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Protocol Steps
View Abstract
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.
1
Maize seed germination
Germinate maize seeds at controlled temperature under red light conditions
2 days24°C
Note: Seeds germinated under red light
View evidence from paper
“seeds of maize (Zea mays L. cv. Honey Bantam 610, Sakata Seed Corp.) were germinated at 24°C under red light for 2 d”
2
Dark growth period
Grow germinated seeds in darkness to promote coleoptile development
1-2 daysNot specified
Note: Darkness is critical for coleoptile development
View evidence from paper
“grown in darkness for 1-2 d”
3
Coleoptile segment preparation
Prepare coleoptile segments of standardized length from seedlings
Not specifiedNot specified
Note: Segments must be 20 mm long; preparation method as previously reported by Nishimura et al. 2009
View evidence from paper
“Coleoptile segments (20 mm long) were prepared as previously reported (Nishimura et al. 2009)”
4
NPA transport inhibition test
Apply NPA solution to coleoptile tip region to inhibit auxin transport
1 hourNot specified
Note: 2.5 μl of 200 μM NPA solution placed in tip region inside each coleoptile
View evidence from paper
“A solution of 2.5 μl of NPA (200 μM NPA) was placed in the tip region inside each coleoptile”
5
Gravitropic stimulation test
Subject coleoptile segments to gravitropic stimulation to assess response
1 hourNot specified
Note: Conducted after NPA treatment or as separate test
View evidence from paper
“used for NPA-dependent transport inhibition (1 h) and gravitropic stimulation (1 h) tests”
6
RNA extraction
Isolate total RNA from coleoptile tissue samples
Not specifiedNot specified
Note: Use RNeasy Plant Mini Kit from Qiagen
View evidence from paper
“total RNA was isolated from plants using the RNeasy Plant Mini Kit (Qiagen)”
7
cDNA synthesis
Synthesize complementary DNA from isolated total RNA
Not specifiedNot specified
Note: Use 1 μg aliquot of total RNA with QuantiTect Reverse Transcription kit
View evidence from paper
“A 1 μg aliquot of total RNA was used for cDNA synthesis using a QuantiTect Reverse Transcription kit (Qiagen)”
8
Quantitative RT-PCR
Measure expression levels of VT2 and SPI1 genes in maize coleoptiles
Not specifiedNot specified
Note: Use 7500 Real-time PCR system with THUNDERBIRD SYBR qPCR mix; 18S rRNA used as internal standard
View evidence from paper
“Quantitative RT-PCR was performed on a 7500 Real-time PCR system (Applied Biosystems) using a THUNDERBIRD SYBR qPCR mix (Toyobo)”
Subjects / Specimens
Species
Maize (Zea mays)
Strain
Honey Bantam 610
Age
Seeds germinated at 24°C for 2 days under red light, then grown in darkness for 1-2 days