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, 35 (1), 39-49

A Genetic Link Between Epigenetic Repressor AS1-AS2 and DNA Replication Factors in Establishment of Adaxial-Abaxial Leaf Polarity of Arabidopsis

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A Genetic Link Between Epigenetic Repressor AS1-AS2 and DNA Replication Factors in Establishment of Adaxial-Abaxial Leaf Polarity of Arabidopsis

Toan Quy Luong et al. Plant Biotechnol (Tokyo).

Abstract

Balanced development of adaxial and abaxial domains in leaf primordia is critical for the formation of flat symmetric leaf lamina. Arabidopsis ASYMMETRIC LEAVES1 (AS1) and AS2 proteins form a complex (AS1-AS2), which acts as key regulators for the adaxial development by the direct repression of expression of the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). Many modifier mutations have been identified, which enhance the defect of as1 and as2 mutations to generate abaxialized filamentous leaves without adaxial traits, suggesting that the development of the adaxial domain is achieved by cooperative repression by AS1-AS2 and the wild-type proteins corresponding to the modifiers. Mutations of several genes for DNA replication-related chromatin remodeling factors such as Chromatin Assembly Factor-1 (CAF-1) have been also identified as modifiers. It is still unknown, however, whether mutations in genes involved in DNA replication themselves might act as modifiers. Here we report that as1 and as2 mutants grown in the presence of hydroxyurea, a known inhibitor of DNA replication, form abaxialized filamentous leaves in a concentration-dependent manner. We further show that a mutation of the INCURVATA2 (ICU2) gene, which encodes the putative catalytic subunit of DNA polymerase α, and a mutation of the Replication Factor C Subunit3 (RFC3) gene, which encodes a protein used in replication as a clamp loader, act as modifiers. In addition, as2-1 icu2-1 double mutants showed increased mRNA levels of the genes for leaf abaxialization. These results suggest a tight link between DNA replication and the function of AS1-AS2 in the development of flat leaves.

Keywords: ASYMMETRIC LEAVES2; DNA replication; ICU2; RFC3; leaf development.

Figures

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Figure 1. Effects of hydroxyurea on as1-1 and as2-1 mutants. (A) Phenotypes of Col-0, as1-1, and as2-1 plants grown on agar plates in the presence and the absence of 4 mM, 6 mM hydroxyurea. Red arrowheads on as1-1 and as2-1 plants show filamentous leaves. Scale bars=2 mm. (B) Frequencies of plants that have true leaves grown in the presence and in the absence of hydroxyurea. Frequency is defined as the ratio of the number of plants having expanded leaves to the total number (n=30) of plants examined. Plants were grown at 22°C. Observations were performed at 21 days after sowing. Bars represent the s.d. from three biological replicates. (C) Frequencies of plants with filamentous leaves grown in the presence and in the absence of hydroxyurea. Frequency is defined as the ratio of the number of plants with more than one filamentous leaf to the total number (n=30) of plants examined. Plants were grown at 22°C. Observations were performed at 21 days after sowing. Bars represent the s.d. from three biological replicates. (D) as1-1/FILp:GFP and as2-1/FILp:GFP plants were grown on medium with and without 4 mM hydroxyurea for 21 days. Expression patterns of FILp:GFP in transverse sections of leaves are shown. Green signals due to GFP; red, autofluorescence. Yellow signals were due to both GFP and autofluorescence. Scale bars=50 µm. Significant differences from wild type were evaluated by Student’s t-test and are represented by asterisks (* p<0.01) in (B) and (C).
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Figure 2. The inc2-1 mutation enhanced the leaf-phenotype in as1-1 and as2-1 mutants. Gross morphology at 21 days after sowing. Plants indicated below pictures were grown as described in Materials and methods. The as1-1 icu2-1 and as2-1 icu2-1 double mutants exhibited filamentous leaves. Arrowheads indicate filamentous leaves. Scale bars=5 mm.
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Figure 3. Transcript levels of genes involved in the determination of leaf polarity and class 1 KNOX genes. Levels of relative expression of (A) class 1 KNOX genes, (B) genes involved in leaf abaxialization (ETT and ARF4), (C) other genes that are involved in leaf abaxialization (KAN2, FIL, YAB5), and a gene that is involved in leaf adaxialization (PHB), respectively, relative to those levels in the wild-type (Col-0) plants. Total RNA was extracted from shoot apices of 14-day-old Col-0, as2-1, icu2-1, and as2-1 icu2-1. Each value was normalized by reference to the level of ACTIN2 (ACT2, at3g18780) transcripts. Light brown, light green and light blue show class 1 KNOX genes, abaxial determinant genes and an adaxial determinant gene, respectively. The values from wild-type plants were arbitrarily set at 1.0. Bars indicate the s.d. among more than three biological replicates. Significant differences from wild type were evaluated by Student’s t-test and are represented by asterisks (* p<0.05 and ** p<0.01).
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Figure 4. The rfc3-1 mutation enhanced the leaf-phenotype in as1-1 and as2-1 mutants. The as1-1 rfc3-1 double mutants exhibited trumpet-like leaves, while the as2-1 rfc3-1 double mutants exhibited filamentous leaves. Gross morphology at 21 days after sowing. Arrowheads indicate higher magnification views of trumpet-like leaves or filamentous leaves. Scale bars=5 mm.
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Figure 5. Roles of AS1–AS2, ICU2, and RFC3 in leaf development in A. thaliana. The AS1–AS2 complex and DNA replication-related factors ICU2 (or RFC3) act cooperatively to repress expression of the leaf abaxial determinant gene ETT/ARF3 and ARF4. Repression of ARFs is crucial for development of the adaxial domain of leaves and then formation of flat and symmetric leaves.

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