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The plant hormone auxin, indole-3-acetic acid (IAA), plays important roles in plant growth and development. The signaling response to IAA is largely dependent on the local concentration of IAA, and this concentration is regulated by multiple mechanisms in plants. Therefore, the precise quantification of local IAA concentration provides insights int...
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... The purification of IAA samples involvs a two-step process of solid phase extraction (SPE) with an amino (NH 2 ) resin followed by a subsequent step using polymethylmethacrylate epoxide (PMME) resin. For the first step, ion exchange TopTips (Glygen, TT2EMT) were prepared with Bondesil-NH 2 resin (Agilent, 12213020; suspended in water, 1:4 w:v) for SPE according to Liu et al. [22]. Each TopTip was prepared with 20 μL resin suspension, then washed with 50 μL each: hexane, acetonitrile, ethyl acetate; finally conditioned with 50 μL 0.2 M imidazole (pH 7.0) followed by 2 × 100 μL water. ...
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.
... IBA movement itself accounted for less than 1% of the bulk transport from exogenous IBA; thus it is not likely that longer distant transport contributes substantially to IBA pools at sites distant from where the IBA is applied. These results show that by using specific stable labeled compounds and mass spectrometry analysis, the movement and metabolic changes of the labeled compounds can be accurately profiled in mutant and wildtype plants and suggests the value of monitoring of metabolic networks in contrast to single compound studies (Liu et al. 2012b). ...
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 highlighting 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.
... Many solvents are used for extraction, such as organic solvents or aqueous buffers (reviewed in Du et al., 2012). Recently, sample purification by solid-phase extraction (SPE) has become the most used method of auxin purification with the tendency to minimize the amount of solvents and analytes (Liu et al., 2007;Liu et al., 2012;Novaḱ et al., 2012;Porfıŕio et al., 2016;Peňcǐḱ et al., 2018;Wang et al., 2020). Until now, a wide range of sorbents have been used for purification: reverse phase columns C18 (Rolcǐḱ et al., 2005;Tivendale et al., 2012), and HLB , poly (styrene-divinylbenzene) copolymer SDB-XC (Peňcǐḱ et al., 2018) or mixed-mode ion-exchange polymeric sorbents (Dobrev et al., 2005;Izumi et al., 2009). ...
Auxins are a group of phytohormones that play a key role in plant growth and development, mainly presented by the major member of the family - indole-3-acetic acid (IAA). The levels of free IAA are regulated, in addition to de novo biosynthesis, by irreversible oxidative catabolism and reversible conjugation with sugars and amino acids. These conjugates, which serve as inactive storage forms of auxin and/or degradation intermediates, can also be oxidized to form 2-oxindole-3-acetyl-1-O-ß- d -glucose (oxIAA-glc) and oxIAA-amino acids (oxIAA-AAs). Until now, only oxIAA conjugates with aspartate and glutamate have been identified in plants. However, detailed information on the endogenous levels of these and other putative oxIAA-amino acid conjugates in various plant species and their spatial distribution is still not well understood but is finally getting more attention. Herein, we identified and characterized two novel naturally occurring auxin metabolites in plants, namely oxIAA-leucine (oxIAA-Leu) and oxIAA-phenylalanine (oxIAA-Phe). Subsequently, a new liquid chromatography–tandem mass spectrometry method was developed for the determination of a wide range of IAA metabolites. Using this methodology, the quantitative determination of IAA metabolites including newly characterized oxIAA conjugates in roots, shoots and cotyledons of four selected plant models - Arabidopsis thaliana , pea ( Pisum sativum L.), wheat ( Triticum aestivum L.) and maize ( Zea mays L.) was performed to compare auxin metabolite profiles. The distribution of various groups of auxin metabolites differed notably among the studied species as well as their sections. For example, oxIAA-AA conjugates were the major metabolites found in pea, while oxIAA-glc dominated in Arabidopsis. We further compared IAA metabolite levels in plants harvested at different growth stages to monitor the dynamics of IAA metabolite profiles during early seedling development. In general, our results show a great diversity of auxin inactivation pathways among angiosperm plants. We believe that our findings will greatly contribute to a better understanding of IAA homeostasis.
... The latter is known to stimulate short-term stress responses such as closure of stomata (Verma et al. 2016). These data are supported by our previous results on stomata density and stomata length regulating transpiration in strawberry plantlets at the acclimation stage during MC treatment (Ambros et al. 2018b;unpublished (Liu et al. 2012;Saeedipour and Moradi 2012;Dierck et al. 2016;Benazzouk et al. 2020;Arif et al. 2021). Si may help to overcome abiotic stress by engaging in signaling pathways and transduction mechanisms through cross talk with phytohormones (Arif et al. 2021;Tripathi et al. 2021). ...
The purpose was to assess effects of a mechanocomposite (MC) based on biogenic silica and green tea flavonoids in terms of alleviation of the impact of abiotic stressors on strawberry plantlets at different stages of clonal micropropagation. An experiment was set up with or without MC treatment at multiplication, in vitro rooting, and ex vitro acclimation stages. We quantified hydrogen peroxide (H2O2) as a marker of oxidative stress along with antioxidants (non- and enzymatic), phytohormones, and biomass parameters. At the multiplication stage, MC treatment increased the level of endogenous H2O2 (2.3-fold), the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase), and the total phenolic content. Nonetheless, the MC decreased levels of phytohormones (indole-3-acetic acid, trans-zeatin, and abscisic acid). The addition of MC at the in vitro rooting stage lowered the H2O2 content 1.6-fold, while the activity of antioxidant enzymes significantly increased, compared to the MC-free group; in the MC group, the studied phytohormones showed maximum values. Effects of the MC on phenolic compounds varied. Concentrations of some compounds significantly increased, while the majority of these compounds showed a decline. At the ex vitro acclimation stage, MC treatment raised the level of H2O2 in microshoots in comparison to in vitro stages (this effect was attributed to the enhancement of antioxidant enzymes’ activities), accompanied by downregulation of the phytohormones and upregulation of most of phenolic compounds. The MC-induced effects on growth and development were confirmed by greater shoot biomass. During micropropagation, MC application allows plants to overcome oxidative stress through modulation of activities of antioxidant enzymes and phytohormone levels and a realignment of the profile of phenolic compounds.
... These can be problematic to use if the goal of extraction is to analyze either the endogenous esters or IAA methyl or ethyl esters. These issues are greatly reduced by employing secondary alcohols, for example using a solvent-soluble anti-oxidant buffer like imidazole with aqueous 2-propanol for extraction (Liu, Hegeman, Gardner, & Cohen, 2012). ...
... This correction (R) is included to account for minor isotopologues not analyzed at the selected monoisotopic ion monitored by the MS (Cohen et al., 1986). However, if the amount of standard was determined by reverse isotope dilution analysis without correction for the isotopic envelope (Liu et al., 2012), the calculations are self-correcting. R must be determined empirically for each lot of [ 13 C 6 ]IAA purchased or synthesized, although it has remained relatively constant for the last 30 years. ...
... (2) IBA can be made using a variety of labeled indoles, the most effective is fully labeled [ 13 C 8 , 15 N]indole, as shown (see Liu et al., 2012;Sutter & Cohen, 1992). (3) IAA-amino acid conjugates are readily synthesized in high yield by a simple dicyclohexylcarbodiimide (DCC) coupling to an esterified amino acid followed by base hydrolysis. ...
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.
... Several methods allow us to track auxin within a tissue. For instance, auxin-inducible genes fused with reporter constructs [32,33], antibodies against auxin or its carriers [34,35], or mass-spec-based measurements [36,37] have been successfully employed to detect or at least infer the auxin content in plant tissues. Each technique has its advantages and disadvantages; some are indirect, such as the approach to visualize auxin-induced promoter activities, while others are destructive, such as that it is not possible to follow fluctuations over time. ...
The complexity of auxin signaling is partially due to multiple auxin receptors that trigger differential signaling. To obtain insight into the subcellular localization of auxin-binding sites, we used fluorescent auxin analogs that can undergo transport but do not deploy auxin signaling. Using fluorescent probes for different subcellular compartments, we can show that the fluorescent analog of 1-naphthaleneacetic acid (NAA) associates with the endoplasmic reticulum (ER) and tonoplast, while the fluorescent analog of indole acetic acid (IAA) binds to the ER. The binding of the fluorescent NAA analog to the ER can be outcompeted by unlabeled NAA, which allows us to estimate the affinity of NAA for this binding site to be around 1 μM. The non-transportable auxin 2,4-dichlorophenoxyacetic acid (2,4-D) interferes with the binding site for the fluorescent NAA analog at the tonoplast but not with the binding site for the fluorescent IAA analog at the ER. We integrate these data into a working model, where the tonoplast hosts a binding site with a high affinity for 2,4-D, while the ER hosts a binding site with high affinity for NAA. Thus, the differential subcellular localization of binding sites reflects the differential signaling in response to these artificial auxins.
... Samples for IAA quantitation by LC-MS/MS were prepared and analyzed essentially as described by (Tang et al., 2019), using imidazole buffered aqueous 2-propanol for extraction. These procedures have five advantages: speed, small sample size, polar enzyme inactivating solvents, and a solvent-soluble buffer/antioxidant (Liu et al., 2012b). Imidazole has been shown to have antioxadant activitiy (Kohen et al., 1988) and, as noted by Tivendale et al. (2015), IPyA is, at neutral pH in methanol, relatively stable. ...
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.
... The samples were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) at high resolution. [ 13 C 6 ]IAA internal standard was added with 2-propanol/ buffer (56) to plant tissue samples weighing 7 to 21 mg, which were then homogenized and incubated for 1 hour on ice. Samples were then diluted with water, centrifuged, and IAA was extracted from the supernatant using amino and polymethylmethacrylate epoxide (PMME) solid phase extraction resins in Top Tips spin tips (Glygen, Columbia, MD, USA). ...
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.
... Endogenous auxin levels in hypocotyls were quantified as described previously (Liu et al., 2012). Col-0 and clc2-1 clc3-1 seedlings were grown (22°C/20°C day/night temperature, 16h-light/8-h-dark photoperiod) for 5 d, and~200 mg hypocotyl tissue was excised from~10,000 Col-0 and clc2-1 clc3-1 seedlings and stored in liquid nitrogen for IAA extraction and measurement. ...
PIN‐FORMED (PIN)‐dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2‐1 clc3‐1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live‐cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2‐1 clc3‐1 mutants to redirect auxin toward the epidermal cell layers. Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2‐1 clc3‐1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.
... Auxin and its precursors orchestrate developmental and morphological plasticity in members of land plants including angiosperms, gymnosperms, spikemoss, moss and liverworts (Casanova-Saez and Voss, 2019). High-throughput biochemical tools have been used to quantify auxins accumulation in diverse plant tissues such as seeds, pollen, and young vegetative organs (Liu et al., 2012;Ludwig-Muller and Cohen, 2002;Muñoz-Sanhueza et al., 2018). ...
Auxin is a key hormone that regulates plant growth and development. Naturally occurring auxins are indole compounds, but synthetic auxins used in agriculture can have diverse chemical structures. Decades of genetic and biochemical studies have led to our current understanding of how auxin is biosynthesized, perceived, and transported in planta. Genomic studies have demonstrated that nuclear auxin signaling events result in widespread transcriptional responses and proteome remodeling, consistent with conventional wisdom that auxin touches nearly every aspect of plant biology. Future directions for the field of auxin biology will include multi-scale interdisciplinary approaches to deepen our understanding of auxin-mediated processes.
