Source Paper
Satoko Sugawara, Kiyoshi Mashiguchi, Keita Tanaka, Shojiro Hishiyama, Tatsuya Sakai et al.
Plant and Cell Physiology • 2015
The phytohormone auxin plays a central role in many aspects of plant growth and development. IAA is the most studied natural auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin for >40 years, but its role in plant growth and development remains unclear. In this study, we show that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. PAA is widely distributed in vascular and non-vascular plants. Although the biological activities of PAA are lower than those of IAA, the endogenous levels of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin-responsive genes through the TIR1/AFB pathway in Arabidopsis. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA (YUC) genes increases PAA metabolite levels in Arabidopsis, indicating that YUC flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by different types of auxins.
Objective: Assessment of gravitropic response in maize coleoptile segments through auxin transport inhibition and gravitropic stimulation tests
This is a Coleoptile Gravitropic Stimulation protocol using Maize (Zea mays) as the model organism. The procedure involves 8 procedural steps, 1 equipment items, 7 materials. Extracted from a 2015 paper published in Plant and Cell Physiology.
Model and subjects
Maize (Zea mays) • Honey Bantam 610 • unknown • Seeds germinated at 24°C for 2 days under red light, then grown in darkness for 1-2 days • Not specified
Study window
~2 day study window | ~2 hours hands-on
Core workflow
Maize seed germination • Dark growth period • Coleoptile segment preparation
Primary readouts
Key equipment and reagents
Verified items
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Germinate maize seeds at controlled temperature under red light conditions
Note: Seeds germinated under red light
“seeds of maize (Zea mays L. cv. Honey Bantam 610, Sakata Seed Corp.) were germinated at 24°C under red light for 2 d”
Grow germinated seeds in darkness to promote coleoptile development
Note: Darkness is critical for coleoptile development
“grown in darkness for 1-2 d”
Prepare coleoptile segments of standardized length from seedlings
Note: Segments must be 20 mm long; preparation method as previously reported by Nishimura et al. 2009
“Coleoptile segments (20 mm long) were prepared as previously reported (Nishimura et al. 2009)”
Apply NPA solution to coleoptile tip region to inhibit auxin transport
Note: 2.5 µl of 200 µM NPA solution placed in tip region inside each coleoptile
“A solution of 2.5 µl of NPA (200 µM NPA) was placed in the tip region inside each coleoptile”
Subject coleoptile segments to gravitropic stimulation to assess response
Note: Conducted after NPA treatment or as separate test
“used for NPA-dependent transport inhibition (1 h) and gravitropic stimulation (1 h) tests”
Isolate total RNA from coleoptile tissue samples
Note: Use RNeasy Plant Mini Kit from Qiagen
“total RNA was isolated from plants using the RNeasy Plant Mini Kit (Qiagen)”
Synthesize complementary DNA from isolated total RNA
Note: Use 1 µg aliquot of total RNA with QuantiTect Reverse Transcription kit
“A 1 µg aliquot of total RNA was used for cDNA synthesis using a QuantiTect Reverse Transcription kit (Qiagen)”
Measure expression levels of VT2 and SPI1 genes in maize coleoptiles
Note: Use 7500 Real-time PCR system with THUNDERBIRD SYBR qPCR mix; 18S rRNA used as internal standard
“Quantitative RT-PCR was performed on a 7500 Real-time PCR system (Applied Biosystems) using a THUNDERBIRD SYBR qPCR mix (Toyobo)”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
Assessment of gravitropic response in maize coleoptile segments through auxin transport inhibition and gravitropic stimulation tests
Objective
Assessment of gravitropic response in maize coleoptile segments through auxin transport inhibition and gravitropic stimulation tests
Subjects
From paperMaize (Zea mays) • Honey Bantam 610 • unknown • Seeds germinated at 24°C for 2 days under red light, then grown in darkness for 1-2 days • Not specified
Cohort notes
From paperSeeds obtained from Sakata Seed Corp.
Maize seed germination (2 days)
Dark growth period (1-2 days)
Coleoptile segment preparation (Not specified)
NPA transport inhibition test (1 hour)
Gravitropic response of coleoptile segments
From paperSemi-quantitative measurement of VT2 and SPI1 gene expression levels as previously described by Gallavotti et al.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Auxin transport inhibition by NPA
From paperSemi-quantitative measurement of VT2 and SPI1 gene expression levels as previously described by Gallavotti et al.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Expression levels of VT2 and SPI1 genes in maize coleoptiles
From paperSemi-quantitative measurement of VT2 and SPI1 gene expression levels as previously described by Gallavotti et al.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Gravitropic response of coleoptile segments
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Auxin transport inhibition by NPA
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Expression levels of VT2 and SPI1 genes in maize coleoptiles
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Semi-quantitative measurement of VT2 and SPI1 gene expression levels as previously described by Gallavotti et al.
Scoring or quantification
Quantify the primary readouts for this experiment: Gravitropic response of coleoptile segments; Auxin transport inhibition by NPA; Expression levels of VT2 and SPI1 genes in maize coleoptiles.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Gravitropic response of coleoptile segments, Auxin transport inhibition by NPA, Expression levels of VT2 and SPI1 genes in maize coleoptiles.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Satoko Sugawara et al. (2015). Distinct Characteristics of Indole-3-Acetic Acid and Phenylacetic Acid, Two Common Auxins in Plants. Plant and Cell Physiology
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Applied Biosystems • 7500 • Not mentioned • Not mentioned
Sakata Seed Corp. • Not applicable • Not mentioned • Not mentioned
Not specified • Not applicable • Not mentioned • Not mentioned
Qiagen • Not applicable • Not mentioned • Not mentioned
Qiagen • Not applicable • Not mentioned • Not mentioned
Toyobo • Not applicable • Not mentioned • Not mentioned
Invitrogen • Not applicable • Not mentioned • Not mentioned
TAKARA • Not applicable • Not mentioned • Not mentioned
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Evidence Quotes
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Evidence
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