, se-1, and ago1-27 vegetative shoot apices. All three samples were stained for the same amount of time in the dark. The wild type was allowed to remain underdeveloped so that the stronger signals in the other two genotypes would not become saturated. (G) and (H) Wild-type and Pro35S:MIM156 vegetative apices. Both samples were stained for the same amount of time in the dark. The wild type was allowed to remain underdeveloped so that the stronger signal in the Pro35S:MIM156 apex would not become saturated. (I) Expression of miR156 in the wild type and se-1 (inset). Shoot apices were dissected from 15-d-old plants grown in short days. Bars = 50 μm.

Figure 4.

Effects of a miR156-Insensitive Form of SPL9. (A) Diagram of the miR156 target sites of the wild-type and modified version of SPL9. Capital letters below indicate encoded amino acids. (B) Forty-day-old (ProSPL9:rSPL9 genotypes) and 20-d-old plants (all other genotypes) grown in short days. Misexpression of rSPL9 with different promoters causes a similar delay in leaf initiation. Numbers indicate the order of leaves, with 1 referring to the oldest leaf. Note that there is no apparent disruption of normal phyllotaxis in plants with decreased or increased SPL activity. (C) Appearance of visible leaves in short-day-grown plants (n = 10). Bars indicate sd.

Figure 5.

cyp78a5 and cyp78a7 Mutant Phenotypes. (A) Diagram of CYP78A5 and CYP78A7 transcribed regions, with thin lines indicating introns. Arrowheads mark T-DNA insertion sites. The cyp78a5 allele has also been described as klu-4 (Anastasiou et al., 2007). (B) Rosettes of 30-d-old plants grown in short days. (C) Appearance of visible leaves in short-day-grown plants (n = 10). Bars indicate sd. (D) and (E) Wild-type embryos at bent-cotyledon stage (D) and at maturity (E). (F) and (G) cyp78a5 cyp78a7 embryos at bent-cotyledon stage (F) and at maturity (G). The latter was dissected from the seed coat. More than 10 siliques were examined, and representative embryos are shown. Note the enlarged shoot apical meristem in (F) and multiple cotyledons in (G). Bars = 150 μm.

Figure 6.

Genetic Interaction between miR156/SPL and CYP78A5. (A) Expression of CYP78A5 and SPL9 in wild-type, mutant, and transgenic plants. Total RNA was extracted from 7-d-old long-day-grown plants and analyzed by real-time RT-PCR with three technical replicates. Expression was normalized relative to that of β-TUBULIN2. Two biological replicates were performed, both with similar results. (B) Leaf initiation rate in wild-type, mutant, and transgenic plants, calculated from 10 short-day-grown individuals (see Supplemental Table 1 online). Bars indicate sd.

Figure 7.

Morphology of Shoot Apical Meristem and Dividing Cells. (A) to (D) Scanning electron micrographs of shoot apices from 30-d-old short-day-grown plants. At least 10 individuals were examined for each genotype, and representative images are shown. (E) to (H) Histone H4 expression in shoot apices of 15-d-old short-day-grown plants. The ProSPL9:rSPL9 plants were grown at a later time point, but the wild-type controls were similar as for the other two genotypes. Bars = 50 μm.