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: Analysis of plant growth and development under controlled light and temperature conditions, with focus on auxin distribution and gene expression across various plant species and tissues
This is a Plant Growth and Development protocol using Multiple plant species as the model organism. The procedure involves 24 procedural steps, 1 equipment items, 17 materials. Extracted from a 2015 paper published in Plant and Cell Physiology.
Model and subjects
Multiple plant species • Varies by species: 2-week-old Arabidopsis seedlings, 7-day-old oat and barley shoots, 3-week-old P. patens, 2-week-old M. polymorpha, 3-month-old dry seeds to 5-week-old Arabidopsis plants
Study window
~1 week study window | ~26 hours hands-on
Core workflow
Arabidopsis seed stratification • Arabidopsis seedling growth on MS medium • Oat and barley growth in soil
Primary readouts
Key equipment and reagents
Verified items
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Arabidopsis thaliana ecotype Col-0 seeds were stratified in darkness
“Arabidopsis seeds were stratified at 4°C for 2 d in the dark”
Stratified seedlings were grown vertically on MS medium with sucrose under continuous light
Note: Continuous light conditions
“Seedlings were grown vertically on Murashige and Skoog (MS) medium with 1% sucrose under continuous light conditions at 20–22°C”
Oats and barley were grown in soil with day-night temperature cycle and photoperiod
Note: 16 h photoperiod
“Oats and barley were grown in soil at 20°C during the day and 15°C at night, with a 16 h photoperiod”
Maize seeds were germinated under red light then grown in darkness
“seeds of maize (Zea mays L. cv. Honey Bantam 610, Sakata Seed Corp.) were germinated at 24°C under red light for 2 d and grown in darkness for 1–2 d”
Coleoptile segments were prepared from germinated maize seedlings
Note: 20 mm long segments
“Coleoptile segments (20 mm long) were prepared as previously reported”
NPA solution was placed in the tip region of coleoptile segments to test auxin transport inhibition
Note: 2.5 µl of 200 µM NPA solution
“A solution of 2.5 µl of NPA (200 µM NPA) was placed in the tip region inside each coleoptile”
Coleoptile segments were subjected to gravitropic stimulation
“gravitropic stimulation (1 h) tests”
Moss protonemata were maintained on BCDATG medium plates
“The moss P. patens was maintained as protonemata on a plate containing BCDATG medium”
Gametophores were cultured on BCD medium with calcium chloride and gellan gum
“gametophores were cultured on a plate with BCD medium (Nishiyama et al. 2000) containing 0.1 mM CaCl2 and 0.1% gellan gum for 7 d”
Gametophores were incubated with auxin analogs to observe auxin response
Note: 5 µM 2,4-D or PAA
“The gametophores were incubated for 7 d with 5 µM 2,4-D or PAA”
WT seedlings were germinated and grown vertically on MS agar medium
Note: n=10 seedlings
“WT seedlings were germinated and grown vertically on MS agar medium for 5 d”
Seedlings were transferred to auxin-containing medium for continued growth
Note: n=10 seedlings
“and then on auxin-containing medium for 4 d (n = 10)”
yucQ seedlings were grown on vertical MS agar medium with auxins in first gravity direction
“yucQ seedlings were grown in the first gravity direction (1st g) on vertical MS agar medium with auxins for 4 ds”
Seedlings were rotated 90 degrees and cultured in new gravity direction
“then rotated by 90° (2nd g) and cultured for an additional 24 h”
YUC1 and YUC2 cDNA were amplified by PCR and cloned into entry vector pENTR/D-TOPO, then shuttled into pMDC7 vector
Note: Using high fidelity Pyrobest DNA polymerase from TAKARA and LR clonase reaction from Invitrogen
“Gateway cloning was used to construct pMDC7::YUC1 and pMDC7::YUC2”
Constructs were transformed into Col-0 plants using Agrobacterium tumefaciens liquid cultures
“The resulting constructs were transformed into Col-0 plants by floral dipping in Agrobacterium tumefaciens liquid cultures”
Seedlings were grown vertically on MS agar media
“seedlings were grown vertically on MS agar media for 7 d”
Seedlings were transferred to MS agar media containing β-estradiol for gene induction
Note: 10 µM β-estradiol
“then transferred to MS agar media containing 10 µM β-estradiol and grown for another 3 d”
Total RNA was isolated from plant tissues using RNeasy Plant Mini Kit
“total RNA was isolated from plants using the RNeasy Plant Mini Kit (Qiagen)”
1 µg of total RNA was converted to cDNA using QuantiTect Reverse Transcription kit
“A 1 µg aliquot of total RNA was used for cDNA synthesis using a QuantiTect Reverse Transcription kit (Qiagen)”
Gene expression levels were measured using real-time PCR with SYBR qPCR mix on Applied Biosystems 7500 system
Note: 18S rRNA used as internal standard, specific primers listed in Supplementary Table S2
“Quantitative RT–PCR was performed on a 7500 Real-time PCR system (Applied Biosystems) using a THUNDERBIRD SYBR qPCR mix (Toyobo)”
Triple knockout mutants (yuc1 yuc2 yuc6) were identified from progeny of yuc1 yuc2/+ yuc4/+ yuc6 plants by genotyping
“we identified triple knockout mutants from the progeny of yuc1 yuc2/ + yuc4/ + yuc6 plants by genotyping”
Genomic sequence of GH3.9 was amplified and cloned into pER8 vector
“To construct pER8::GH3.9, the genomic sequence was amplified using the primers shown in Supplementary Table S2”
pER8::GH3.9 construct was transformed into Arabidopsis DR5::GFP plants using floral dipping
“The resulting construct, pER8::GH3.9, was transformed into Arabidopsis DR5::GFP using floral dipping in A. tumefaciens liquid culture”
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.
Analysis of plant growth and development under controlled light and temperature conditions, with focus on auxin distribution and gene expression across various plant species and tissues
Objective
Analysis of plant growth and development under controlled light and temperature conditions, with focus on auxin distribution and gene expression across various plant species and tissues
Subjects
From paperMultiple plant species • Varies by species: 2-week-old Arabidopsis seedlings, 7-day-old oat and barley shoots, 3-week-old P. patens, 2-week-old M. polymorpha, 3-month-old dry seeds to 5-week-old Arabidopsis plants
Cohort notes
From paperMultiple tissue types analyzed including roots, stems, leaves, siliques, inflorescences, and coleoptiles
Arabidopsis seed stratification (2 days)
Arabidopsis seedling growth on MS medium
Oat and barley growth in soil
Maize seed germination (2 days under red light, then 1-2 days in darkness)
Auxin distribution across plant tissues and species
From paperSemi-quantitative measurement of VT2 and SPI1 gene expression in maize coleoptiles as previously described.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Lateral root formation
From paperSemi-quantitative measurement of VT2 and SPI1 gene expression in maize coleoptiles as previously described.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Gene expression levels of VT2 and SPI1 in maize coleoptiles
From paperSemi-quantitative measurement of VT2 and SPI1 gene expression in maize coleoptiles as previously described.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
YUC gene expression levels via quantitative RT-PCR
From paperSemi-quantitative measurement of VT2 and SPI1 gene expression in maize coleoptiles as previously described.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Auxin distribution across plant tissues and species
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
Lateral root formation
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
Gene expression levels of VT2 and SPI1 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
YUC gene expression levels via quantitative RT-PCR
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 in maize coleoptiles as previously described.
Scoring or quantification
Quantify the primary readouts for this experiment: Auxin distribution across plant tissues and species; Lateral root formation; Gene expression levels of VT2 and SPI1 in maize coleoptiles; YUC gene expression levels via quantitative RT-PCR.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Auxin distribution across plant tissues and species, Lateral root formation, Gene expression levels of VT2 and SPI1 in maize coleoptiles, YUC gene expression levels via quantitative RT-PCR.
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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Invitrogen
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TAKARA
Qiagen
Qiagen
Toyobo
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Sakata Seed Corp.
Arabidopsis Biological Resource Center (ABRC)
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