REGULATION OF AUXIN RESPONSE AND METABOLISM

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: TERMINATED
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 1000338
• Project No.: MIN-21-021
• Project Start Date: Jul 10, 2013
• Project End Date: Jun 30, 2018

Project Director
Cohen, JE, DA.

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Horticultural Science

Non Technical Summary
Summary: Answers to the questions posed above will have important implications for understanding the mechanisms involved in effective plant propagation, seedling and root growth and the control of both plant growth and form throughout the life cycle. An understanding of these processes will lead to more uniform plant production methods, higher yields and methods to regulate plant responses to adverse conditions. Auxin is a key hormone during early stages of plant embryogenesis, defining the polar axis and subsequent events in shaping the embryo's development. It is involved in early seedling growth, thus critical for establishment of a newly planted crop. Auxin is important in establishment of plant form, from leaf and stem angle to phyllotaxis, as well as the development of lateral and adventitious roots. It is also involved in the shade avoidance response, which has implications for permissible crop density in the field. These actions of auxin during development are critical for other aspects of the development of highly productive crops plants as plant form was a major aspect of the green revolution and is likely to play a key role in future cropping system improvements. Finally, auxins are involved in the process of fruit ripening, where elevated auxin levels appear to reduce ethylene responsiveness delaying senescence while permitting many of the events associated with ripening to proceed. Understanding of the fundamentals of auxin biosynthesis, regulation and responses to stress and the environment will allow future advances to be informed by identification of target genes and their gene products.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	2499	1000	100%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

plant hormones

auxin

shade avoidance

polar auxin transport

metabolomics

proteomics

Goals / Objectives
OBJECTIVES: Naturally occurring and synthetic auxins have numerous uses in modern agriculture. For instance, the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is one of the world's most commonly used herbicides due to its selectivity against broadleaf plants. Naphthaleneacetic acid (NAA) and indolebutyric acid (IBA) form the active ingredients in products that stimulate root formation on plant cuttings. Auxins are also used to enhance fruit production or harvesting of specific crops. Endogenous auxin manipulations offer promise for altering both quantity and quality of crop plants. However, many of the practical uses of auxins are limited because the effects, treatment methods, and dosage required are often species specific and, to date, experiments on genetic manipulation of auxins often give plant-specific results. It would be predicted, therefore, that a more detailed understanding of auxin metabolism, regulation, and transport will provide the foundation for expanded uses of auxins and auxin genetics for commercial agriculture and for crop improvement. Classic studies from the late 1940's and 1950's, in which tissue sections were incubated on tryptophan and investigators measured the resulting IAA produced, led to the concept of IAA production from its closely related amino acid, eclipsing conflicting in vivo data on intact tissues. An abundance of evidence now supports the idea of redundant pathways to IAA (see Cohen et al., 2003; Woodward and Bartel, 2005), including at least one tryptophan-independent (Trp-I) pathway. Since the first description of the Trp-I pathway, the general concept of indole as a biosynthetic precursor independent of tryptophan in plant metabolism has expanded. Several reports of indole-3-glycerol (IGP) lyase (also called indole synthase) in plants have appeared and, in addition to IAA, natural products such as camalexin, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), and free indole have been attributed to indole derived from IGP in this way. The concept that multiple tryptohan-dependent (Trp-D) pathways also exist within the same plant is a more recent addition, relying on the discovery of genes that alter IAA levels when constitutively overexpressed. The effects of light, biological, and environmental stress on indolic intermediates have received considerable attention over the last several years thanks to an abundance of useful Arabidopsis mutants and concerted efforts to look at light response signaling as well as pathogen and other stress responses in this model system. This has resulted in sufficient information to propose that plant responses to both light and stress involve a remodeling of indolic pathways. For example, Zhao and Last (1996) showed that in the cell death mutant acd2, spontaneous lesions and the accumulation of the indolic phytoalexin camalexin, induced by abiotic elicitors and plant pathogens, were accompanied by the coordinate induction of mRNAs for the tryptophan biosynthetic pathway enzymes. This was confirmed in a report (Hagemeir et al., 2001) that showed a range of tryptophan-derived secondary products accumulate following infection of Arabidopsis by Pseudomonas. This is an important finding since camalexin, like tryptophan and Trp-I-derived IAA, may also be produced from indole directly as well as through tryptophan (Zook 1998; Glawischnig 2007). In maize, which does not produce camalexin, the remodeling of indolic metabolism also occurs, and two genes, bx1 and igl, produce free indole independent of tryptophan biosynthesis. The first yields the defense compound DIMBOA (Frey et al., 1997), and the second responds to an herbivore elicitor (Frey et al., 2000). Other indole pathway secondary products, such as vinblastine, have been shown to be regulated by light (St-Pierre et al. 1999) as is a key early step in indole biosynthesis (Henstrand et al. 1992). We seek to define in metabolic detail when a seedling plant transitions from stored auxin reserves to making its own auxin (a change from auxin heterotrophy to auxin autotrophy), the observed effects of light, physical stresses, and developmental stage on the multiple inputs into IAA biosynthesis, which tissues are biosynthetic sources of auxin, and how auxin is made in plants. Also, shade-intolerant plants, including Arabidopsis, when exposed to light with a reduced red/far-red (R/FR) ratio, as encountered under a plant canopy, initiate a suite of responses known as the shade avoidance response (SAR). Recent results from two labs have suggested that growth under reduced R/FR leads to a rapid increase in auxin levels by activation of auxin biosynthesis dependent on TAA1, an aminotransferase that catalyses the formation of indole-3-pyruvate (IPyA; Stepanova et al, 2008; Tao et al, 2008). We seek to understand the relative role of this process for the overall IAA biosynthesis under shade and non-shade conditions. Another important aspect of plant auxin relationships has been the study of native auxins other than IAA. First discovered as a "synthetic auxin" active in the induction of rooting on woody plant cuttings (Cooper, 1935), IBA's natural occurrence remains relatively poorly acknowledged in modern textbooks and popular publications. IBA was first shown to be a natural product during the early days of paper chromatography (Blommaert, 1954), and its presence in a number of plant species has been confirmed by its analysis by full scan GC-MS (reviewed in Ludwig- Müller, 2000). As we have shown (Epstein et al., 1989), the levels, and indeed the very presence of IBA, can be profoundly different in various cultivars of the same species. The ability to synthesize IBA is also apparently variable within different tissues of the same plant, the relative levels also appear to be cultivar-specific (Ludwig-Müller and Epstein, 1992) and the ability to make IBA is under environmental regulation (Ludwig-Müller et al., 1995; Ludwig-Müller et al., 2000; Ludwig-Müller, 2007). Unfortunately, as summarized by Ludwig-Müller (2000), studies on the presence of IBA in plants have been relatively few in number and most studies have not involved methods that allow accurate quantitative measurements. Recent work from our group has shown that IAA and IBA behave in fundamentally different ways to determine plant developmental responses (Liu et al. 2012). We have been the first to use a mass spectrometry (MS)-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [13C1]IBA and the [13C1]IAA derived from [13C1]IBA. We found that the amount of transported IBA was dramatically lower than IAA, and that IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Significant amounts of the applied [13C1]IBA were converted to [13C1]IAA during transport, but [13C1]IBA transport was independent of IBA-to-IAA conversion. In a related study (Strader et al. 2010), we showed that mutants defective in IBA to IAA conversion displayed shorter root hairs and smaller cotyledons than wild type and that these cell expansion defects are suggestive of low IAA levels in certain tissues. Our data suggest that IBA β-oxidation provides IAA for partially distinct developmental processes and specifically that IBA-derived IAA plays a major role in driving root hair and cotyledon cell expansion during seedling development.

Project Methods
Objective 1. Elucidate the metabolism of the plant hormone auxin. A. Characterize the tryptophan-independent pathway for the biosynthesis of auxin (with J.Normanly, J.P. Slovin). We propose to use in vivo stable isotopic methods coupled with in vitro systems to analyze the Trp-I biosynthetic pathway. Our working hypothesis is that Trp-IAA biosynthesis involves an indole lyase/transferase type enzyme activity. To test this hypothesis we propose to: a. Test possible side chain donors. b. Determine if the isolated enzyme from maize catalyzes deuterium exchange from the medium to indole, as predicted for a lyase-type mechanism. c. Determine if a pyridoxal phosphate-dependent enzyme is required. d. Test available mutants from maize, Arabidopsis and rice for Trp-I activity using in vitro enzyme reparations. B.Elucidate tryptophan-dependent pathways for the biosynthesis of auxin (with A. Hegeman, J. Celenza, J. Normanly). We will employ two basic approaches to this problem: a. Determine the reaction mechanism for the YUCCA flavin monooxygenase involved in the conversion of indole-3- pyruvate to IAA. We will test potential indoyl substrates for turnover with GC-MS/MS and LC-MS/MS. The role of diatomic oxygen in the mechanism will be confirmed by comparing reaction mixtures in the steady state spectrophotometrically under aerobic and anaerobic. We will next perform the reaction with 18O2. b. Develop in vivo and in vitro methods for determination of the relative contribution of the YUCCA flavin monooxygenase to overall IAA biosynthesis Once conditions are optimized, 18O2 incorporation into IAA by YUC1 will be evaluated using GC-MS (Barkawi et al., 2010). Recent work in our laboratory has increased sensitivity by 100x (Liu et al., 2012). Objective 2. Characterize environmental regulation of auxin transport and metabolism (with G. Gardner). A. Determine the phytohormonal signaling pathways for the shade avoidance response in Arabidopsis (with G. Gardner). We have re-examined the shade avoidance process and show that while there is indeed an initial auxin increase following exposure to reduced R/FR, the auxin increase is transitory while the growth persists well after the auxin levels return to the basal level. This result suggests a working hypothesis that auxin acts as a transition signal initiating a response cascade leading to prolonged hypocotyl growth in the absence of elevated auxin. Thus, an important goal of this proposal is to define the SAR signaling system. B. Determine the interactions among auxin transport, auxin synthesis, and light (with G. Gardner). Although light has been shown to affect aspects of auxin biosynthesis and polar auxin transport (PAT), how auxin transport is regulated by light has not been extensively analyzed. We found profound changes in PAT following light treatment (Liu et al. 2011). We will extend the characterization of the response in the indolic metabolome as altered by light. We will utilize two model systems, Arabidopsis and tomato. Objective 3. Identify methods by which understanding regulation of plant hormone homeostasis can help to manage crop growth (with G. Gardner, B. Bartel and L. Strader). A. Understand the relationship and regulation between the native auxins indole-butyric acid (IBA) and IAA and their role in plant rooting and propagation In order to advance the study of IBA and related compounds in plants, we propose four lines of study that will establish the tools necessary for future progress in extending our understanding of auxin relationships. Specifically, we will: 1 .Develop the necessary labeled compounds and analytical procedures for high throughput measurement of concentration and metabolism of indolealkanoic acids. 2. Determine the chemical identity and levels of conjugated forms of indolealkanoic acids other than IAA in a range of plant species. 3. Determine the genes responsible for the synthesis of IBA from IAA in Arabidopsis by analysis of related insertional mutants or isolate the enzyme(s) responsible from either maize or Arabidopsis and microsequence tryptic peptides of the proteins(s). As a working hypothesis, we have proposed that the activation of IAA for its conjugation to amino acids and its chain extension proceed by related mechanisms. Thus our goal is to test this directly by revisiting the isolation of the activated intermediate. Using the analytical and genetic tools developed in our prior studies (Liu et al. 2012; Strader et al. 2010) we propose to continue studies to elucidate the specific roles of IBA in plant development, with a focus on its agriculturally important role as a root organogenic signal. Objective 4. Analyze the dynamic changes in plant proteins and metabolites (with W. Gray and A. Hegeman). A. Develop methods for the global measurement of protein turnover to determine the relationship between auxin response and the stability of specific proteins. The goals of the proposed research are to develop robust methods for labeling plant proteins for the purpose of determining absolute rates of turnover for a wide number of different proteins under changing developmental and environmental conditions, as well as for determining the effects of specific mutations on rates of protein turnover. B.Develop improved methods for whole plant metabolomics flux analysis. Robust methods that connect observable macroscopic or physiological phenotypes of plants with their genetic determinants are badly needed (Benfey and Mitchell-Olds, 2008). Measurement of the metabolome and metabolic flux are the most direct molecular indicators of an organism's phenotype (Roscher et al., 2000; Goodenowe, 2004; Allwood et al., 2011). While metabolic flux analysis (MFA) has been used to great effect in microbial systems (Yuan et al., 2006; Munger et al., 2008; Yuan et al., 2008; Amador-Noguez et al., 2010), it has been less widely employed in plants (Allen et al., 2009). We will improve the applicability of dynamic MFA to intact plants by 1) optimizing dynamic MFA methods; 2) create an MFA-based workflow for metabolic model validation and pathway discovery; and 3) developing microsampling metabolomic approaches.

Progress 07/10/13 to 06/30/18

Outputs
Target Audience:The work accomplished is primarily directed to active plant biology researchers interested in the complexity of signaling systems in plants as they relate to stress adaptation and long distance communication within the plant. However, a secondary audience would be students and educators interested in modern approaches to long-studied problems in this area. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided educational opportunities for 4 graduate students, 7 undergraduate students, 4 high school students and 2 postdoctoral associates. This research has resulted in a significant number of scientific publications in high-rated journals, presentational at national and international conferences and several invited talks at major universities. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The accomplishments from this period can be divided into two main areas, those repated to auxin biochemistry and general advances in understanding metabolomics in relation to plant growth. In our 2013 paper on "Flower development master regulator LEAFY controls auxin response pathways in floral primordia formation" we showed using exacting methods that LEAFY operates by changes in auxin senstivity and not by regulation of auxin levels. This was accomplished using an auxin reporter system and mass spectral quantification. These results were extended by development of methods for absolute protein quantification (one example is: A proteome scale-protein turnover analysis using high resolution mass spectrometric data from stable-isotope labeled plants) that can tell the stability of auxin response pathway proteins unter different growth conditions. We also used our stable isotope methods to examine the conversion of indole-3-butyric acid to indole-3-acetic acid in relation to root formation (see, for example, Conversion of indole-3-butyric acid to indole-3-acetic acid in shoot tissue of hazelnut (Corylus) and elm (Ulmus)). Finally, we developed advanced high resolution methods to discovery of new auxins and related compounds in plants quickly and with absolute identification (see A facile means for the identification of indolic compounds from plant tissues). More general metabolomic studies improved our understanding of plat resins and their importance in honey bee health (see: Metabolomics reveals the origins of antimicrobial plant resins collected by honey bees, Regional variation in composition and antimicrobial activity of U.S. propolis against Paenibacillus larvae and Ascopheara apis, and 3-acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis). We also developed improved methods for the analysis and identification of polyphenolics (see, as an example, Targeted deuteration of polyphenolics for their qualitative and quantitative metabolomic analysis in plant-derived extracts) as well as carotenoids (see: An improved method for fast and selective separation of carotenoids by LC-MS). In summary, we advanced the analytical and developmental biology of auxin biochemistry and used that understanding to describe better how plants interact with their environment.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wilson M, Pawlus AD, Brinkman D, Gardner G, Hegeman AD, Spivak M, Cohen JD 3-acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis. Phytochemistry 138:83-92
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Abate-Pella D, Freund DM, Slovin JP, Hegeman AD, and Cohen JD An improved method for fast and selective separation of carotenoids by LC-MS. J Chromatography B 1067:34-37
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Freund DM, Martin AC, Cohen JD, Hegeman AD Direct detection of surface localized specialized metabolites from Glycyrrhiza lepidota (American licorice) by leaf spray mass spectrometry. Planta 247:267-275
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Tivendale N, Jewett EM, Hegeman AD, Cohen JD Extraction, purification, methylation and GC-MS analysis of short-chain carboxylic acids for metabolic flux analysis. Journal of Chromatography B 1928:165-174

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The work accomplished is primarily directed to active plant biology researchers interested in the complexity of signaling systems in plants as they relate to stress adaptation and long distance communication within the plant. However, a secondary audience would be students and educators interested in modern approaches to long-studied problems in this area. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided educational opportunities for 3 graduate students, 5 undergraduate students, 2 high school students and 2 postdoctoral associates. This research has resulted in a significant number of scientific publications in high-rated journals, presentational at national and international conferences and several invited talks at major universities. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Our plans remain unchanged from those originally submitted. Our research is currently on track with our stated expectations.

Impacts
What was accomplished under these goals? The ability to correctly measure the actual auxin amount in a tissue is complimentary to molecular methods measuring the expression of auxin related genes. Improved methods for analyzing ultra-small amounts of tissue are now available. Using laser dissection microscope (LMD) also for auxin analysis would be valued. However, harvesting the tissue and still maintaining the auxin in a pristine state during the harvesting process is not trivial. Current analytical methods quantifying auxin have pushed the limit of detection to such an extent, that auxin can be routinely quantified at the pictogram level. The amount of tissue needed to perform these kind of studies, is reduced to amounts never imagined before. In parallel, the development of technologies like LMD has allowed scientists to harvest specific cells from discrete tissues without including the adjacent cells. This method is popular for transcriptome profiling, enabling analysis of the complexity of biological systems with higher degree of spatial resolution. As with other quantitative measurements, including hormone quantifications, sampling using traditional LMD is still challenging, because the sample preparation clearly compromises the preservation of the analytes. We have developed and validated a sample preparation protocol combining cryosectioning with freeze-drying, capturing with LMD, to provide high quality and well-preserved plant materials suitable for an ultrasensitive spatially-resolved quantification of auxin. We developed a new method to provide discrete plant tissues for indole-3-acetic acid (IAA) quantification, while at the same time 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. As a proof of concept, freeze dried cryosections of the plant tissue were suitable for LMD harvest, to provide high quality material for quantification of the phytohormone auxin content using GC-MS/MS. We expect that the ability to resolve auxin levels will increase our knowledge of the role of auxins in plant development. Another important development was leaf spray-MS that minimizes tissue manipulation by effectively and quickly assessing in vivo specialized metabolites from intact plant tissue surfaces, including trichome metabolites. Intact leaves of American licorice were analyzed by direct electrospray leaf spray-MS, an ambient ionization technique. Comparison of metabolites detected by leaf spray-MS to those from LC-MS of bulk tissue and trichome enriched extracts showed dramatic differences. Leaf spray-MS results suggest that in specific situations this approach could complement traditional LC-MS analysis of bulk extracts. Leaf spray-MS as a metabolomics technique eliminates sample pretreatment and preparation allowing for rapid sampling in real time of living intact tissues. Specialized metabolites on the surface of tissues such as glandular trichomes metabolites are detected by leaf spray-MS. Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC-MS). In order to address these issues, we implemented the use of LC-MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC-MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC-MS conditions.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Freund DM, Martin AC, Cohen JD, Hegeman AD Direct detection of surface localized specialized metabolites from Glycyrrhiza lepidota (American licorice) by leaf spray mass spectrometry. Planta (online: DOI 10.1007/s00425-017-2782-9) (2017)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wilson M, Pawlus AD, Brinkman D, Gardner G, Hegeman AD, Spivak M, Cohen JD 3-acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis. Phytochemistry 138:83-92 (2017)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Abate-Pella D, Freund DM, Slovin JP, Hegeman AD, and Cohen JD An improved method for fast and selective separation of carotenoids by LC-MS. J Chromatography B 1067:34-37 (2017)

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The work accomplished is primarily directed to active plant biology researchers interested in the complexity of signaling systems in plants as they relate to stress adaptation and long distance communication within the plant. However, a secondary audience would be students and educators interested in modern approaches to long-studied problems in this area. Changes/Problems:This project has provided educational opportunities for 3 graduate students, 5 undergraduate students and 2 postdoctoral associates. This research has resulted in a significant number of scientific publications in high-rated journals, presentational at national and international conferences and several invited talks at major universities. Our plans remain unchanged from those originally submitted. Our research is currently on track with our stated expectations. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Our current studies have confirmed and continue to extend our longer-term studies on the relationship between environmental and stress responses of the plant hormone auxin. Understanding the unique changes in growth hormone metabolism that result from wounding, heat, light and other stress events has allowed development of more advanced strategies for the potential improvement of crop growth under adverse conditions. In addition, using the methods developed for the analysis of IAA biosynthesis in highly defined tissues will allow a detailed analysis of the pathways in operation in a given tissue under defined conditions. This has the potential to uncovered unique finding about the interrelationship of IAA, its biosynthetic pathways and the role of IBA and IAA-conjugates not revealed by previous technologies and could ultimately have important implications in the area of plant propagation as well as extending our knowledge of auxin relationships in planta. What do you plan to do during the next reporting period to accomplish the goals?Our plans remain unchanged from those originally submitted. Our research is currently on track with our stated expectations.

Impacts
What was accomplished under these goals? 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 ß-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. We investigated this process in hybrid hazelnut (Corylus Americana, C. avellana), American elm (Ulmus americana), and Cathedral hybrid elm (U. pumila, 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 isotope-labeled 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. 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. 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. The understanding of the actual distribution of auxin within the plant tissues is crucial to comprehend the role of this hormone in plant growth and development. The current analytical methods to quantify auxin have pushed the limit of detection to such an extent, that auxin can be routinely quantified at the pg level. This has enabled the reduction of the amount of tissue needed to perform these kind of studies, to amounts never imagined a few years ago. In parallel, the development of technologies like laser microdissection microscope (LMD) has allowed scientists to harvest specific cells from discrete tissues without including the adjacent cells. This method has gained popularity in the recent years, especially in transcriptomics profiling, enabling analysis of the complexity of biological systems with a higher degree of spatial resolution. As with other quantitative measurements, including hormone quantifications, sampling using traditional LMD is still challenging because the traditional sample preparation clearly compromises the preservation of the analytes. Nevertheless, the possibility of detecting the auxin gradients in plant tissues with a higher resolution is intriguing. Thus, we developed a sample preparation protocol using LMD to provide high quality and well-preserved plant materials for an ultrasensitive spatially-resolved quantification of indole-3-acetic acid. We also developed a new protocol to provide discrete plant tissues for indole-3-acetic acid (IAA) quantification: the method combines the use of cryosectioning (CS), freeze drying (FD) and LMD. The protocol may also be used for other applications that require small molecule analysis with high tissue-specificity.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Fan K-T, Rendahl A, Chen W-P, Freund D, Gray W, Cohen JD, Hegeman A Proteome scale-protein turnover analysis using high resolution mass spectrometric data from stable-isotope labeled plants. Journal of Proteome Research 15:851867 (2016)
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kreiser M, Giblin C, Murphy R, Fiesel P, Braun L, Johnson G, Wyse D, Cohen JD Conversion of indole-3-butyric acid to indole-3-acetic acid in shoot tissue of hazelnut (Corylus) and elm (Ulmus). J Plant Growth Regulation 35:710-721 (2016)
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Tivendale N, Jewett EM, Hegeman AD, Cohen JD Extraction, purification, methylation and GC-MS analysis of short-chain carboxylic acids for metabolic flux analysis. Journal of Chromatography B 1928:165-174 (2016)
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Wilson M, Pawlus AD, Brinkman D, Gardner G, Hegeman AD, Spivak M, Cohen JD 3-acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis. Phytochemistry

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The work accomplished is primarily directed to active plant biology researchers interested in the complexity of signaling systems in plants as they relate to stress adaptation and long distance communication within the plant. However, a secondary audience would be students and educators interested in modern approaches to long-studied problems in this area. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We presented a national workshop on Plant Metabolomics (http://hegemanlab.cfans.umn.edu/plant-metabolomics-workshop-2015/) during the summer of 2015 in partnership with Dr. Adrian Hegeman. The workshop was sponsored by the National Science Foundation but included research supported by this project as an important part of the lecture and lab experience. How have the results been disseminated to communities of interest?Our current studies have confirmed and continue to extend our longer-term studies on the relationship between environmental and stress responses of the plant hormone auxin. Understanding the unique changes in growth hormone metabolism that result from wounding, heat, light and other stress events has allowed development of more advanced strategies for the potential improvement of crop growth under adverse conditions. In addition, using the methods developed for the analysis of IAA biosynthesis in highly defined tissues will allow a detailed analysis of the pathways in operation in a given tissue under defined conditions. This has the potential to uncovered unique finding about the interrelationship of IAA, its biosynthetic pathways and the role of IBA and IAA-conjugates not revealed by previous technologies and could ultimately have important implications in the area of plant propagation as well as extending our knowledge of auxin relationships in planta. We have in the past two years published significant reviews of this area of research to reach a wider group of interested scientists and will continue to do so this mext year. What do you plan to do during the next reporting period to accomplish the goals?Work is continuing according to the original goals but additional effort funded by the ForeverGreen Initiative and the Minnesota Depoartment of Agriculture have increased our research in woody plant research.

Impacts
What was accomplished under these goals? Analytical studies have demonstrated that the majority of cellular auxin is conjugated to simple sugars, cyclitols, glycans, amino acids, and other biomolecules. A number of studies have confirmed the enzymatic systems responsible for the synthesis and hydrolysis of a number of such conjugates in Arabidopsis thaliana and some of these compounds have been identified in situ. However, our work has demonstrated that the major method of estimating the amount of unknown IAA conjugates-base hydrolysis-can be significantly complicated by chemical artifacts such as glucobrassicin or protein degradation. The concept of 'bound auxin' traces its origin back to more than 80 years ago and has driven research on the sources and forms of these plant hormones since. However, the amount of indole-3-acetic acid (IAA) released upon treating Arabidopsis tissue extracts with base, a commonly employed technique for estimating the amount of IAA conjugates, greatly exceeded the summation of all the IAA conjugates known individually to be present in Arabidopsis. This discrepancy has remained as an unsolved question. In our studies, however, we found that a significant portion of the IAA found after base treatment could be attributed to chemical conversions other than conjugate hydrolysis. Specifically, we showed that glucobrassicin conversion, previously thought to occur at insignificant levels, actually accounted for the majority of solvent soluble IAA released and that proteinaceous tryptophan degradation accounted for a large portion of solvent insoluble IAA. These studies clearly demonstrated the limits associated with using a harsh technique like base hydrolysis in determining IAA conjugates and support using more direct approaches such as mass spectrometry-based strategies for unambiguous characterizations of the total complement of IAA conjugates in new plant materials under study. Plant drought stress responses lead to modified gene expression that result in changes of metabolism, the direct signature of biochemical activity. A mass spectrometry-based untargeted metabolomics approach was used to study whole plant metabolic changes induced by drought stress and other abiotic stresses including heat, cold, and high light. Seedings were grown vertically on agar plates at 22°C under a 16-h-light/8-h-dark photoperiod of 80 μmolm-2s-1 cool-white fluorescent for 11 days. Seedlings were treated with drought stress (desiccation for 2h), basal heat stress (45°C for 5h), acquired heat stress (38°C for 1.5h , 22°C for 2h, 45°C for 5h), basal cold stress (3°C for 3h), acquired cold stress (3°C for 3h, -20°C for 1h), and high light stress (902 μmolm-2s-1 high light for 1h). Each stress group has a corresponding recovery group where the plants were moved to non-stress conditions for 2 days after the stress treatment. Metabolic profiles of control, stress groups, and stress recovery groups were acquired using ultra performance liquid chromatography high resolution mass spectrometry (UPLC-HRMS) on a hybrid quadrupole orbitrap instrument. Thousands of metabolic features (m/z, retention time, intensity) were analyzed by SIEVE software for principal component (PCA) and single metabolite t-test analyses. Hundreds of metabolites were significantly altered in stress groups or recovery groups compared with the control group (p-value < 0.05 from t-test). Among them, 30 metabolites of interests were confidently identified by comparison to authentic standards. These included amino acids, tricarboxylic acid cycle intermediates, sugars, and other plant metabolites, indicating that significant aspects of their metabolism were modified by exposure to different abiotic stress conditions.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Yu P, Lor P, Ludwig-M�ller J, Hegeman AD, Cohen JD Quantitative evaluation of IAA conjugate pools in Arabidopsis thaliana. Planta 241:539-548 (2015)
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Wilson MB, Brinkman D, Spivak M, Gardner G, Cohen JD Regional variation in composition and antimicrobial activity of U.S. propolis against Paenibacillus larvae and Ascopheara apis. Journal of Invertebrate Pathology 124:44-50 (2015)
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Shi Y-F, Wang D-I, Wang C, Culler AH, Kreiser MA, Suresh J, Cohen JD, Pan J, Baker B, and Liu J-Z Loss of GSNOR1 function leads to compromised auxin signaling and polar auxin transport. Molecular Plant 8:1350-1365 (2015)
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Tivendale ND, Cohen JD Analytical history of auxin. J. Plant Growth Regulation 34:708722 (2015)

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The work accomplished is primarily directed to active plant biology researchers interested in the complexity of signaling systems in plants as they relate to stress adaptation and long distance communication within the plant. However, a secondary audience would be students and educators interested in modern approaches to long-studied problems in this area. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Yuan Xu attended the American Society for Mass Spectrometry two-day Short Course on High Resolution Mass Spectrometry prior to attending the national meeting in Baltimore MD on June 14-15, 2014. Dana Freund attended the Software Carpentry Workshop following ASPB in Portland, OR. How have the results been disseminated to communities of interest? The results of our work have resulted in scientific publications, conference posters and presentations, as well as public presentations at other universities. In addition to those reported in the publications section a university seminar was also presented: Cohen JD (July 8, 2014) Norwegian University of Life Science, "Targeted metabolomics in plant hormone research", Graduate Program in Plant Sciences, (invited seminar). What do you plan to do during the next reporting period to accomplish the goals? Our goals remain the same as previously outlined and the project is on target.

Impacts
What was accomplished under these goals? The bulk of the indole-3-acetic acid in plants is found in the form of conjugated molecules, yet past research on identifying these compounds has largely relied on methods that were both laborious and inefficient. Utilizing recent advances in analytical instrumentation, we have developed a simple yet powerful liquid chromatography-mass spectrometry (LC-MS) based method for the facile characterization of the small IAA conjugate profile in plants. The method employs using as the signature ion the well-known quinolinium ion (130.0651 m/z) generated in MS processes with high mass accuracy to query the plant extract for any potential indolic compounds including IAA conjugates. We reinvestigated soybean for its indoles and found indole-3-acetyl-trytophan (IA-Trp) in addition to the already known indole-3-acetyl-aspartic acid (IA-Asp) and indole-3-acetyl-glutamic acid (IA-Glu) conjugates. Surprisingly, several organic acid conjugates of tryptophan were also discovered, most of which are now described for the first time in plants. Our method has proven to be sensitive and versatile toward the identification of novel indolic compounds. It involves minimal sample preparation but can work in conjunction with sample enrichment techniques. This method enables quick screening of IAA conjugates in both previously characterized as well as uncharacterized species and facilitates identification of novel indolic compounds in general. The concept of ‘bound auxin’ traces its origin back to more than eighty years ago and has driven research on the sources and forms of these plant hormones since. Indeed, analytical studies have demonstrated that the majority of cellular auxin is conjugated to simple sugars, cyclitols, glycans, amino acids, and other biomolecules. A number of studies have confirmed the enzymatic systems responsible for the synthesis and hydrolysis of a number of such conjugates in Arabidopsis thaliana and some of these compounds have been identified in situ. However, the amount of indole-3-acetic acid (IAA) released upon treating Arabidopsis tissue extracts with base, a commonly employed technique for estimating the amount of IAA conjugates, greatly exceeded the summation of all the IAA conjugates known individually to be present in Arabidopsis. This discrepancy has remained as an unsolved question. In this study, however, we found that a significant portion of the IAA found after base treatment could be attributed to chemical conversions other than conjugate hydrolysis. Specifically, we showed that glucobrassicin conversion, previously thought to occur at insignificant levels, actually accounted for the majority of solvent soluble IAA released and that proteinaceous tryptophan degradation accounted for a large portion of solvent insoluble IAA. These studies clearly demonstrated the limits associated with using a harsh technique like base hydrolysis in determining IAA conjugates and support using more direct approaches such as mass spectrometry based strategies for unambiguous characterizations of the total complement of IAA conjugates in new plant materials under study.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Yu P, Hegeman AD, Cohen JD(2014) A facile means for the identification of indolic compounds from plant tissues. Plant Journal 79:10651075
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Yu P, Lor P, Ludwig-Mueller J, Hegeman AD, Cohen JD (2014) Quantitative evaluation of IAA conjugate pools in Arabidopsis thaliana. Planta DOI 10.1007/s00425-014-2206-z
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Spiess GM, Hausman A, Yu P, Cohen JD, Rampey RA, Zolman BK (2014) Auxin input pathway disruptions are mitigated by changes in auxin biosynthetic gene expression in Arabidopsis. Plant Physiol. 165:1092-1104
• Type: Book Chapters Status: Published Year Published: 2014 Citation: Roe MR, Cohen JD, and Hegeman AD (2014) Targeted deuteration of polyphenolics for their qualitative and quantitative metabolomic analysis in plant-derived extracts, Sriram G. Ed. Methods Mol Biol.1083:17-29
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Tivendale ND, Ross JJ, Cohen JD (2014) The shifting paradigms of auxin biosynthesis. Trends in Plant Science 19:44-51
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Roe M, Cohen JD, Hegeman AD (2014) Regioselective solvent-phase deuteration of polyphenolic compounds informs their identification by mass spectrometry. Anal Biochem. 452:76-85
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Cohen JD, Yu P, Kreiser M, Chen J, Hegeman A (June 29  July 4, 2014) Targeted metabolomics in auxin research (Abstract 01-2, page 19), International Symposium on Auxins and Cytokinins in Plant Development - ACPD 2014, Prague (invited oral).
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Yu P, Hegeman A, Cohen J (June 29  July 4, 2014) A facile means for the identification of indolic compounds from plant tissues (Abstract 01-5, page 21), International Symposium on Auxins and Cytokinins in Plant Development - ACPD 2014, Prague (invited oral).
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Freund DM, Cohen JD and Hegeman AD (2014) Direct Tissue Spray Ionization of Living Plants by Mass Spectrometry for Metabolomics, American Society of Plant Biologists, Annual Meeting Plant Biology 2014, Portland, OR (Minisymposium talk).
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Yu P. (2014) New analytical methodologies in the study of auxin biochemistry. Ph.D. dissertation, University of Minnesota
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Wilson, MB (2014) Origin, Composition, and Role of Antimicrobial Plant Resins Collected by Honey Bees, Apis mellifera. Ph.D. dissertation, University of Minnesota

Progress 07/10/13 to 09/30/13

Outputs
Target Audience: The work accomplished is primarily directed to active plant biology researchers interested in the complexity of signaling systems in plants as they relate to stress adaptation and long distance communication within the plant. However, a secondary audience would be students and educators interested in modern approaches to long-studied problems in this area. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided educational opportunities for 3 graduate students, 2 undergraduate students and 1 postdoctoral associate. How have the results been disseminated to communities of interest? This research has resulted in a significant number of scientific publications in high-rated journals, presentations at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals? This project is its initial phase and our plans remain unchanged from those originally submitted. Our research is currently on track with our stated expectations.

Impacts
What was accomplished under these goals? Earlier work identified indole-3-butyric acid (IBA) as an endogenous auxin in Arabidopsis and other plant species with identification typically based on full scan gas chromatography-mass spectrometry (GC-MS) on a single quadrupole system. Subsequently, IBA concentrations within a species were shown to vary greatly depending on the cultivar being analyzed and the environment in which plants was grown. A recent study by Novák et al. (Plant J 72:523, 2012) used a method combining solid phase extraction (SPE) purification with liquid chromatography-selected reaction monitoring-mass spectrometry (LC-SRM-MS/MS) analysis and failed to detect measurable levels of endogenous IBA in Col-0 Arabidopsis grown under various conditions and postulated that prior reports were in error. These results were in contrast to prior reports as well as different from recent work from our laboratory (Liu et al., Plant Methods 8:31, 2012) using GC-SRM-MS/MS which was able to measure significant levels (1.05 +0.15 ng/g FW) of IBA in Col-0 Arabidopsis roots. Because various groups have utilized isotope dilution analysis for their studies, the postulated differences poised by Novák et al. in light of their negative results all seemed problematic and could have been due to plant culture differences or the inherent higher background of LC-MS techniques. Thus, we confirmed the presence of endogenous IBA in Col-0 Arabidopsis by isolation of IBA from both unlabeled and 15N-labeled plants and confirmed its identity by GC-MS/MS selected reaction monitoring. Most current research on auxin biosynthesis has focused on studying individual precursors within a single pathway. Many methods have been developed to quantify these precursors together with the active hormone to gain insights into the roles of particular compounds. The isolated studies of specific pathways present a static picture of auxin synthesis from a precursor. However, such analyses fail to capture the dynamic changes in the metabolic responses following environmental or developmental signaling event and do not measure the interplay between different hormonal pathways. The intricate network of reactions involved in auxin biosynthesis requires a more dynamic and comprehensive analytical approach in order to understand the link between hormone metabolism and the developmental events they regulate. We have developed a method to simultaneously measure the metabolic flux of most of the known indole-3-acetic acid (IAA) precursors in Arabidopsis thaliana. The procedure involves creating 13C labeled plant seedlings, feeding 15N labeled anthranilic acid (the primary indole pathway precursor), and measuring the process of label incorporation into different precursors over time as well as their absolute concentration via isotope dilution assays. Based on this analyses we are able to calculate the flux through each precursor and thus reconstruct the full metabolic network that is operating and leading to IAA. Such analyses should reveal what the relevant biosynthesis pathways are that are active under particular physiological conditions and bring us a step further towards understanding the mechanism by which plants regulate auxin homeostasis in order to cope with environmental stresses and respond to developmental needs. Preliminary information showed that indole-3-pyruvic acid and indole-3-acetaldehyde were labeled much more quickly relative to other precursors, partly agrees with previous findings. The labeling kinetics of IAA and tryptophan further supported the existence of tryptophan independent auxin biosynthesis. We are currently constructing the detailed metabolic network model to interrogate qualitatively and quantitatively the role of each precursor in the framework of such a network.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Yerramsetty V, Roe M, Cohen JD, Hegeman A, Ismail B Development of a simple, fast and accurate method for the direct quantification of selective estrogen receptor modulators in biological fluids using stable isotope dilution mass spectrometry, J Agric and Food Chem 61: 7028-7037 (2013)
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Li W, Zhou Y, Liu X, Yu P, Cohen JD, Meyerowitz EM Flower development master regulator LEAFY controls auxin response pathways in floral primordia formation. Science Signaling 6: ra23 (2013) [DOI: 10.1126/scisignal.2003937]
• Type: Journal Articles Status: Published Year Published: 2013 Citation: DeMason DA, Chetty V, Barkawi LS, Liu X, Cohen JD Unifoliata-Afila interactions in pea leaf morphogenesis. American J Botany 100:478-495 (2013)
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Yu H, Karampelias M, Robert S, Peer WA, Swarup R, Ye S, Ge L, Cohen JD, Murphy A, Friml J, Estelle M ROOT ULTRAVIOLET B-SENSITIVE1/WEAK AUXIN RESPONSE3 is essential for polar auxin transport in Arabidopsis. Plant Physiology 162:965-976 (2013)
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Mazhar S, Cohen JD, Hasnain S Auxin producing non-heterocystous Cyanobacteria and their impact on the growth and endogenous auxin homeostasis of wheat. Journal of Basic Microbiology?(in press, 2013) DOI: 10.1002/jobm.201100563.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Wilson MB, Spivak M, Hegeman AD, Rendahl A, Cohen JD Metabolomics reveals the origins of antimicrobial plant resins collected by honey bees. PLoS ONE 8(10): e77512. doi:10.1371/journal.pone.0077512 (2013)
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Fan K-T, Cohen JD, Gray WM, and Hegeman AD (2013) Absolute quantification of TIR1/AFB proteins in Arabidopsis using the QconCAT strategy, J. Am. Soc. Mass Spec. 24(S1), Minneapolis, MN, (poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Gentle C, Roe MR, Hegeman AD, and Cohen JD (2013) Mild Base Catalyzed Deuteration of Polyphenolics for Improving their Quantification in Cold Hardy Wines by Multiple Reaction Monitoring Mass Spectrometry, J. Am. Soc. Mass Spec. 24(S1), Minneapolis, MN, (poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Yu P, Ludwig-M�ller J, Hegeman AD, and Cohen JD (2013) Identification of indole-3-acetic acid modified proteins of Arabidopsis, J. Am. Soc. Mass Spec. 24(S1), Minneapolis, MN, (poster).
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Roe MR, Cohen JD, and Hegeman AD (2013) Solvent- and gas-phase deuteration of polyphenolics informs their identification by mass spectrometry, J. Am. Soc. Mass Spec. 24(S1), Minneapolis, MN, (oral report).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Kreiser MA, Yu P, and Cohen JD. Identification of IBA in Arabidopsis using high-resolution mass spectrometry techniques, Poster PS01-09, Abstracts, 21st Conference of the International Plant Growth Substances Association, Shanghai, China, June 18th-22nd, 2013, pp 39 (poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Yu P, Hegeman AD, and Cohen JD (2013) Indole Metabolomics: Identification and quantification of indole-3-acetic acid pathway related compounds, Poster PS01-20, Abstracts, 21st Conference of the International Plant Growth Substances Association, Shanghai, China, June 18th-22nd, 2013, pp 45 (poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Cohen JD, Yu P, Liu X, Hageman AD, Gardner GM (2013). Toward single cell targeted metabolomics in plant hormone research. Session C01, Abstracts, 21st Conference of the International Plant Growth Substances Association, Shanghai, China, June 18th-22nd, 2013, pp 33 (invited talk).