Leaf Positioning of Arabidopsis in Response to Blue Light Inoue Shin-ichiro , Kinoshita Toshinori , Takemiya Atsushi , Doi Michio , Shimazaki Ken-ichiro   Molecular Plant  Volume 1, Issue 1, Pages 15-26 (January 2008) DOI: 10.1093/mp/ssm001 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Leaf Positioning in Response to a Very Low Intensity of Blue Light. Wild-type (Col-0) plants of Arabidopsis were grown under white light (50 μmol m−2 s−1) for 7 d and then transferred to red light (25 μmol m−2 s−1) with or without blue light (0.1 μmol m−2 s−1). The plants were further grown for 5 d. The supplemental blue light was applied from above (A–D) or from the side (E). White solid arrowheads show the first true leaves. White open arrowheads show cotyledons. White arrows show the direction of blue light. (A) Side view of plants after growth for 5 d with or without blue light. The white bar represents 1 cm. (B) Angles (θ) of petioles from the horizontal line. Values presented are means of 25 seedlings with standard errors. (C) Pictures taken from above. The black bar represents 1 cm. (D) Area of light perception in the first leaf. Areas of projections by the first leaves were measured by taking pictures from above. Bars represent means ± SE (n = 32). (E) Side view of plants after growth for 5 d. Side view is perpendicular to the applied blue light. Right view is from the same direction as the blue light source. Molecular Plant 2008 1, 15-26DOI: (10.1093/mp/ssm001) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Leaf Positioning Mediated by phot1. Wild-type (gl1 and WS), phot1-5 phot2-1 phot1-5 pho2-1 and hy4-3 cry2-1 plants were grown and transferred as described in Figure 1. (A) Plants grown under red light at 25 μmol m−2 s−1. (B) Plants grown under red light with blue light at 0.1 μmol m−2 s−1. (C) Angles of petioles in these plants. The measurements were done as in Figure 1. Values are the means of 25–38 seedlings with standard errors. White bars represent 1 cm. Molecular Plant 2008 1, 15-26DOI: (10.1093/mp/ssm001) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Involvement of NPH3 in Leaf Positioning. (A) Isolation of mutants impaired in upward petiole growth under the low blue light condition. The picture shows mutant plants grown under red light with low blue light. The white bar represents 1 cm. (B) Determination of the mutated gene in the isolated mutants. The genomic structure of NPH3 on chromosome 5 is shown. Black boxes and bold lines represent exons and introns, respectively. An nph3-201 mutant has a C-to-T nucleotide substitution in the last exon. This nucleotide change causes the substitution of Gln681 by the stop codon. T-DNA insertion in nph3-202 was identified in the fifth exon. (C) Expression of NPH3 and TUB2 (β-tubulin) mRNAs analyzed by RT-PCR in 2-week-old seedlings of wild-type (Col and WS) plants and of two nph3 mutants (nph3-201 and nph3-202). (D) Functional complementation of nph3-201 and nph3-202 mutants with wild-type genomic NPH3 genes. Plants of nph3-201 nph3-201 transformed with wild-type genomic NPH3 (201-G), nph3-202 and nph3-202 transformed with wild-type genomic NPH3 (202-G) were grown as in Figure 1. The white bar represents 1 cm. Molecular Plant 2008 1, 15-26DOI: (10.1093/mp/ssm001) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Expression of NPH3 mRNAs and Subcellular Localization of NPH3 Protein. (A) Expression of NPH3 mRNAs in guard cell protoplasts (GCPs), mesophyll cell protoplasts (MCPs), leaves, stems, and roots from 4-week-old plants analyzed by RT-PCR. The purities of GCPs and MCPs were 98 and 99%, respectively, on a cell number basis. ACT8 was used as an internal standard for cDNA amounts. Two separate experiments gave similar results. (B) Transient expression of NPH3–GFP proteins in Vicia epidermal cells and guard cells. The primary structure of NPH3 protein and structures of fusion proteins are illustrated. Four conserved domains in the NPH3/RPT2 family are shown in light gray open blocks as described in Liscum (2002). The BTB (broad complex, tramtrack, bric à brac)/POZ (pox virus and zinc finger) domain and the coiled-coil domain are shown in the dark gray block and black block, respectively. The full length and fragments of NPH3 proteins were fused in-frame to the N-terminal end of sGFP and were expressed transiently by particle bombardment under the control of the CaMV 35S promoter. Full length, full-length NPH3 protein fused to GFP; NPH3-201, NPH3 fragment of the N-terminus fused to GFP on Met680; Coiled-coil-C, NPH3 fragment of Phe645 to the C-terminus fused to GFP; Coiled-coil, NPH3 fragment from Phe645 to Ser696 fused to GFP; C-terminus, NPH3 fragment from Thr693 to the C-terminus fused to GFP; sGFP, GFP protein. Epidermal cells and guard cells expressing these proteins were inspected by GFP fluorescence using a confocal laser microscope. All pictures are cross-sectional. Molecular Plant 2008 1, 15-26DOI: (10.1093/mp/ssm001) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Rescue of Leaf Positioning Under a Relatively High Intensity of Blue Light in phot1-5 and nph3 Mutants. Wild-type (gl1 Col-0, and WS) plants and phot1-5 phot2-1 phot1-5 phot2-1 nph3-201 and nph3-202 plants were grown under white light at 50 μmol m−2 s−1 from fluorescent lamps for 7 d and then transferred under red light (25 μmol m−2 s−1) with blue light and allowed to grow for an additional 5 d for the determination of the petiole angles. (A) Pictures indicate the leaf positioning in the mutant plants under 5 μmol m−2 s−1 of blue light. (B) Angles of petioles were measured under 0.1 or 5 μmol m−2 s−1 of blue light as in Figure 1. Values are means of 21–28 seedlings with standard errors. Molecular Plant 2008 1, 15-26DOI: (10.1093/mp/ssm001) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Leaf Flattening in Wild Type and Various Mutants in Response to Low and High Intensities of Blue Light. Plants of the wild types (gl1 Col-0, and WS), phot1-5 phot2-1 phot1-5 phot2-1 nph3-201 and nph3-202 were initially grown under white light at 50 μmol m−2 s−1 from fluorescent lamps for 7 d. The plants were then transferred under red light (25 μmol m−2 s−1) with blue light of two different intensities and allowed to grow for an additional 10 d to determine the leaf flattening. (A) Leaf flattening of the wild types and mutants with blue light at 0.1 μmol m−2 s−1. (B) Leaf flattening of wild-types and mutants with blue light at 5 μmol m−2 s−1. White bars represent 1 cm. Molecular Plant 2008 1, 15-26DOI: (10.1093/mp/ssm001) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 7 Growth Enhancement, Chloroplast Accumulation, and Stomatal Opening in Response to Low Intensity of Blue Light. Wild-type (gl1), phot1-5 nph3-201 and nph3-6 plants were grown for 5 weeks under red light (25 μmol m−2 s−1) with or without blue light (0.1 μmol m−2 s−1). The growth was determined as fresh weight of green tissues. (A) Growth enhancement by blue light in wild-type and mutant plants. Plants grown under red light (left) and red light with blue light (right). (B) Fresh weights of green tissues of plants. Bars represent means ± SE (n = 25). Asterisks show significant statistical differences by t-test (P <0.05) in fresh weights. (C) Distribution of chloroplasts in mesophyll cells of wild-type and mutant leaves under our growth conditions. (D) Stomatal aperture in leaves of the wild type and mutants under our growth conditions. Apertures are expressed as the ratio of width to length of the guard cell pair, as described in Takemiya et al. (2005). Bars represent means ± SE (n = 25). Molecular Plant 2008 1, 15-26DOI: (10.1093/mp/ssm001) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 8 Changes in Leaf Position in Response to Blue Light. Wild-type (gl1) plants were grown under white light (50 μmol m−2 s−1) for 7 d and then transferred to red light (25 μmol m−2 s−1) from above with blue light (0.1 μmol m−2 s−1) from the plant side, and were grown for 5 d, as indicated in Figure 1E. The plants were then transferred again and irradiated with blue light (0.1 μmol m−2 s−1) from above under the red light, and growth was allowed for an additional 5 d. (A) Side view of the plants after the second transfer. Pictures were taken at the indicated times from the perpendicular to the direction of the first applied blue light, which had been derived from the left (upper panels), and taken from the same direction of the blue light (lower panels). White solid arrowheads show the first true leaves. White open arrowheads show cotyledons. The black arrow indicates the direction of the first blue light treatment. The white arrow shows the direction of the second blue light treatment. (B) Angle of the first leaf from the vertical (θL) and that of the first leaf petiole from the vertical (θP). Typical changes in these angles in response to blue light are shown. The left illustration indicates the change of angles during 8 h with high time resolution. The right illustration shows the change of angles during 5 d. Gray ovals represent the first leaves. White ovals show the cotyledons. (C) Rotation of the first leaves which occurred after the initial leaf orientation. Pictures were taken at the indicated times from above. White solid arrowheads show the first true leaves. Black arrows indicate the direction of blue light applied previously. (D) Petiole rotation. Typical changes in the angles of petioles (θR) in response to blue light are shown. Gray ovals represent the first leaves. White ovals show the cotyledons. Molecular Plant 2008 1, 15-26DOI: (10.1093/mp/ssm001) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions