A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

Lana S. Barkawi; Yuen Yee Tam; Julie A. Tillman; Ben Pederson; Jessica Calio; Hussein Al-Amier; Michael Emerick; Jennifer Normanly; Jerry D. Cohen

doi:10.1016/j.ab.2007.08.009

A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

Lana S. Barkawi, Yuen Yee Tam, Julie A. Tillman, Ben Pederson, Jessica Calio, Hussein Al-Amier, Michael Emerick, Jennifer Normanly, Jerry D. Cohen

Horticultural Science

Research output: Contribution to journal › Article › peer-review

83 Scopus citations

Abstract

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.

Original language	English (US)
Pages (from-to)	177-188
Number of pages	12
Journal	Analytical Biochemistry
Volume	372
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2007.08.009
State	Published - Jan 15 2008

Bibliographical note

Funding Information:
This work was supported by NSF DBI 0077769 to J.D.C. and J.N., and in part by USDA-NRI 2005-35318-16197 to J.D.C. and USDA-NRI 2002-03555 to J.N. We are grateful to Camille Jensen, Vikesh Patel, and Yazmin Mohammad for conducting many trials of the extraction protocols. We thank the many visitors to our laboratories over the past 5 years who tested our procedures as they were developed. We also thank Dan Bensen for the design and production of the custom metal bead dropper, insert plate, and diazomethane generator.

Keywords

4-Chloro-indole-3-acetic acid
4-Cl-IAA
Auxin
High-throughput methylation
High-throughput purification
IAA
IBA
IPA
Indole-3-acetic acid
Indole-3-butyric acid
Indole-3-propanoic acid
Organic acids
Plant hormones

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title = "A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue",

abstract = "To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.",

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author = "Barkawi, {Lana S.} and Tam, {Yuen Yee} and Tillman, {Julie A.} and Ben Pederson and Jessica Calio and Hussein Al-Amier and Michael Emerick and Jennifer Normanly and Cohen, {Jerry D.}",

note = "Funding Information: This work was supported by NSF DBI 0077769 to J.D.C. and J.N., and in part by USDA-NRI 2005-35318-16197 to J.D.C. and USDA-NRI 2002-03555 to J.N. We are grateful to Camille Jensen, Vikesh Patel, and Yazmin Mohammad for conducting many trials of the extraction protocols. We thank the many visitors to our laboratories over the past 5 years who tested our procedures as they were developed. We also thank Dan Bensen for the design and production of the custom metal bead dropper, insert plate, and diazomethane generator.",

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AU - Barkawi, Lana S.

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AU - Pederson, Ben

AU - Calio, Jessica

AU - Al-Amier, Hussein

AU - Emerick, Michael

AU - Normanly, Jennifer

AU - Cohen, Jerry D.

N1 - Funding Information: This work was supported by NSF DBI 0077769 to J.D.C. and J.N., and in part by USDA-NRI 2005-35318-16197 to J.D.C. and USDA-NRI 2002-03555 to J.N. We are grateful to Camille Jensen, Vikesh Patel, and Yazmin Mohammad for conducting many trials of the extraction protocols. We thank the many visitors to our laboratories over the past 5 years who tested our procedures as they were developed. We also thank Dan Bensen for the design and production of the custom metal bead dropper, insert plate, and diazomethane generator.

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N2 - To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.

AB - To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.

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A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

Abstract

Bibliographical note

Keywords

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