Analytical methods for stable isotope labeling to elucidate rapid auxin kinetics in Arabidopsis thaliana.

The phytohormone auxin plays a critical role in plant growth and development. Despite significant progress in elucidating metabolic pathways of the primary bioactive auxin, indole-3-acetic acid (IAA), over the past few decades, key components such as intermediates and enzymes have not been fully characterized, and the dynamic regulation of IAA metabolism in response to environmental signals has not been completely revealed. In this study, we established a protocol employing a highly sensitive liquid chromatography-mass spectrometry (LC-MS) instrumentation and a rapid stable isotope labeling approach. We treated Arabidopsis seedlings with two stable isotope labeled precursors ([13C6]anthranilate and [13C8, 15N1]indole) and monitored the label incorporation into proposed indolic compounds involved in IAA biosynthetic pathways. This Stable Isotope Labeled Kinetics (SILK) method allowed us to trace the turnover rates of IAA pathway precursors and product concurrently with a time scale of seconds to minutes. By measuring the entire pathways over time and using different isotopic tracer techniques, we demonstrated that these methods offer more detailed information about this complex interacting network of IAA biosynthesis, and should prove to be useful for studying auxin metabolic network in vivo in a variety of plant tissues and under different environmental conditions.

Complexity of the auxin biosynthetic network in Arabidopsis hypocotyls is revealed by multiple stable-labeled precursors.

Auxin is a key regulator of plant development and in Arabidopsis thaliana can be synthesized through multiple pathways; however, the contributions of various biosynthetic pathways to specific developmental processes are largely unknown. To trace the involvement of various biosynthetic routes to indole-3-acetic acid (IAA) under conditions that induce adventitious root formation in Arabidopsis hypocotyls, we treated seedlings with three different stable isotope-labeled precursors ([13C6]anthranilate, [15N1]indole, and [13C3]serine) and monitored label incorporation into a number of proposed biosynthesis intermediates as well as IAA. We also employed inhibitors targeting tryptophan aminotransferases and flavin monooxygenases of the IPyA pathway, and treatment with these inhibitors differentially altered the labeling patterns from all three precursors into intermediate compounds and IAA. [13C3]Serine was used to trace utilization of tryptophan (Trp) and downstream intermediates by monitoring 13C incorporation into Trp, indole-3-pyruvic acid (IPyA), and IAA; most 13C incorporation into IAA was eliminated with inhibitor treatments, suggesting Trp-dependent IAA biosynthesis through the IPyA pathway is a dominant contributor to the auxin pool in de-etiolating hypocotyls that can be effectively blocked using chemical inhibitors. Labeling treatment with both [13C6]anthranilate and [15N1]indole simultaneously resulted in higher label incorporation into IAA through [15N1]indole than through [13C6]anthranilate; however, this trend was reversed in the proposed precursors that were monitored, with the majority of isotope label originating from [13C6]anthranilate. An even greater proportion of IAA became [15N1]-labeled compared to [13C6]-labeled in seedlings treated with IPyA pathway inhibitors, suggesting that, when the IPyA pathway is blocked, IAA biosynthesis from labeled indole may also come from an origin independent of the measured pool of Trp in these tissues.

Biphasic control of cell expansion by auxin coordinates etiolated seedling development.

Seedling emergence is critical for food security. It requires rapid hypocotyl elongation and apical hook formation, both of which are mediated by regulated cell expansion. How these events are coordinated in etiolated seedlings is unclear. Here, we show that biphasic control of cell expansion by the phytohormone auxin underlies this process. Shortly after germination, high auxin levels restrain elongation. This provides a temporal window for apical hook formation, involving a gravity-induced auxin maximum on the eventual concave side of the hook. This auxin maximum induces PP2C.D1 expression, leading to asymmetrical H+-ATPase activity across the hypocotyl that contributes to the differential cell elongation underlying hook development. Subsequently, auxin concentrations decline acropetally and switch from restraining to promoting elongation, thereby driving hypocotyl elongation. Our findings demonstrate how differential auxin concentrations throughout the hypocotyl coordinate etiolated development, leading to successful soil emergence.

Protocol: analytical methods for visualizing the indolic precursor network leading to auxin biosynthesis.

BACKGROUND: The plant hormone auxin plays a central role in regulation of plant growth and response to environmental stimuli. Multiple pathways have been proposed for biosynthesis of indole-3-acetic acid (IAA), the primary auxin in a number of plant species. However, utilization of these different pathways under various environmental conditions and developmental time points remains largely unknown. RESULTS: Monitoring incorporation of stable isotopes from labeled precursors into proposed intermediates provides a method to trace pathway utilization and characterize new biosynthetic routes to auxin. These techniques can be aided by addition of chemical inhibitors to target specific steps or entire pathways of auxin synthesis. CONCLUSIONS: Here we describe techniques for pathway analysis in Arabidopsis thaliana seedlings using multiple stable isotope-labeled precursors and chemical inhibitors coupled with highly sensitive liquid chromatography-mass spectrometry (LC-MS) methods. These methods should prove to be useful to researchers studying routes of IAA biosynthesis in vivo in a variety of plant tissues.

Indole-3-acetylaspartate and indole-3-acetylglutamate, the IAA-amide conjugates in the diploid strawberry achene, are hydrolyzed in growing seedlings.

MAIN CONCLUSION: Indole-3-acetylaspartate and indole-3-acetylglutamate are the stored auxin amino acid conjugates of the achene of the diploid strawberry and serve as sources of auxin during seedling growth. The edible part of the strawberry, a pseudocarp, has long been known to enlarge in response to auxin produced by the developing achenes, the botanical true fruit. Auxin homeostasis involves a complex interaction between biosynthesis, conjugate formation and hydrolysis, catabolism and transport. Strawberry tissues are capable of synthesizing auxin conjugates, and transcriptome data support the expression of genes involved in IAA conjugate formation and hydrolysis throughout embryo development and subsequent seedling growth. Using a highly sensitive and selective mass spectrometric method, we identified all the low molecular weight indole-auxin amino acid conjugates in achenes of F. vesca as consisting of indole-3-acetylaspartate (IAasp) and indole-3-acetylglutamate (IAglu). In contrast to what has been proposed to occur in Arabidopsis, we determined that IAasp and IAglu are hydrolyzed by seedlings to provide a source of free IAA for growth.

Auxin analysis using laser microdissected plant tissues sections.

BACKGROUND: Quantitative measurement of actual auxin levels in plant tissue is complimentary to molecular methods measuring the expression of auxin related genes. Current analytical methods to quantify auxin have pushed the limit of detection to where auxin can be routinely quantified at the pictogram (pg) level, reducing the amount of tissue needed to perform these kinds of studies to amounts never imagined a few years ago. In parallel, the development of technologies like laser microdissection microscopy (LMD) has allowed specific cells to be harvested from discrete tissues without including adjacent cells. This method has gained popularity in recent years, especially for enabling a higher degree of spatial resolution in transcriptome profiling. As with other quantitative measurements, including hormone quantifications, sampling using traditional LMD is still challenging because sample preparation clearly compromises the preservation of analytes. Thus, we have developed and validated a sample preparation protocol combining cryosectioning, freeze-drying, and capturing with a laser microdissection microscope to provide high-quality and well-preserved plant materials suitable for ultrasensitive, spatially-resolved auxin quantification. RESULTS: We developed a new method to provide discrete plant tissues for indole-3-acetic acid (IAA) quantification while preserving the plant tissue in the best possible condition to prevent auxin degradation. The method combines the use of cryosectioning, freeze-drying and LMD. The protocol may also be used for other applications that require small molecule analysis with high tissue-specificity where degradation of biological compounds may be an issue. It was possible to collect the equivalent to 15 mg of very specific tissue in approximately 4 h using LMD. CONCLUSIONS: We have shown, by proof of concept, that freeze dried cryosections of plant tissue were suitable for LMD harvest and quantification of the phytohormone auxin using GC-MS/MS. We expect that the ability to resolve auxin levels with both spatial- and temporal resolution with high accuracy will enable experiments on complex processes, which will increase our knowledge of the many roles of auxins (and, in time, other phytohormones) in plant development.