Auxin analysis using laser microdissected plant tissues sections

Abstract

BackgroundQuantitative 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.ResultsWe 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.ConclusionsWe 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.

Laser Microdissection of Specific Stem-Base Tissue Types from Olive Microcuttings for Isolation of High-Quality RNA

“…Despite such constrains, LM applied to plants [ 17 ] has been optimized and today, this technique has successfully been applied on different research areas of plant biology. A relatively high number of studies have been reported so far on transcriptomic, proteomic and metabolomic analysis, mainly focused on individual plant tissue and cell types, with various aims, such as to study cell specialization for growth and development, protection and stress responses [ 18 , 19 , 20 , 21 , 22 ], to quantify phytohormones at a tissue level [ 23 ], to understand the network of transcriptional regulators controlling processes like inflorescence development and fruit development and ripening [ 10 , 24 , 25 ], or to study the embryogenesis process [ 26 ]. Worthy of note is the fact that in all these works there is not an available standard protocol suitable for all plant species and cell types, implying that LM protocols must be developed and optimized for each tissue type.…”

A roadmap for research in octoploid strawberry

“…However, the increase in expression of FaTAR2 encoding the auxin biosynthesis enzyme tryptophan amino transferase, and genes encoding proteins involved in auxin perception (FaAux/IAA11, FaAux/IAA14b, and FaAux/IAA33) together with expression of genes involved in auxin signaling (FaARF6a and FaARF16c) in ripening receptacles strongly suggests cell-autonomous auxin synthesis and cell-specific response in the receptacle at ripening 112 . Laser capture microdissection and newer methodologies in mass spectral analysis of very small amounts of tissues 113 , for example, are needed to specify which types of cells (cortex, pith, and vasculature) are engaged in hormone metabolism in the ripening receptacle.…”