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Calorespirometric measurements proved to be useful for phenotyping temperature response in terms of optimum temperatures for growth and low temperature limits for growth respiration in diverse carrot genotypes. High and low-temperature tolerance is an important trait in many breeding programs, but to date, improvement strategies have had limited success. Developing new, cost efficient and reliable screening tools to identify and select the most tolerant crop plant genotypes is necessary to assist plant breeding on cold and heat tolerance, and calorespirometry is proposed for this. Calorespirometry is a technique to simultaneously measure metabolic heat rates and CO2 emission rates of respiring tissues and can be used as a rapid method to determine how changes in the environment (e.g., temperature) influence plant growth. The main aim of this work was, therefore, to test the usefulness of calorespirometry as a phenotyping tool for carrot taproot growth in response to temperature. Calorespirometric measurements in the carrot taproot meristems of plants from eight carrot inbred lines allowed identification of optimum and minimum temperatures for growth of plants and to distinguish between phenotypes based on those characteristics. The technique proved to be useful for predicting yield-determining temperature responses in diverse carrot genotypes. Preliminary screening of new crop plant genotypes with calorespirometry based on their temperature adaptation and acclimation capability could make the screening process much less laborious by allowing selection of genotypes presenting the best growth performance under particular biotic or abiotic conditions before field tests.

Calorespirometric measurements of metabolic heat rates and CO2 emission rates of respiring tissues as functions of temperature enable rapid determination of the temperatures that plants are adapted to without growing them in different environmental temperatures. However, the correct choice of target material for measurements that enable prediction of growth temperature responses is crucial, and needs to be identified in a species‐ and trait‐specific manner. In this study, different carrot materials were tested: a primary culture system proposed as an in vitro test system for carrot yield potential, taproots of young plants, and the root meristem of actively growing plants during secondary root growth. The central root meristem is the most suitable for studying temperature response by calorespirometry for genotype comparison. Calorespirometric methods for predicting genotype‐specific temperature responses of crop plant cultivars can be used to predict productivity in environments with differing temperature conditions.

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.

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