Data from: Coding-sequence evolution does not explain divergence in petal anthocyanin pigmentation between Mimulus luteus var. luteus and M. l. variegatus
Data files
Jun 16, 2023 version files 16.82 KB
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README.md
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Sgreen_pigment_data_for_Dryad.xlsx
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Aug 29, 2024 version files 3.84 GB
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Dataset1_All_Tobacco_leaf_images_Brightness_30percent.pptx
209.10 MB
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Dataset1_Sgreen.csv
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Dataset2_Infiltrated_Leaves_Data_Summer_2024_ForDataDryad.csv
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Dataset2_LeafPunchImages.zip
1.84 GB
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Dataset2_WholeLeafImages.zip
1.79 GB
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README.md
3.65 KB
Abstract
Biologists have long been interested in understanding genetic constraints on the evolution of development. For example, noncoding changes in a gene might be favored relative to coding changes due to being less constrained by pleiotropic effects. Here we evaluate the importance of coding-sequence changes to the recent evolution of a novel anthocyanin pigmentation trait in the monkeyflower genus Mimulus. The magenta-flowered Mimulus luteus var. variegatus recently gained petal lobe anthocyanin pigmentation via a single-locus Mendelian difference from its sister taxon, the yellow-flowered M. l. luteus. Previous work showed that the differentially expressed transcription factor gene MYB5a/NEGAN is the single causal gene. However, it was not clear whether MYB5a coding-sequence evolution (in addition to the observed patterns of differential expression) might also have contributed to increased anthocyanin production in M. l. variegatus. Quantitative image analysis of tobacco leaves, transfected with MYB5a coding sequence from each taxon, revealed robust anthocyanin production driven by both alleles. Counter to expectations, significantly higher anthocyanin production was driven by the allele from the low-anthocyanin M. l. luteus. Together with previously-published expression studies, this supports the hypothesis that petal pigment in M. l. variegatus was not gained by protein-coding changes, but instead solely via non-coding cis-regulatory evolution. Finally, while constructing the transgenes needed for this experiment, we unexpectedly discovered two sites in MYB5a that appear to be post-transcriptionally edited – a phenomenon that has been rarely reported, and even less often explored, for nuclear-encoded plant mRNAs.
Anthocyanin pigmentation values obtained from Nicotiana tabacum leaves that had been infiltrated with one of three overexpression transgenes (containing the coding regions of the MYB5a gene from Mimulus luteus var. luteus or from M. l. variegatus; or a negative control of pGFPGUSPlus).
Description of the data and file structure
Damaged leaves were excluded from the dataset prior to analysis.
Dataset1_Sgreen.csv: Infiltrated areas of the leaf were digitally defined and analyzed for color.*
Pair: Each pair of luteus and variegatus transgenes that were infiltrated into the same leaf also share an ID number.
Treatment: Leaf infiltrated with the coding regions of the MYB5a gene from Mimulus luteus var. luteus ("luteus") or from M. l. variegatus ("variegatus"); or a negative control of pGFPGUSPlus ("pGFP").
Vis**-red:* 1 indicates pigment intensity higher than the average for the negative controls and represents plants where red pigment was visibly produced. A value of 0 corresponds to no visible pigment produced. "n/a" corresponds to the negative controls which were not assessed.
Sgreen: Obtained by digital image analysis of the infiltrated region as described in the manuscript. A lower Sgreen value corresponds to a greater intensity of red (anthocyanin) pigmentation.
Redness: 1-Sgreen of the infiltrated region.
Dataset2_Infiltrated_Leaves_Data_Summer_2024_ForDataDryad.csv: Leaf discs of uniform size were excised from infiltrated leaves and were analyzed in two ways: digitally, as in Dataset 1, and also by extracting their anthocyanins and quantifying anthocyanin concentration via spectrophotometry.
Leaf ID: Each pair of luteus and variegatus transgenes that were infiltrated into the same leaf also share an ID number.
Type: The line of Nicotiana tabacum plant into which the transgenes were infiltrated.
LeafNumber: Which leaf was infiltrated, counting from the bottom up with the first true leaf assigned the number 1.
LeafSize: Length in centimeters.
*Treatment: Leaf infiltrated with the coding regions of the MYB5a gene from Mimulus luteus var. luteus ("l") or from M. l. variegatus ("v"); or a negative control of pGFPGUSPlus ("n").
Infiltration: Indicates which infiltration number the data were obtained from, with the first four infiltrations used as pilots to establish methodology and excluded from the final dataset.
A530: Absorbance at 530 nm of the anthocyanin extraction from a leaf disc.
A653: Absorbance at 653 nm of the anthocyanin extraction from a leaf disc.
Anthocyanin concentration estimated by spectrophotometry, in Absorbance Units: A530-(0.24A653).
Visible Anthocyanin in leaf? 0 = none, 1 = faint, 2 = dark
*Sgreen of Punch: *Obtained by digital image analysis of a leaf disc as described in the manuscript. A lower Sgreen value corresponds to a greater intensity of red (anthocyanin) pigmentation.
Redness of punch: 1-Sgreen of the leaf disc.
Dataset1_All_Tobacco_leaf_images_Brightness_30percent.pptx: Whole-leaf images from Dataset 1, with brightness uniformly increased 30% in PowerPoint for better clarity to the human eye.
Dataset2_LeafPunchImages.zip. Leaf disc images from Dataset 2, unmodified.
Dataset2_WholeLeafImages.zip. Whole-leaf images from Dataset 2, prior to punching out a leaf disc, unmodified.
Code/Software
Image analysis code is at https://github.com/WhitmanOptiLab/PigmentSpotting.
A 1-mL disposable syringe with the needle removed was used to deliver A. tumefaciens cells containing overexpression transgenes to the leaves of young (1–3 months) N. tabacum. The top of the leaf was held firmly while the leaf underside was injected until liquid had visibly spread past the site of injection (100–200μL). The M. l. luteus and M. l. variegatus alleles of transcription factor gene MYB5a were infiltrated in pairs, alternating which transgene was injected into the left versus the right side of each leaf. pGFPGUSPlus negative controls were evenly distributed between the two sides of other leaves.
Leaves were imaged 3–12 days after infiltration in a dark room with a Nikon D3500 DSLR camera with an 18–55mm lens, illuminated by a Sylvania Ceramic Metal Halide bulb. VGG Image Annotator was used on .jpeg images to demarcate the infiltration boundaries and injection site.
A custom Python program imported RAW image files for processing, using a modified version of the MacDuff color chart detection algorithm (https://github.com/mathandy/python-macduff-colorchecker-detector) to automatically detect a reference color chart. Image pixel values were converted into normalized reflectance values based on a linear fit of the red, green, and blue signal strengths to the color chart.
No special software is required to open the data files, but data may be analyzed using the image analysis code at https://github.com/WhitmanOptiLab/PigmentSpotting
- Orr, Walker E et al. (2022), Coding-sequence evolution does not explain divergence in petal anthocyanin pigmentation between Mimulus luteus var. luteus and M. l. variegatus, [], Posted-content, https://doi.org/10.1101/2022.12.11.519905