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First published online 3 July 2006
doi: 10.1242/dev.02491

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Trans-acting siRNA-mediated repression of ETTIN and ARF4 regulates heteroblasty in Arabidopsis

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.

View larger version (44K): [in a new window]   Fig. 1. The structure of wild-type and mutant alleles of ETT and ARF4. (A) Genomic structure of ETT and ARF4, and the nature and position of mutant alleles of these genes. Grey box, exon; blue, conserved DNA binding domain; red, tasi-ARF target site; green, domains III and IV; triangle, T-DNA insertion. (B) RT-PCR analysis of ARF4 mRNA in arf4 mutants. PCR amplification was performed with exon primers that flanked the site of the mutation.

View larger version (51K): [in a new window]   Fig. 2. ett and arf4 mutations suppress the zip phenotype. Photographs of 11-day-old plants showing the effect of ett-15, ett-7 and arf4-2 on leaf shape. In a wild-type background, these mutations cause a slight rounding and flattening of the first two leaves. In a zip background, they partially suppress the elongation of the lamina and the epinasty that results from the premature expression of adult traits in this mutant.

View larger version (39K): [in a new window]   Fig. 3. ett and arf4 affect leaf shape and trichome distribution. (A) Successive rosette and inflorescence leaves from wild-type and mutant plants, arranged from the base (left) to the tip (right) of the stem. ett and arf4 suppress the narrow, elongated phenotype of the first two zip leaves. Later leaves of double mutants have rounder tips than zip-2, and the distal portion of the leaf blade is often wider than the proximal portion. ett and arf4 do not affect the serration of the leaf blade. (B) The L:W ratio of the leaf blade of successive leaves of wild-type, ett-7 and arf4-2 plants (n=10 plants of each genotype; ±s.e.m.). In wild-type plants, this ratio increases gradually until leaf 8, after which it remains constant until flowering. ett-7 and arf4-2 cause leaves 4-7 to be slightly rounder than normal. (C) ett-15, ett-7 and arf4-2 increase the number of leaves without abaxial trichomes (black) in both a wild-type and a zip background. This is associated with a compensatory decrease in the number of adult leaves (grey; n18, ±s.e.m.). The number of cauline leaves is indicated by the white bar. The numbers above each bar represent the percentage of flowers with a split septum, based on an analysis of the first 10 flowers of five plants of each genotype. ett-15 and arf4 partially suppress the septum splitting observed in zip. Other arf4 mutations also delay abaxial trichome production in a zip-2 background. (D) The number of leaves without abaxial trichomes among plants segregating ett-7 and zip-2. ett-7 has a semi-dominant effect on abaxial trichome production.

View larger version (70K): [in a new window]   Fig. 4. ett has an adaxialized phenotype. (A) The morphology of 3-week-old wild-type, ett-7 and kan-12 plants. kan-12 has flat leaves, whereas ett-7 is not dramatically different from wild type. (B) Camera lucida drawings of adaxial and abaxial mesophyll cells of leaf 6 of wild-type and mutant plants. The adaxialized phenotype of kan1-12 is shown for comparison. The phenotype of ett-7 is slightly weaker than that of kan1-12. ett-15 and arf4-2 have a much less significant effect, if any, on the shape of abaxial mesophyll cells.

View larger version (63K): [in a new window]   Fig. 5. zip reduces tasiR-ARF levels and increases ETT and ARF4 expression. (A) Northern analysis of zip-2, sgs3-11 and rdr6-11 shows that all three reduce levels of tasiR-ARF, whereas only sgs3 and rdr6 reduce levels of the TAS2 product siR1511. 5'RLM-RACE demonstrates that the reduction in tasiR-ARF correlates with a reduction in the cleavage of the ETT transcript. (B) Northern analysis of tasiR-ARF and ETT RNA at different times in rosette development in continuous light. (C) RT-PCR analysis of ETT and ARF4 RNA from successive 3-mm leaves of wild-type and zip plants grown in short days. zip-2 causes a consistent level of increase in both ETT and ARF4 expression, but both genes are expressed uniformly in successive leaves. The ratio of the signal relative to the loading control eIF4a is shown. (D) RT-PCR analysis of ETT and ARF4 RNA from leaves of wild-type and zip-2 plants grown in continuous light.

View larger version (60K): [in a new window]   Fig. 6. Phenotype of plants overexpressing ETT. (A) Sequence of the mutated tasi-ARF target sites in the 35S::ETTmAB construct. The amino acid sequence of ARF3 is unaffected. (B) Abaxial trichome production in primary transformants expressing 35S::ETT (48 plants), 35S::ETTmAB (76 plants) or the empty vector control (101 plants). 35S::ETTmAB produces an increase in the number of plants with precocious abaxial trichomes and with unusually late abaxial trichomes; the latter phenotype mimics the effect of ett mutations and probably results from a co-suppression of ETT. (C) RT-PCR analysis of ETT mRNA in rosettes of wild type, ett-15, zip-2 and `wild-type', zip-like and as2-like primary transgenics expressing 35S::ETTmAB. The ratio of the ETT signal relative to the loading control eIF4a is shown. (D) The phenotypes of 18-day-old plants overexpressing 35S::ETTmAB. Phenotypes ranged from zip-like plants with elongated, curled-down leaves, and 2 or 3 leaves without abaxial trichomes (middle), to more severely affected plants (right) with small tightly-curled leaves, all of which produced abaxial trichomes. The vector control is shown for comparison (left). (E) Leaves of the most severely affected transgenics were tightly in-rolled and extensively lobed, and produced leaflets (arrow) from the petiole. This phenotype became more severe in later leaves, and resembles the phenotype of as2-1 mutants, shown on the right. (F) Flowers from zip-like 35S::ETTmAB plants had short stamens and split septa (top), whereas those from more severely affected plants had narrow, mispositioned petals and sepals, unfused carpels and very short stamens. zip and as2-1 flowers are shown for comparison.

View larger version (22K): [in a new window]   Fig. 7. A model for temporal regulation of leaf morphology. (A) Heteroblasty is regulated by the interaction between factors that change temporally during shoot development (developmental clocks) and by the pathways controlling leaf morphology. ETT and ARF4 regulate a subset of the traits associated with vegetative phase change and heteroblasty. Their expression is repressed by the tasiR-ARF, the production of which requires ZIP, SGS3, RDR6 and DCL4. Other heteroblastic traits, such as leaf serration and hydathode number, are regulated by another, as-yet-unknown, target of these four genes. (B) Expression of adult traits requires ETT and ARF4. tasiR-ARF creates a threshold for entry into the adult phase by constitutively limiting levels of ETT and ARF4 transcripts. This threshold is lowered by mutations that block tasiR-ARF production (zip, rdr6, sgs3, dcl4). The developmental clock may progress to the adult phase by promoting ETT and ARF4 translation or activity.