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First published online 11 February 2009
doi: 10.1242/dev.028613

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The more and smaller cells mutants of Arabidopsis thaliana identify novel roles for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes in the control of heteroblasty

Takeshi Usami¹, Gorou Horiguchi^2,*,, Satoshi Yano¹ and Hirokazu Tsukaya^1,2

¹ National Institute for Basic Biology, Nishigo-naka 38, Myodaiji-cho, Okazaki, Aichi, 444-8585 Japan.
² Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0033 Japan.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Phenotypes of Arabidopsis msc mutants. (A) Morphology of the shoots of seedlings 25 days after sowing. (B-D) Leaf area (B), total cell number (C) and average cell area (D) of the first leaves of 30-day-old seedlings. Subepidermal palisade mesophyll cells were observed. The mean ±s.d. from six individual plants is indicated. (E) Seedling morphology of the wild type and msc2 mutant 10 days after sowing. (F) Seedling morphology of the wild type and msc3 mutant 18 days after sowing. Note that the wild type has seven visible true leaves, whereas the msc3 mutant has five. Arrowheads indicate first leaves. Scale bars: 1 cm in A,F; 5 mm in E.

View larger version (29K): [in this window] [in a new window]   Fig. 2. msc1-D, msc2 and msc3 have genetic lesions in the heteroblasty-related genes SPL15, PAUSED and SQUINT, respectively. (A) Diagram of Arabidopsis SPL15, PAUSED (PSD) and SQUINT (SQN) genes. Coding and non-coding regions of exons are denoted by black and white boxes, respectively, and introns by lines between boxes. The C-to-T nucleotide substitution in the codon of Leu 226 (CTG to TTG) found in the msc1-D mutant does not cause an amino acid change. A T-DNA insertion and an 870 bp deletion found in msc2, and a 127 bp deletion in msc3, are indicated. (B,C)5'RLM-RACE of SPL15 indicates that the efficiency of miR156-directed cleavage of SPL15 mRNA was reduced in msc1-D. Shown are the 5'RLM-RACE products of SPL15 (B) and the cleavage sites identified by 5'RLM-RACE (C). (D) SPL15 mRNA levels in the first and second leaves of 14-day-old seedlings (14d), and third and fourth leaves of 21-day-old seedlings (21d) of the wild type and msc1-D mutant as determined by quantitative RT-PCR. (E) Heteroblasty in msc mutants. The number of rosette leaves without abaxial trichomes (black) or with abaxial trichomes (gray) and cauline leaves (white) were counted. The mean ±s.d. (n=9-10) is indicated.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Cell number increases and cell size decreases in leaves at higher nodes. Total cell number (A) and average cell area (B) of the first, third and fifth leaves of the wild type and msc mutants 30 days after sowing. The mean ±s.d. from six individual plants is indicated. *P<0.05 and **P<0.01 versus wild type (Student's t-test).

View larger version (47K): [in this window] [in a new window]   Fig. 4. Heteroblastic changes in cell number and cell size are controlled predominantly by miR156-dependent regulation of SPL genes. Total cell number (A) and average cell area (B) of rdr6-11, sgs3-11 and zip-1 mutants. Total cell number (C) and average cell area (D) of arf3-2 and arf4-2, and the arf3-2 arf4-2 double mutant. Total cell number (E) and average cell area (F) of the transgenic plants overexpressing miR156 (35S:miR156), SPL3 with a mutation in the miR156 target site (35S:SPL3m), or SPL3, SPL4 or SPL5 with a deletion in the miR156 target site (35S:SPL3, 35S:SPL4 and 35S:SPL5). All samples were observed 30 days after sowing. The mean ±s.d. from six individual plants is indicated. *P<0.05 and **P<0.01 versus wild type (Student's t-test).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Quantification of the expression levels of miR156 and of ten SPL genes with the miR156 target site. Total RNAs were extracted from the first and second leaves of 15-day-old wild-type and msc1-msc3 mutant Arabidopsis seedlings. (A) Northern blot of miR156. Relative intensities of signals, normalized to U6, are indicated. (B) Quantitative RT-PCR of SPL genes. Relative expression levels in msc1-msc3 as compared with the wild type are indicated.

View larger version (16K): [in this window] [in a new window]   Fig. 6. The phenotypes of msc2 and msc3 Arabidopsis mutants are suppressed by overexpression of miR156. Total cell number (A) and average cell area (B) of msc2 35S:miR156 and msc3 35S:miR156 double mutants 30 days after sowing. The mean ±s.d. from six individual plants is indicated. *P<0.05 and **P<0.01 versus wild type (Student's t-test).

View larger version (24K): [in this window] [in a new window]   Fig. 7. Heteroblasty in compensation-exhibiting mutants and the axr1 mutant. (A) The number of rosette leaves without abaxial trichomes (black), with abaxial trichomes (gray) and cauline leaves (white) were counted in an3, ant and fugu1-fugu5 mutants. The mean ±s.d. (n=4-10) is indicated. Total cell number (B) and average cell area (C) of the wild type, axr1-3 and axr1-3 msc1-D mutants 30 days after sowing. The mean ±s.d. from six individual plants is indicated. *P<0.05 and **P<0.01 versus wild type (Student's t-test).

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