DNA adducts are central in the carcinogenic process because they can cause miscoding leading to permanent mutations in important genes involved in carcinogenesis. While it is known that tobacco smoking leads to increased levels of multiple DNA adducts, most DNA adducts detected to date in humans cannot be explicitly attributed to smoking but instead have various possible exogenous and endogenous sources. We plan to probe the tobacco source of DNA adducts by providing carbon-13 labelled ([13C]-labelled) cigarettes to smokers and analyzing [13C]-labelled DNA adducts in their oral cells to determine which adducts arise from smoking. Prior to conducting studies in humans, we first report here proof-of-principle machine smoking experiments to evaluate carbon isotopologues of (a) selected carbonyls and (b) DNA adducts resulting from direct exposure of cigarette smoke vapour-phase to calf-thymus DNA. The smoke of the study cigarettes, made from a 50:50 mixture of [13C]-labelled tobacco and a popular commercial tobacco, yielded similar concentrations of carbonyl compounds and their respective DNA adducts compared with the smoke of 1R6F reference cigarettes and the popular brand of cigarettes. We detected [13C]-isotopologues of DNA adducts such as 1,N6-etheno-dA, (8R/S)-3-(2’-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dG), and (6S,8S and 6R,8R)-3-(2’-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)-one [(6S,8S)-γ-OH-Cro-dG and (6R,8R)-γ-OH-Cro-dG], proving that they have a direct source from tobacco smoke and providing important new insights regarding their mechanisms of formation. These unique results form the basis for further studies in cell culture and in cigarette smokers to establish how carcinogens in tobacco smoke cause DNA adduct formation.

Brian Larkins died in January 2025 at the age of 78 leaving a legacy of seminal scientific contributions that were fundamental to the establishment of the field of plant molecular biology. He elucidated the key processes in maize endosperm development and delivered technical breakthroughs in isolation of polysomes and translation of associated messenger RNAs. His models for protein body structure and his work to understand the molecular basis for restoring transparency to mutant maize seeds provided fundamental insights to improving nutritional quality of maize. Brian was an inspirational, beloved, and effective mentor to students, postdocs, and colleagues, leading by example and service. His visionary efforts during challenging financial times strengthened professional societies through exceptional fund-raising to support early career scientists thus providing a pathway for continued success.

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.

The first reports that auxins promoted root formation in cuttings and that indole-3-butyric acid (IBA) was a particularly effective treatment date from the early 1930s. Since its introduction into horticultural practice, the focus on improvements in the rooting of plants has been largely on the proper use of auxins to enhance adventitious rooting (AR) as well as to increase the range of plants where it can be effective. In this review, we focus on new ideas that might build on what is known about auxin induction of AR. We explore what the evolution in chemical biology has opened through novel high-throughput screening tools to explore auxin regulation of plant development and what it might add to our understanding and potential to produce new tools for the manipulation of AR. The potential for using stronger auxin analogues, alternative indolealkanoic acids, compounds that alter β-oxidation of IBA and other indolealkanoic acids, auxin conjugates, inhibitors of auxin conjugation, inhibitors of endogenous auxin biosynthesis, as well as other plant hormones and compounds that inhibit the production or mimic the effects of signals that might be involved in AR are all discussed. The expectation is that a summary of these advances in our understanding of the chemical biology important to AR might increase the use and exploration of new ideas for the improvement in the practical approaches to advance horticultural rooting methods.

Cigarette smoking is the acknowledged major cause of cancers of the lung and oral cavity and is an established important risk factor for multiple other cancers. DNA addition products (DNA adducts) caused by cigarette smoking are critical factors in its mechanism of carcinogenesis. However, most DNA adducts detected to date in humans cannot be specifically ascribed to smoking but rather have multiple exogenous and endogenous sources. In the study reported here, we prepared [13C]-labeled tobacco to address this problem. We report for the first time the successful growth from seeds to flowering under hydroponic conditions of highly [13C]-labeled tobacco in a controlled 13CO2 environment. The standard growth procedure with optimized conditions is described in detail. The [13C]-enrichment rate was assessed by quantifying nicotine and sugars and their [13C]-isotopologues in this tobacco using high-resolution mass spectrometry, reaching >94% in the tobacco leaves. The [13C]-labeled leaves after curing will be used to make cigarettes, allowing investigation of the specific contributions of tobacco smoke carcinogens to identified DNA adducts in smokers.

In recent years, the combined stress of drought and aluminum (Al) has received considerable attention due to the widespread distribution of acidic soils worldwide, exacerbated by climate change. The responses of plants to drought and Al stress are intricately governed by phytohormones, which serve as key regulators for a range of resistance mechanisms. However, the role of combined drought-Al stress remains largely unexplored. Therefore, it is important to critically examine the most recent advances in plant resistance mechanisms to combined drought and Al stress, with a specific focus on the potential involvement of phytohormonal mechanisms. It is postulated that phytohormones such as abscisic acid, auxins, cytokinins, gibberellins, brassinosteroids, and jasmonates are also involved in triggering the activation of resistance mechanisms against combined drought-Al stress, such as induction of stomatal closure, increased exudation of organic acids, and increased antioxidant activity. This review aims to deepen our understanding of the intricate interplay between phytohormonal signaling, combined Al-drought stress, and the implications of climate change. By shedding light on the underlying mechanisms that govern how plants respond to these combined stresses, we aim to pave the way for targeted strategies to mitigate the adverse effects of climate change on plant performance.

The phytohormone auxin is at times called the master regulator of plant processes and has been shown to be a central player in embryo development, the establishment of the polar axis, early aspects of seedling growth, as well as growth and organ formation during later stages of plant development. The Plant Cell has been key, since the inception of the journal, to developing an understanding of auxin biology. Auxin-regulated plant growth control is accomplished by both changes in the levels of active hormones and the sensitivity of plant tissues to these concentration changes. In this historical review, we chart auxin research as it has progressed in key areas and highlight the role The Plant Cell played in these scientific developments. We focus on understanding auxin-responsive genes, transcription factors, reporter constructs, perception, and signal transduction processes. Auxin metabolism is discussed from the development of tryptophan auxotrophic mutants, the molecular biology of conjugate formation and hydrolysis, indole-3-butyric acid metabolism and transport, and key steps in indole-3-acetic acid biosynthesis, catabolism, and transport. This progress leads to an expectation of a more comprehensive understanding of the systems biology of auxin and the spatial and temporal regulation of cellular growth and development.

There are growing doubts about the true role of the common mycorrhizal networks (CMN or wood wide web) connecting the roots of trees in forests. We question the claims of a substantial carbon transfer from 'mother trees' to their offspring and nearby seedlings through the CMN. Recent reviews show that evidence for the 'mother tree concept' is inconclusive or absent. The origin of this concept seems to stem from a desire to humanize plant life but can lead to misunderstandings and false interpretations and may eventually harm rather than help the commendable cause of preserving forests. Two recent books serve as examples: The Hidden Life of Trees and Finding the Mother Tree.

Salicylic acid (SA) application is a promising agronomic tool. However, studies under field conditions are required, to confirm the potential benefits of SA. Thus, SA application was evaluated under field conditions for its effect on abscisic acid levels, antioxidant related-parameters, fruit quality, and yield in Aristotelia chilensis subjected to different levels of irrigation. During two growing seasons, three-year-old plants under field conditions were subjected to full irrigation (FI: 100% of reference evapotranspiration (ETo), and deficit irrigation (DI: 60% ETo). During each growth season, a single application of 0.5 mM SA was performed at fruit color change by spraying fruits and leaves of both irrigation treatments. The results showed that DI plants experienced moderate water stress (−1.3 MPa), which increased ABA levels and oxidative stress in the leaves. The SA application facilitated the recovery of all physiological parameters under the DI condition, increasing fruit fresh weight by 44%, with a 27% increase in fruit dry weight, a 1 mm increase in equatorial diameter, a 27% improvement in yield per plant and a 27% increase in total yield, with lesser oxidative stress and tissue ABA levels in leaves. Also, SA application significantly increased (by about 10%) the values of fruit trait variables such as soluble solids, total phenols, and antioxidant activity, with the exceptions of titratable acidity and total anthocyanins, which did not vary. The results demonstrated that SA application might be used as an agronomic strategy to improve fruit yield and quality, representing a saving of 40% regarding water use.

Temperature, water, and light are three abiotic stress factors that have major influences on plant growth, development, and reproduction. Plants can be primed by a prior mild stress to enhance their resistance to future stress. We used an untargeted metabolomics approach to examine Arabidopsis thaliana 11-day-old seedling's abiotic stress responses including heat (with and without priming), cold (with and without priming), water-deficit and high-light before and after a 2-day-recovery period. Analysis of the physiological phenotypes showed that seedlings with stress treatment resulted in a reduction in fresh weight, hypocotyl and root length but remained viable. Several stress responsive metabolites were identified, confirmed with reference standards, quantified, and clustered. We identified shared and specific stress signatures for cold, heat, water-deficit, and high-light treatments. Central metabolism including amino acid metabolism, sugar metabolism, glycolysis, TCA cycle, GABA shunt, glutathione metabolism, purine metabolism, and urea cycle were found to undergo changes that are fundamentally different, although some shared commonalities in response to different treatments. Large increases in cysteine abundance and decreases in reduced glutathione were observed following multiple stress treatments highlighting the importance of oxidative stress as a general phenomenon in abiotic stress. Large fold increases in low-turnover amino acids and maltose demonstrate the critical role of protein and starch autolysis in early abiotic stress responses.

Drought stress reduces plant growth and crop yields. Plants activate defense mechanisms, such as stomatal closure, phenolic compounds biosynthesis, compatible solutes, protein, amino acids, and abscisic acid to cope with drought stress. Most studies on drought stress effects have been focused only on fully expanded leaves, while information on young leaves is scarce. In this study, we investigated the differential photosynthetic performance as well as the primary and secondary metabolic responses in young and fully expanded leaves of Aristotelia chilensis plants subjected to drought stress. We found that drought stress negatively affected net CO2 assimilation (AN), stomatal conductance (gs), and transpiration (E) more drastically in young leaves than fully expanded leaves of A. chilensis plants. On the other hand, young leaves accumulated higher total protein than fully expanded leaves of plants at day 10 of drought stress. Likewise, the total amino acid was increased in young leaves of stressed plants on days 5, 10, and 20, showing higher values at the end of the experiment. Starch was reduced in both young and fully expanded leaves of drought-stressed plants, while sucrose and fructose levels increased around 2-fold in young leaves at days 10 and 20 of drought stress. We observed that leucine and valine increased in young leaves at days 10 and 20 of drought stress, while in contrast, no changes were observed in the fully expanded leaves. PCA analysis clearly separated the young from fully expanded leaves at 10 and 20 days under drought stress, where anthocyanins and related genes (UFGT and NCED) were stimulated in fully expanded leaves, while in young leaves was amino acids, sucrose, and fructose. Our results demonstrate that young leaves stimulate branched-chain amino acid and phenylalanine accumulation, while fully expanded leaves stimulate anthocyanins and related genes, showing different physiological, biochemical, and molecular mechanisms to respond to drought stress.

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.

The micropropagation of hybrid hazelnut (Corylus americana × Corylus avellana) has been limited in its agricultural application due to the lack of efficient procedures for tissue culture and root organogenesis. We established an efficient sterilization protocol for hazelnut micropropagation suitable for in vitro root induction that permitted us to approach the poor root organogenesis experienced with the micropropagation of hazelnut hybrids in vitro. Typically, with standard protocols, root organogenesis had yields of well under 20%. We found that cuttings grown in vitro to the four-leaf stage can be successfully rooted using a combination of indole-3-butyric acid (IBA) pretreatment, followed by a one-week growth period in absolute darkness. The dark period was then followed by a transition to continuous light. In many cases, prolific visible roots formed between 12 and 14 days.

The plant hormone auxin plays important roles throughout the entire life span of a plant and facilitates its adaptation to a changing environment. Multiple metabolic pathways intersect to control the levels and flux through indole-3-acetic acid (IAA), the primary auxin in most plant species. Measurement of changes in these pathways represents an important objective to understanding core aspects of auxin signal regulation. Such studies have become approachable through the technologies encompassed by targeted metabolomics. By monitoring incorporation of stable isotopes from labeled precursors into proposed intermediates, it is possible to trace pathway utilization and characterize new biosynthetic routes to auxin. Chemical inhibitors that target specific steps or entire pathways related to auxin synthesis aid these techniques. Here we describe methods for obtaining stable isotope labeled pathway intermediates necessary for pathway analysis and quantification of compounds. We describe how to use isotope dilution with methods employing either gas chromatography or high performance liquid chromatography mass spectrometry techniques for sensitive analysis of IAA. Complete biosynthetic pathway analysis in seedlings using multiple stable isotope-labeled precursors and chemical inhibitors coupled with highly sensitive liquid chromatography-mass spectrometry methods are described that allow rapid measurement of isotopic flux into biochemical pools. These methods should prove to be useful to researchers studying aspects of the auxin metabolic network in vivo in a variety of plant tissues and during various environmental conditions.

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.

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.

Perennial crops are constantly exposed to fungi and bacteria in their environment, and thus explants from field-grown plants are difficult to disinfect for micropropagation because of both endophytic and epiphytic microbes. Field grown S. integrifolium plants were potted and grown in the greenhouse for five weeks; new stems and anthers along with seeds were tested for in vitro sterilization. We report successful sterilization protocols that involved disinfecting seeds with isothiazolone biocides (PPM) and NaClO and sterilizing stems and anthers through vacuum infiltration of PPM. The establishing of the protocols is useful for subsequent micropropagation and would in this way facilitate Silphium breeding and domestication processes.

Non-heterocystous nitrogen fixing strains of cyanobacteria were screened by their ability to grow in nitrogen deficient media. The selected nitrogen fixing cyanobacterial cells were then cultured in BG11 media supplemented with [15N]-labeled sodium nitrate. Under these growth conditions any organic [14N] found in the cyanobacterial cells would simply come from nitrogen fixation because [15N] was the only available source of nitrogen in the medium. Amino acids extracted after different time periods (after 15, 30, 40, 50 and 60 days of inoculation) were used for the determination of the 14N/15N ratio using GC-MS. Results from the present study support the conclusion that at stationary phase of growth cyanobacterial nitrogen fixation was no longer supplying a significant amount of nitrogen. This approach not only provided a detailed method for the evaluation of the nitrogen fixing potential of the cyanobacteria in culture, but also suggests novel approaches for the assessment of the ability of the strains to provide nitrogen enrichment to plants under co-cultivation conditions.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Hybrid hazelnut has enormous potential as a perennial crop plant for Minnesota with the promise of reduced nutrient runoff, a nutritious farm product and a social benefit as a derivative of the plant that formed an important aspect of the diet of the original peoples of this state. Hybrid hazelnuts have many desirable traits as a new cropping system including the fact that they may be grown on marginal ground not necessarily conducive to row-crop production, such as sites with poor soil, highly erodible slopes, or poor drainage – conditions often found in water quality buffer strips statewide. Hybrid hazelnuts have, for example, shown good productivity in soils ranging from heavy clay to sand. Once the plants are well established, they are capable of withstanding water availability changes from drought to standing water. Although hazelnuts do best in highly productive areas, the use of marginal lands or the buffer zones between row crop fields and our lakes and streams, areas that are frequently the most environmentally sensitive and runoff-prone, may be a way of reaping an economic return from these lands without contributing to and perhaps reversing further environmental degradation. Several physiological factors previously inhibited tissue culture micropropagation of hybrid hazelnuts and this project was undertaken because root initiation was not at levels that allowed in vitro methods to become economical, with success rates below 20% in vitro in most cases. The major part of this project therefore was focused on studies of the auxin class of plant hormones, indoleacetic acid and indolebutyric acid, and their metabolism as the biochemical limitation on the induction of roots critical to large scale propagation. Our goal was to identify the hormonal roadblocks to adventitious root formation in hazelnuts, and we showed that a plant’s low ability to convert indole-3-butyric acid (IBA) to free indole-3-acetic acid (IAA) is an important factor in the failure of some germplasm to respond to IBA application. The initial focus was on development of exacting methods to study the critical step in the rooting process where applied IBA is converted to the active hormone IAA via peroxisome-mediated beta-oxidation. Rapid screens for germplasm with optimized activity were developed should allow selection of plants much better adapted to standard propagation and micro-propagation technologies. In a related project using a reference plant species, our lab showed an involvement of phytochrome in the induction of adventitious roots and we were able to take advantage of that new information to devise a system of dark/light transitions to test these concepts in hazelnut cuttings. A simple 7-day dark period followed by IBA treatment produced dramatic increases in rooting in cultured materials where the rooting success in some cases increased from less than 20% to over 60%. We have experimented with variations on this process, changing the time and sequence of treatments, levels of IBA, etc. and have optimize many aspects of the process, thus making the establishment of rooted cutting in vitro a routine operation and setting the stage for increased use of micropropagation methods to increase hazelnut propagation of elite selections. Project description: A. Background of the project: As woody perennials, hazelnuts are an important piece of the Forever Green initiative, providing continuous living cover to protect soil quality and reduce agricultural runoff. Because of a highly variable genetic background of hazelnut, seed propagation is not an effective method for increasing plant numbers. Thus, adventitious root formation is the preferred method for propagation from elite plants selection, but this process is also a critical and limiting step in all methods of vegetative propagation used in hybrid and American hazelnuts. Because of the shortcomings of current propagation methods, widespread adoption of hazelnuts in the Midwest is limited by a lack of quality plants. Our initial goal was to identify the hormonal roadblocks to adventitious root formation in hazelnuts using stable isotope methods of analysis of plant hormone changes. Identifying these limitations provided knowledge that can be used to make more informed decisions on how to optimize the rooting phase and also to identify which genotypes are most likely to form adventitious roots. The major outcomes include 1) creation of a screen to identify genotypes likely to have poor rooting ability to exclude from breeding programs; 2) determination of changes in primary metabolism, peroxisome function and the potential for metabolic manipulation to effect rooting ability in response to rooting hormone treatments; 3) based on these initial studies to expand on the light requirements for root initiation. These results could greatly accelerate the process of propagating many genetically uniform plants that can then be strategically placed on the Minnesota landscape. B. Progress made toward the original goals of the project: Indole-3-butyric acid (IBA) is an endogenous compound that appears to regulate both lateral and adventitious root formation in many plant species and is also the auxin most available commercially for application to promote rooting. IBA is converted to indole-3-acetic acid (IAA) by beta-oxidation in the peroxisomes. This process has been observed in a number of plant species and has been shown to be critical for normal root development in response to treatment with IBA. In our studies, we investigated this process in hybrid hazelnut (Corylus americana x C. avellana), and compared this to what was occurring with American elm (Ulmus americana), and Cathedral hybrid elm (U. pumila x U. davidiana var. japonica ‘Cathedral’), in which adventitious rooting is a major bottleneck for vegetative propagation, and the efficacy of IBA treatment is highly variable across different cultivars and at different collection times. Using differentially stable isotopelabeled IBA and IAA tracer and internal standard, respectively, and using gas chromatography coupled with selected reaction monitoring mass spectrometry, IBA-derived IAA was measured in shoot tissue treated with stable isotope-labeled IBA (Figure 1). In elm, higher levels of IBA-to-IAA conversion were generally observed in cultivars which formed adventitious roots most easily in softwood stem cutting trials. IBA-to-IAA conversion was observed in hazelnut genotypes with different rooting abilities and suggested a complex relationship exists between IBA conversion and root organogenesis. In both hazelnut and elm, endogenous free IAA levels were not significantly different across the genotypes examined (Table 1). High rates of root formation is a key trait for establishment of large-scale production systems. Screening for optimal rates of IBA-to-IAA conversion may facilitate selection against genotypes which respond poorly to exogenous IBA and are thus difficult to propagate using hormone treatment. Critical to the success of this work was to develop a novel assay, based on advanced analytical and stable isotope tracer methods for determination of IBA to IAA conversion. [C8 N1]IBA was synthesized from [C8 N1]indole and was further purified by preparative HPLC. 400 uL of 0.63 mg/mL [C8 N1]IBA solution in 50 % isopropanol was injected onto a 21.2 9 250 mm ZORBAX Eclipse XDB-C18 Prep HT column with a 7-um particle size. A gradient starting with 10 % methanol, increasing to 90 % methanol over 20 min, and holding at 90 % methanol for 2 min with a constant flow rate of 20 mL/min was used to separate IBA from IAA with an Agilent 1200 Series G1361A preparative pumping system. Absorbance at 280 nm was monitored with an Agilent 1200 series G1365B multiple wavelength detector to visualize elution of indolic com-pounds; [C8 N1]IBA eluted at approximately 16 min and was collected in an acid-washed glass bottle. HPLC purification was repeated several times to purify approximately 10 mL of the original stock solution. [C8 N1]IBA fractions were pooled and reduced to a volume of approximately 50 mL on a SpeedVac vacuum concentrator. Concentration of the final purified solution was determined by measuring the absorbance of [C8 N1]IBA at 282 nm and using an extinction coefficient of 5541, which was determined by measuring absorbances of IBA solutions of known concentrations using a HP 8453 UV-Visible spectrophotometer. Hazelnut stems were rinsed with sterile deionized water, cut into 7–12 mg internodal segments, weighed, and placed basal end-up into wells of an untreated 384-well plate each containing 40 uL of 55 uM [[C8 N1]IBA in distilled water. After incubating in the 55 uM [C8 N1]IBA solution at room temperature for 6 h, shoot segments were removed from wells, blotted on a laboratory tissue, transferred to 1.5 mL microcentrifuge tubes, frozen by dipping in liquid nitrogen or dry ice in isopropanol, and stored at -80 C until extraction. 0.85 ng of [C6]IAA internal standard (Cambridge Isotope Laboratories) was added to each sample prior to extraction, and 0.4 ng of [C1]IBA was added to each hazelnut sample (in addition to [13C6]IAA internal standard) to quantify overall [C8 N1]IBA uptake. In vitro-grown shoots were assayed under sterile conditions. Plant material was cut and weighed in a laminar flow hood using sterile technique. [C8 N1]IBA solution was filter sterilized with an Acrodisc 13-mm syringe filter with a 0.2 lM PTFE membrane, and a sterile 384-well plate with a lid was used. The development of propagation methods can be a labor and time-consuming process. Nevertheless, establishment of a woody plant cropping system is highly dependent on the ability to obtain uniform genetic materials expressing a set of desirable traits. We showed that an important aspect of hormonally induced adventitious rooting is likely the conversion of applied IBA to IAA and, in order to accomplish that developed a rapid and facile method to screen germp