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Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips.

ABSTRACT: To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related compounds, including tryptophan, indole-3-acetamide, and conjugated-IAA was observed. Immunolocalization showed that the IAA signal was intense in the approximately 1 mm region and the outer epidermis at the approximately 0.5 mm region from the top of coleoptiles treated with 1-N-naphthylphthalamic acid. By contrast, the IAA immuno-signal in the outer epidermis almost disappeared after 5-methyl-tryptophan treatment. Immunogold labeling of IAA with an anti-IAA-N-polyclonal antibody in the outer-epidermal cells showed cytoplasmic localization of free-IAA, but none in cell walls or vacuoles. These findings indicated that IAA is synthesized in the 0–2.0 mm region of maize coleoptile tips from Trp, in which the outer-epidermal cells of the 0.5 mm tip are the most active IAA synthetic cells.

SUBMITTER: Nishimura T

PROVIDER: S-EPMC3337196 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips.

Nishimura Takeshi T Toyooka Kiminori K Sato Mayuko M Matsumoto Sachiko S Lucas M Mercedes MM Strnad Miroslav M Baluska Frantisek F Koshiba Tomokazu T

Plant signaling & behavior 20111201 12

To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related c ...[more]

PMID: 22112455

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Project description:Arabidopsis seedlings (Col-0) were grown in suspension in half-strength MS medium with agitation at ~100 rpm at ~22 C under ~50 microeinsteins m-2s-1 cool white fluorescent continuous illumination as described by (Xiao et al., Plant Physiol. 2002 Dec;130(4):2118-28). Seedlings were treated at 10-12 days by addition of freshly made IAA (0.1 or 1.0uM) to each flask, and harvested after a 1 or 3 hour incubation. Controls were not treated and harvested at 0hr. All tissue harvested. Total RNA was extracted using TRIzol (Invitrogen) as described by the manufacturer and then filtered using QIAGEN RNeasy columns. cDNA was synthesized from total RNA using a Superscript double-stranded cDNA synthesis kit (Invitrogen) and a T7-dT24 primer. cRNA was synthesized using the Enzo BioArray HighYield RNA Transcript Labeling kit (Affymetrix p/n 900182) and fragmented by Mg2+ hydrolysis. 15ug per ATH1 array was hybridized overnight at 45 C. Arrays were then washed and scanned using the GeneChip FS400 fluidics station and Agilent GeneArray scanner. Images were analyzed using Affymetrix Microarray Suite 5.0, scaling to a target average intensity of 500. Spiking controls were added to the total RNA before cDNA synthesis and additional spiking samples were added to the resulting cDNA prior to cRNA synthesis.,In comparison table below:,SIGNAL_LOG_RATIO = Mean of log to base two of the experimental divided by control signal ratios across all probe pairs in a set.,CHANGE = Qualitative measurement indicating whether the probe set signal is increased (I), marginally increased (MI), not changed (NC), marginally decreased (MD), or decreased (D) as compared to a control hybridization across all probe pairs, based on a p-value calculation.,change_p-value = Measures the probability that all probe pairs in the set indicate a change, with 0 indicating strong likelyhood for increase, 0.5 indicating little probability for difference, and 1 indicating strong probability for decrease.

Dataset Information

Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips.

Publications

Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips.

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