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Hyperspectral imaging techniques for rapid identification of Arabidopsis mutants with altered leaf pigment status.

Matsuda O, Tanaka A, Fujita T, Iba K - Plant Cell Physiol. (2012)

Bottom Line: The 'non-targeted' mode highlights differences in reflectance spectra of leaf samples relative to reference spectra from the wild-type leaves.Analysis of these and other mutants revealed that the RI-based targeted pigment estimation was robust at least against changes in trichome density, but was confounded by genetic defects in chloroplast photorelocation movement.Notwithstanding such a limitation, the techniques presented here provide rapid and high-sensitive means to identify genetic mechanisms that coordinate leaf pigment status with developmental stages and/or environmental stress conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan. matsuda.osamu.084@m.kyushu-u.ac.jp

ABSTRACT
The spectral reflectance signature of living organisms provides information that closely reflects their physiological status. Because of its high potential for the estimation of geomorphic biological parameters, particularly of gross photosynthesis of plants, two-dimensional spectroscopy, via the use of hyperspectral instruments, has been widely used in remote sensing applications. In genetics research, in contrast, the reflectance phenotype has rarely been the subject of quantitative analysis; its potential for illuminating the pathway leading from the gene to phenotype remains largely unexplored. In this study, we employed hyperspectral imaging techniques to identify Arabidopsis mutants with altered leaf pigment status. The techniques are comprised of two modes; the first is referred to as the 'targeted mode' and the second as the 'non-targeted mode'. The 'targeted' mode is aimed at visualizing individual concentrations and compositional parameters of leaf pigments based on reflectance indices (RIs) developed for Chls a and b, carotenoids and anthocyanins. The 'non-targeted' mode highlights differences in reflectance spectra of leaf samples relative to reference spectra from the wild-type leaves. Through the latter approach, three mutant lines with weak irregular reflectance phenotypes, that are hardly identifiable by simple observation, were isolated. Analysis of these and other mutants revealed that the RI-based targeted pigment estimation was robust at least against changes in trichome density, but was confounded by genetic defects in chloroplast photorelocation movement. Notwithstanding such a limitation, the techniques presented here provide rapid and high-sensitive means to identify genetic mechanisms that coordinate leaf pigment status with developmental stages and/or environmental stress conditions.

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Isolation and characterization of mutants with irregular reflectance (iref) phenotypes. Pseudocolor images in D and E were produced by HSD Visualizer (Fig. 2D), while those in F were produced by PPM (Fig. 2C). The hyperspectral image data used in E and F are provided as Supplementary File S2 of this article. (A) Reflectance spectra of the wild-type (WT) and iref mutant leaves. The reflectance was calculated by linear regression against the 50% reflectance standard (as in Fig. 3B). Data are the mean ± SD (SD is shown by a vertical bar) derived from measurements of five different leaves of Arabidopsis plants from each genotype grown under normal conditions (as defined in Table 1). (B) Ratio reflectance spectra of iref mutants relative to the wild type. Data are derived from the spectra shown in A. As an example, maximum and average deviations of spectral reflectance in a wavelength range of 600–650 nm are shown for each iref mutant. (C) RGB image of iref mutants grown under normal conditions. (D) Visualization of iref phenotypes. The plants shown in C were subjected to a hyperspectral imaging procedure. The deviation of spectral reflectance in iref mutants relative to averaged reference spectra from the wild-type leaves (indicated by open triangles in C and D) is displayed in a color gradient from black (smallest) to pink (largest). Here, the values of maximum deviation in a wavelength range of 600–650 nm were used for visualization. (E) Confirmation of iref1 and iref3 loci through an allelism test and genetic complementation. The image was produced by the same procedure as in D, except detached leaves from the indicated plant lines were used. iref1 T-DNA1, WiscDsLox457-460P9 homozygous line; iref1 T-DNA2, SAIL_574_B09 homozygous line; iref3/At1g09520 Nos. 1 and 2, T2 individuals from transgenic iref3 lines carrying a wild-type genomic fragment of the At1g09520 locus. (F) Visualization of predicted pigment status in iref mutants. Individual concentrations and compositional parameters of pigments indicated on the left were calculated using Equations 3 (Chl a), 5 (Chl b) and 11 (Car). The range of parameters displayed in a color gradient from black (smallest) to pink (largest) is indicated alongside the scale bar on the right.
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pcs043-F7: Isolation and characterization of mutants with irregular reflectance (iref) phenotypes. Pseudocolor images in D and E were produced by HSD Visualizer (Fig. 2D), while those in F were produced by PPM (Fig. 2C). The hyperspectral image data used in E and F are provided as Supplementary File S2 of this article. (A) Reflectance spectra of the wild-type (WT) and iref mutant leaves. The reflectance was calculated by linear regression against the 50% reflectance standard (as in Fig. 3B). Data are the mean ± SD (SD is shown by a vertical bar) derived from measurements of five different leaves of Arabidopsis plants from each genotype grown under normal conditions (as defined in Table 1). (B) Ratio reflectance spectra of iref mutants relative to the wild type. Data are derived from the spectra shown in A. As an example, maximum and average deviations of spectral reflectance in a wavelength range of 600–650 nm are shown for each iref mutant. (C) RGB image of iref mutants grown under normal conditions. (D) Visualization of iref phenotypes. The plants shown in C were subjected to a hyperspectral imaging procedure. The deviation of spectral reflectance in iref mutants relative to averaged reference spectra from the wild-type leaves (indicated by open triangles in C and D) is displayed in a color gradient from black (smallest) to pink (largest). Here, the values of maximum deviation in a wavelength range of 600–650 nm were used for visualization. (E) Confirmation of iref1 and iref3 loci through an allelism test and genetic complementation. The image was produced by the same procedure as in D, except detached leaves from the indicated plant lines were used. iref1 T-DNA1, WiscDsLox457-460P9 homozygous line; iref1 T-DNA2, SAIL_574_B09 homozygous line; iref3/At1g09520 Nos. 1 and 2, T2 individuals from transgenic iref3 lines carrying a wild-type genomic fragment of the At1g09520 locus. (F) Visualization of predicted pigment status in iref mutants. Individual concentrations and compositional parameters of pigments indicated on the left were calculated using Equations 3 (Chl a), 5 (Chl b) and 11 (Car). The range of parameters displayed in a color gradient from black (smallest) to pink (largest) is indicated alongside the scale bar on the right.

Mentions: Using these equations, the software PPM (Plant Pigment Monitor), which allows 2D monitoring of leaf pigment status, was constructed (Fig. 2C). In addition to the individual concentrations, this also allows visualization of compositional parameters of pigments such as the sum and/or ratio of two or more constituents (e.g. see Fig. 7F). The software is provided as Supplementary File S1 of this article (see Supplementary Text S1 for legends and methods of operation).Fig. 7


Hyperspectral imaging techniques for rapid identification of Arabidopsis mutants with altered leaf pigment status.

Matsuda O, Tanaka A, Fujita T, Iba K - Plant Cell Physiol. (2012)

Isolation and characterization of mutants with irregular reflectance (iref) phenotypes. Pseudocolor images in D and E were produced by HSD Visualizer (Fig. 2D), while those in F were produced by PPM (Fig. 2C). The hyperspectral image data used in E and F are provided as Supplementary File S2 of this article. (A) Reflectance spectra of the wild-type (WT) and iref mutant leaves. The reflectance was calculated by linear regression against the 50% reflectance standard (as in Fig. 3B). Data are the mean ± SD (SD is shown by a vertical bar) derived from measurements of five different leaves of Arabidopsis plants from each genotype grown under normal conditions (as defined in Table 1). (B) Ratio reflectance spectra of iref mutants relative to the wild type. Data are derived from the spectra shown in A. As an example, maximum and average deviations of spectral reflectance in a wavelength range of 600–650 nm are shown for each iref mutant. (C) RGB image of iref mutants grown under normal conditions. (D) Visualization of iref phenotypes. The plants shown in C were subjected to a hyperspectral imaging procedure. The deviation of spectral reflectance in iref mutants relative to averaged reference spectra from the wild-type leaves (indicated by open triangles in C and D) is displayed in a color gradient from black (smallest) to pink (largest). Here, the values of maximum deviation in a wavelength range of 600–650 nm were used for visualization. (E) Confirmation of iref1 and iref3 loci through an allelism test and genetic complementation. The image was produced by the same procedure as in D, except detached leaves from the indicated plant lines were used. iref1 T-DNA1, WiscDsLox457-460P9 homozygous line; iref1 T-DNA2, SAIL_574_B09 homozygous line; iref3/At1g09520 Nos. 1 and 2, T2 individuals from transgenic iref3 lines carrying a wild-type genomic fragment of the At1g09520 locus. (F) Visualization of predicted pigment status in iref mutants. Individual concentrations and compositional parameters of pigments indicated on the left were calculated using Equations 3 (Chl a), 5 (Chl b) and 11 (Car). The range of parameters displayed in a color gradient from black (smallest) to pink (largest) is indicated alongside the scale bar on the right.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3367163&req=5

pcs043-F7: Isolation and characterization of mutants with irregular reflectance (iref) phenotypes. Pseudocolor images in D and E were produced by HSD Visualizer (Fig. 2D), while those in F were produced by PPM (Fig. 2C). The hyperspectral image data used in E and F are provided as Supplementary File S2 of this article. (A) Reflectance spectra of the wild-type (WT) and iref mutant leaves. The reflectance was calculated by linear regression against the 50% reflectance standard (as in Fig. 3B). Data are the mean ± SD (SD is shown by a vertical bar) derived from measurements of five different leaves of Arabidopsis plants from each genotype grown under normal conditions (as defined in Table 1). (B) Ratio reflectance spectra of iref mutants relative to the wild type. Data are derived from the spectra shown in A. As an example, maximum and average deviations of spectral reflectance in a wavelength range of 600–650 nm are shown for each iref mutant. (C) RGB image of iref mutants grown under normal conditions. (D) Visualization of iref phenotypes. The plants shown in C were subjected to a hyperspectral imaging procedure. The deviation of spectral reflectance in iref mutants relative to averaged reference spectra from the wild-type leaves (indicated by open triangles in C and D) is displayed in a color gradient from black (smallest) to pink (largest). Here, the values of maximum deviation in a wavelength range of 600–650 nm were used for visualization. (E) Confirmation of iref1 and iref3 loci through an allelism test and genetic complementation. The image was produced by the same procedure as in D, except detached leaves from the indicated plant lines were used. iref1 T-DNA1, WiscDsLox457-460P9 homozygous line; iref1 T-DNA2, SAIL_574_B09 homozygous line; iref3/At1g09520 Nos. 1 and 2, T2 individuals from transgenic iref3 lines carrying a wild-type genomic fragment of the At1g09520 locus. (F) Visualization of predicted pigment status in iref mutants. Individual concentrations and compositional parameters of pigments indicated on the left were calculated using Equations 3 (Chl a), 5 (Chl b) and 11 (Car). The range of parameters displayed in a color gradient from black (smallest) to pink (largest) is indicated alongside the scale bar on the right.
Mentions: Using these equations, the software PPM (Plant Pigment Monitor), which allows 2D monitoring of leaf pigment status, was constructed (Fig. 2C). In addition to the individual concentrations, this also allows visualization of compositional parameters of pigments such as the sum and/or ratio of two or more constituents (e.g. see Fig. 7F). The software is provided as Supplementary File S1 of this article (see Supplementary Text S1 for legends and methods of operation).Fig. 7

Bottom Line: The 'non-targeted' mode highlights differences in reflectance spectra of leaf samples relative to reference spectra from the wild-type leaves.Analysis of these and other mutants revealed that the RI-based targeted pigment estimation was robust at least against changes in trichome density, but was confounded by genetic defects in chloroplast photorelocation movement.Notwithstanding such a limitation, the techniques presented here provide rapid and high-sensitive means to identify genetic mechanisms that coordinate leaf pigment status with developmental stages and/or environmental stress conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan. matsuda.osamu.084@m.kyushu-u.ac.jp

ABSTRACT
The spectral reflectance signature of living organisms provides information that closely reflects their physiological status. Because of its high potential for the estimation of geomorphic biological parameters, particularly of gross photosynthesis of plants, two-dimensional spectroscopy, via the use of hyperspectral instruments, has been widely used in remote sensing applications. In genetics research, in contrast, the reflectance phenotype has rarely been the subject of quantitative analysis; its potential for illuminating the pathway leading from the gene to phenotype remains largely unexplored. In this study, we employed hyperspectral imaging techniques to identify Arabidopsis mutants with altered leaf pigment status. The techniques are comprised of two modes; the first is referred to as the 'targeted mode' and the second as the 'non-targeted mode'. The 'targeted' mode is aimed at visualizing individual concentrations and compositional parameters of leaf pigments based on reflectance indices (RIs) developed for Chls a and b, carotenoids and anthocyanins. The 'non-targeted' mode highlights differences in reflectance spectra of leaf samples relative to reference spectra from the wild-type leaves. Through the latter approach, three mutant lines with weak irregular reflectance phenotypes, that are hardly identifiable by simple observation, were isolated. Analysis of these and other mutants revealed that the RI-based targeted pigment estimation was robust at least against changes in trichome density, but was confounded by genetic defects in chloroplast photorelocation movement. Notwithstanding such a limitation, the techniques presented here provide rapid and high-sensitive means to identify genetic mechanisms that coordinate leaf pigment status with developmental stages and/or environmental stress conditions.

Show MeSH
Related in: MedlinePlus