In recent years, substantial progress has been made in exploring auxin conjugation and metabolism, primarily aiming at indole-3-acetic acid (IAA). However, the metabolic regulation of another key auxin, phenylacetic acid (PAA), remains largely uncharacterized. Here, we provide a comprehensive exploration of PAA metabolism in land plants. Through LC-MS screening across multiple plant species and their organs, we identified four previously unreported endogenous PAA metabolites: phenylacetyl-leucine, phenylacetyl-phenylalanine, phenylacetyl-valine, and phenylacetyl-glucose. Enzyme assays, genetic evidence, crystal structures, and docking studies demonstrate that PAA and IAA share core metabolic machinery, revealing a complex regulatory network that maintains auxin homeostasis. Furthermore, our study of PAA conjugation with amino acids and glucose suggests limited compensatory mechanisms within known conjugation pathways, pointing to the existence of alternative metabolic routes in land plants. These insights advance our knowledge of auxin-specific metabolic networks and highlight the unique complexity within plant hormone regulation.

Jasmonates are a family of oxylipin phytohormones regulating plant development and growth and mediating "defense versus growth" responses. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-OPDA) acts independently of CORONATIVE INSENSITIVE 1-mediated JA signaling in several stress-induced and developmental processes. However, its perception and metabolism are only partially understood. An isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected years ago in wounded leaves of flowering plants, opening up the possibility that conjugation of cis-OPDA to amino acids might be a relevant mechanism for cis-OPDA regulation. Here, we extended the analysis of amino acid conjugates of cis-OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp accumulating in response to biotic and abiotic stress in Arabidopsis (Arabidopsis thaliana). The OPDA amino acid conjugates displayed cis-OPDA-related plant responses in a JA-Ile-dependent manner. We also showed that the synthesis and hydrolysis of cis-OPDA amino acid conjugates are mediated by members of the amidosynthetase GRETCHEN HAGEN 3 and the amidohydrolase INDOLE-3-ACETYL-LEUCINE RESISTANT 1/ILR1-like families. Thus, OPDA amino acid conjugates function in the catabolism or temporary storage of cis-OPDA in stress responses instead of acting as chemical signals per se. Amide conjugate synthesis and hydrolysis control the homeostasis of a jasmonate precursor during stress responses in Arabidopsis.

Seasonal dynamics of root growth play an important role in large-scale ecosystem processes; they are largely governed by growth regulatory compounds and influenced by environmental conditions. Yet, our knowledge about physiological drivers of root growth is mostly limited to laboratory-based studies on model plant species. We sampled root tips of Eriophorum vaginatum and analyzed their auxin concentrations and meristem lengths biweekly over a growing season in situ in a subarctic peatland, both in surface soil and at the permafrost thawfront. Auxin concentrations were almost five times higher in surface than in thawfront soils and increased over the season, especially at the thawfront. Surprisingly, meristem length showed an opposite pattern and was almost double in thawfront compared with surface soils. Meristem length increased from peak to late season in the surface soils but decreased at the thawfront. Our study of in situ seasonal dynamics in root physiological parameters illustrates the potential for physiological methods to be applied in ecological studies and emphasizes the importance of in situ measurements. The strong effect of root location and the unexpected opposite patterns of meristem length and auxin concentrations likely show that auxin actively governs root growth to ensure a high potential for nutrient uptake at the thawfront.

Metabolic degradation of plant hormones cytokinins (CKs) co-regulates their homeostasis and signalling. In this work, we employed a large-scale bioinformatical analysis to address a diversity of cytokinin oxidase/dehydrogenase (CKX) substrate specificities previously described in several case studies. We present a three-way correlation of the entire CKX amino acid sequences, a variable motif involved in substrate binding, and subcellular localizations predicted by a deep learning model. This correlation is conserved in monocotyledonous plants, suggesting that the CKX diversity in a single species allows a precise tuning of the CK homeostasis. Following these findings, we detected CKX activity in xylem sap for the first time, using the oat ( Avena sativa ) as a model plant. Further investigation of the substrate specificity and glycosylation of this xylem-located CKX suggested that it originates in roots. We have identified 27 putative CKXs in oats and attributed the xylem-located activity to the extracellular isoforms AsCKX1a,c,d. Finally, we show that the xylem-located CKX activity responds to the nitrate supply, highlighting its physiological relevance. Taken together, we show that CKX directly modulates root-to-shoot CK translocation through metabolic degradation of the transported CKs.

AbstractJasmonates (JAs) are a family of oxylipin phytohormones regulating plant development and growth and mediating ‘defenseversusgrowth’ responses. The upstream JA biosynthetic precursorcis-(+)-12-oxo-phytodienoic acid (cis-OPDA) has been reported to act independently of the COI1-mediated JA signaling in several stress-induced and developmental processes. However, its means of perception and metabolism are only partially understood. Furthermore,cis-OPDA, but not JA, occurs in non-vascular plant species, such as bryophytes, exhibiting specific functions in defense and development. A few years ago, a low abundant isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected in wounded leaves of flowering plants, opening up to the possibility that conjugation ofcis-OPDA to amino acids might be a relevant mechanism forcis-OPDA regulation. Here, we extended the analysis of amino acid conjugates ofcis-OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp in response to biotic and abiotic stress in Arabidopsis. The newly identified OPDA-amino acid conjugates showcis-OPDA-related plant responses in a JAR1-dependent manner. We also discovered that the synthesis and hydrolysis ofcis-OPDA amino acid conjugates are regulated by members of the amidosynthetase GH3 and the amidohydrolase ILR1/ILL families. Finally, we found that thecis-OPDA conjugative pathway already functions in non-vascular plants and gymnosperms. Thus, one level of regulation by which plants modulatecis-OPDA homeostasis is the synthesis and hydrolysis of OPDA-amino acid conjugates, which temporarily storecis-OPDA in stress responses.

Dynamic regulation of the concentration of the natural auxin indole-3-acetic acid (IAA) is essential to coordinate most physiological and developmental processes and responses to environmental changes (reviewed in Friml, 2022). Auxin inactivation plays a crucial role in auxin homeostasis and metabolism. The primary enzymes involved in auxin metabolism have been known for some time. Members of the acyl acid amide synthetases belonging to the GRETCHEN HAGEN 3 (GH3), and the amidohydrolase IAA-LEUCINE RESISTANT 1 (ILR1) and ILR1-like (ILL) families catalyze the conjugation of IAA to amino acids and hydrolysis of the IAA-amino acid conjugates, respectively (LeClere et al., 2002; Staswick et al., 2005). The DIOXIGENASE FOR AUXIN OXIDATION (DAO) enzymes were shown to catalyze the oxidation of IAA to form oxIAA (Porco et al., 2016; Zhang et al., 2016). Albeit these IAA-inactivating enzymes appeared to participate in different catabolic routes, very recently, it was reported that GH3, ILR1, and DAO are part of a single linear pathway rather than two distinct pathways (Hayashi et al., 2021). According to this model, IAA is mainly inactivated by GH3 enzymes, DAO functions as an oxidase of IAA-amino acid conjugates to produce oxIAA-amino acid conjugates downstream of GH3, and oxIAA is produced from oxIAA-amino acid hydrolysis by ILR1. Therefore, DAO and ILR1 enzymes appeared to play a role in this pathway that differs from that assigned initially. Although GH3 enzymes are known to possess catalytic promiscuity accepting various substrates and amino acids, a possible additional role of GH3s in auxin inactivation was not investigated (Staswick et al., 2005; Westfall et al., 2012). Besides, while the new model proposed by Hayashi et al. (2021) was described to occur in angiosperms, whether it operates in nonflowering species remains unknown (Ross & Gélinas-Marion, 2021). GRETCHEN HAGEN 3 proteins are highly conserved all over the plant kingdom, whereas DAO and DAO-like enzymes have specifically evolved with angiosperms (Okrent & Wildermurth, 2011; Brunoni et al., 2020; Kaneko et al., 2020; Takehara et al., 2020). Here, we report the evidence of oxIAA-amino acid conjugation being catalyzed by the group of IAA-conjugating enzymes belonging to Group II of GH3s. Our work suggests that the contribution of this pathway to auxin homeostasis is species-dependent. Arabidopsis thaliana (L.) Heynh. seeds wild-type (WT) Col-0 and gh3 sextuple mutant (gh3.1, gh3.2, gh3.3, gh3.4, gh3.5, and gh3.6; Porco et al., 2016) were gas sterilized with chlorine gas (10 ml of HCl (35%) in 50 ml of bleach) for at least 2 h in an airtight box. Seeds were then sown under sterile conditions on Petri dishes containing ½ Murashige-Skoog (½MS) medium (2.15 g salts including vitamins per 1 l) with 1% sucrose and 0.5 g l−1 MES monohydrate at pH 5.7 and solidified with 0.57% Gellan gum. Stratification was carried out for 3 d at 4°C, and then, plates were transferred to light at 22 ± 1°C, under long-day (LD) conditions (16 h : 8 h, light : dark photoperiod; 100 μmol m−2 s−1). Gametophores from Physcomitrium patens (Hedw.) Mitt. WT and knockout lines (International Moss Stock Center IMSC#40207 GH3-1/B34, IMSC#40208 GH3-2/22, and IMSC#40209 GH3-doKO-A96; Bierfreund et al., 2004; Ludwig-Müller et al., 2009) were obtained from IMSC (www.moss-stock-center.org) and cultured axenically in Knop medium (100 mg l−1 Ca(NO3)2·4H2O, 25 mg l−1 KCl, 25 mg l−1 KH2PO4, 25 mg l−1 MgSO4·7H2O, and 1.25 mg l−1 FeSO4·7H2O, pH 5.8) including 92.05 mg l−1 ammonium tartrate, 0.5 mg l−1 nicotinic acid, 0.125 mg l−1 p-amino benzoic acid, 2.5 mg l−1 thiamine HCl, trace-element solution (0.614 mg l−1 H3BO3, 0.389 mg l−1 MnCl2·4H2O, 0.059 mg l−1 NiCl2·6H2O, 0.055 mg l−1 CoCl2·6H2O, 0.055 mg l−1 CuSO4·5H2O, 0.055 mg l−1 ZnSO4·7H2O, 0.0386 mg l−1 Al2(SO4)3·18H2O, 0.028 mg l−1 KBr, 0.028 mg l−1 KI, 0.028 mg l−1 LiCl, and 0.028 mg l−1 SnCl2·2H2O), and 200 mg l−1 glucose. The medium was solidified with 1.5% plant agar. Plants were cultured in a growth chamber at 22 ± 1°C, under LD conditions, and subcultured onto fresh medium every 3 wk. Picea abies (L. Karst) seeds were provided by SkogForsk (Sävar, Sweden). Seeds were soaked in tap water for 12 h at 4°C and sown in fine wet vermiculite. Germination and seedling growth occurred in a growth chamber at 24°C during the day and 18°C at night, under LD conditions at a light intensity of 100 μmol m−2 s−1. Seven-days-after-germination (DAG), A. thaliana seedlings were transferred to sterile liquid ½MS medium for 24 h and subsequently supplemented with 50 μM IAA, IAA–Asp, IAA–Glu, or oxIAA for 6 and 24 h. Three-week-old moss gametophores were transferred to sterile liquid Knop medium for 24 h and subsequently supplemented with 50 μM IAA, IAA–Glu, or oxIAA for 24 h. Two-week-old spruce seedlings were transferred to ½MS liquid medium for 24 h and subsequently supplemented with 50 μM IAA, IAA–Asp, IAA–Glu, or oxIAA for 6 h; for the inhibition of oxIAA conjugation in spruce, 2-wk-old seedlings were incubated in ½MS liquid medium with 50 μM kakeimide (KKI), 5 μM oxIAA, or a combination of 50 μM KKI and 5 μM oxIAA for 6 h. Mock-treated Arabidopsis, moss, and spruce plants were used as control. All liquid cultures were kept under gentle shaking and in darkness for the whole experiment in a growth chamber at 24°C during the day and 18°C during the night. For each time point, A. thaliana whole seedlings, P. patens gametophores, and P. abies roots were collected in three replicates (10 mg tissue per sample). Sample purification and quantification of oxIAA–Asp and oxIAA–Glu were performed as described previously (Pěnčík et al., 2018). Briefly, samples were extracted with 1 ml of 50 mM sodium phosphate buffer, pH 7.0, containing 0.1% sodium diethyldithiocarbamate. oxIAA–[13C4,15N]Asp and oxIAA–[13C5,15N]Glu were added as internal standards. Two hundred microliters of the extract was acidified with 1 M HCl to pH 2.7 and purified by in-tip micro solid phase extraction (in-tip μSPE). After evaporation under reduced pressure, samples were analyzed using HPLC system 1260 Infinity II (Agilent Technologies, Santa Clara, CA, USA) equipped with Kinetex C18 column (50 mm × 2.1 mm, 1.7 μm; Phenomenex, Torrance, CA, USA) and linked to 6495 Triple Quad detector (Agilent Technologies). Cloning and protein production of AtGH3s (AtGH3.1–5, 7–16, 18, 19) and PpGH3s (PpGH3.1 and PpGH3.2) were performed as described previously (Brunoni et al., 2020). Primers used for cloning are listed in Supporting Information Table S1. Escherichia coli BL21 (DE3) strains expressing recombinant AtGH3.6, AtGH3.17, and PaGH3s (PaGH3.16, PaGH3.17, PaGH3.gII.8, and PaGH3.gII.9) used in this work were previously generated (Brunoni et al., 2019, 2020). Briefly, the production of recombinant proteins was induced by adding 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to cell cultures in a liquid medium with OD600 between 0.6 and 0.8. Cells were grown overnight at 20°C with constant shaking at 180 rpm. Protein expression was tested by western blotting with anti-6x His antibody horseradish peroxidase (HPR) conjugate. Fifty milliliters of GH3-producing bacteria were pelleted and resuspended in an equal volume of 1× phosphate-buffered saline (PBS) buffer. The crude cell lysate was obtained by sonication and clarified by centrifugation (21 325 g at 4°C for 30 min). Five hundred microliters of clarified cell lysate were incubated with 1 mM amino acid mixture, 3 mM ATP, 3 mM MgCl2, and 1 mM auxin substrate (IAA or oxIAA) for 5 h at 30°C with constant shaking at 50 rpm in darkness. Sample preparation and quantification of IAA- and oxIAA-amino acid conjugates were performed as described previously (Brunoni et al., 2020), using [13C6]IAA–Asp, [13C6]IAA–Glu, oxIAA–[13C4,15N]Asp, and oxIAA–[13C5,15N]Glu as internal standards. Two hundred milliliters of overnight-induced AtGH3.2- and AtGH3.6-producing bacterial cultures were used for protein purification. Cell lysis and protein extraction were done using the cell lyser One Shot model (Constant Systems Ltd, Northamptonshire, UK) at 24 000 psi in the presence of cOmplete EDTA-free Protease Inhibitor Cocktail (Roche), followed by treatment with DENARASE (c-LEcta GmbH, Leipzig, Germany). Recombinant AtGH3.2 and AtGH3.6 were purified on a Nickel-HiTrap IMAC FF column (Cytiva Life Sciences, Marlborough, MA, USA) on an NGC Medium-Pressure Liquid Chromatography System into 50 mM Tris–HCl buffer, pH 7.4, 300 mM NaCl, and 5% glycerol. The yield after purification was c. 14.4 mg for GH3.2 and 2.4 mg for GH3.6, per 200 ml of E. coli BL21 (DE3) cells. The enzyme activity and saturation curves were measured in 50 mM Tris–HCl buffer, pH 8.0, 0.1 mM DTT, 30 mM ATP, 30 mM L-aspartic acid, 3 mM MgCl2, and varying concentrations (0.1–8 mM) of auxin substrate (IAA or oxIAA). The reactions were carried out in triplicates at 30°C in the presence of 5 μM enzyme for 30 min in darkness and stopped after adding 100% methanol. Sample preparation and quantification of oxIAA-amino acid conjugates were performed as described previously (Brunoni et al., 2020), using [13C6]IAA–Asp, [13C6]IAA–Glu, oxIAA–[13C4,15N]Asp, and oxIAA–[13C5,15N]Glu as internal standards. Kinetic constants Km and Vmax were determined using the GraphPad Prism 8.0 software (www.graphpad.com). Statistical analysis was carried out using Microsoft Excel 2016 for Windows PC. Quantitative values are presented as mean ± SD. We studied the oxIAA-conjugating activity of recombinant Arabidopsis GH3 enzymes after their production in E. coli to test a possible further involvement of GH3 enzymes in IAA inactivation, using a bacterial assay that was previously adopted to study the activity of several IAA catabolic enzymes (Brunoni et al., 2019; Müller et al., 2021). Arabidopsis possesses 19 different GH3 proteins classified into three groups (I, II, and III) based on substrate specificity and sequence homology (Staswick et al., 2005). Group I GH3s conjugate jasmonic acid with isoleucine (Staswick & Tiryaki, 2004; Delfin et al., 2022); Group II GH3s contain enzymes that conjugate auxins (Staswick et al., 2005); and Group III, specific for Brassicaceae, contains proteins predicted to be active on benzoates (Okrent et al., 2009; Holland et al., 2019; Torrens-Spence et al., 2019). While none of the members of Group I and III GH3s could accept oxIAA as a substrate for conjugation with amino acids, members of the Group II GH3s were able to catalyze the conversion of oxIAA (Dataset S1; Fig. S1). Among Group II, AtGH3.1, AtGH3.2, AtGH3.3, AtGH3.5, AtGH3.6, and AtGH3.17 showed activity with oxIAA (Fig. S1). By contrast, two members, namely AtGH3.4 and AtGH3.9, did not show any activity with this substrate (Dataset S1). AtGH3.2, AtGH3.3, and AtGH3.5 conjugated oxIAA with aspartate (Asp) preferentially, whereas AtGH3.1 and AtGH3.6 conjugated oxIAA exclusively with Asp and AtGH3.17 with glutamate (Glu; Fig. S1). Notably, AtGH3.2 conjugates oxIAA also with leucine (Leu) and phenylalanine (Phe; Dataset S1). To assess the substrate specificity of Group II GH3 enzymes, we studied the enzymatic activity of recombinant AtGH3 enzymes in vitro. Our kinetic data with recombinant AtGH3.2 and AtGH3.6 revealed that both enzymes preferred IAA to oxIAA as a substrate for the conjugation with Asp (Dataset S2; Fig. 1a). AtGH3.6 displayed higher specific activity than AtGH3.2, and Km values for auxin substrates are in low mM range (Dataset S2; Fig. 1b). Calculated catalytic efficiency (Vmax/Km) values showed that conjugation of oxIAA with Asp reached only 2% of that measured with IAA (Dataset S2; Fig. 1b). To investigate whether oxIAA conjugation occurred in planta, we carried out a feeding experiment by incubating WT Col-0 seedlings with oxIAA and sampled after 6 and 24 h (Dataset S2; Fig. 2a). For comparison, we also treated plants with IAA, IAA–Asp, and IAA–Glu (Dataset S2; Fig. 2a). Feeding experiments confirmed that the production of oxIAA–Asp and oxIAA–Glu mainly resulted from the oxidation of IAA–Asp and IAA–Glu, respectively, as described previously (Hayashi et al., 2021), as a high level of oxIAA–Asp and oxIAA–Glu was detected after exogenous application of IAA–Asp and IAA–Glu, respectively (Dataset S2; Fig. 2a). Nonetheless, oxIAA–Asp and oxIAA–Glu also accumulated after feeding with oxIAA, suggesting that, although to a minor extent, oxIAA-amino acid conjugates originated from oxIAA conjugation (Dataset S2; Fig. 2a). To investigate whether the GH3 pathway is involved in oxIAA conjugation, we fed gh3 sextuple mutant with oxIAA (Dataset S2; Fig. 1b). We observed a dramatic drop in oxIAA–Asp level when compared to WT (Dataset S2; Fig. 2b) after treatment with oxIAA, confirming that oxIAA conjugation with Asp is mediated by Group II GH3s. On the contrary, oxIAA–Glu levels were unaltered in WT and gh3 sextuple mutant after feeding with oxIAA, most likely due to remaining GH3.17 activity (Porco et al., 2016; Dataset S2; Fig. S2b). These results, together with previous findings (Hayashi et al., 2021; Müller et al., 2021), suggest that conjugation/oxidation/hydrolysis reactions play multiple roles in the IAA inactivation pathway to fine-tune auxin and auxin metabolite levels (Fig. 1c). Group II of the GH3 family emerged very early during the land plant evolution (Ludwig-Müller et al., 2009; Okrent & Wildermurth, 2011; Brunoni et al., 2020). Intrigued by the evidence collected with Arabidopsis, we undertook a similar experimental approach to elucidate these pathways in evolutionarily distant species. We chose the moss P. patens as a model for bryophytes and the conifer Picea abies as a model for gymnosperms, as these species were used in previous studies to investigate the evolutionary patterns in the auxin metabolism (Ludwig-Müller et al., 2009; Brunoni et al., 2020). Physcomitrium patens possesses only two GH3 enzymes (Ludwig-Müller et al., 2009). In our experimental conditions, recombinant PpGH3.2, but not PpGH3.1, catalyzed the conjugation of oxIAA exclusively with Glu (Dataset S1; Fig. S3). A feeding experiment of moss WT gametophores with IAA, IAA–Glu, and oxIAA revealed that oxIAA–Glu mainly originated from oxidation of IAA–Glu (Dataset S2; Fig. 2c). Nevertheless, oxIAA–Glu also accumulated after feeding with oxIAA (Fig. 2d). Results from the feeding of moss gh3 knockout mutants with oxIAA confirmed that PpGH3.2 catalyzes the conjugation of oxIAA in vivo, as oxIAA conjugation was impaired in the gh3.2 single knockout and gh3.1 gh3.2 double knockout mutants (Dataset S2; Fig. 2d). The conifer P. abies possesses several GH3 proteins that fall within Group II, and we previously characterized four members conjugating IAA (Brunoni et al., 2020). We tested the ability of these recombinant PaGH3s to target oxIAA, and PaGH3.gII.8 and PaGH3.17 were observed to conjugate oxIAA (Dataset S1; Fig. S4). PaGH3.gII.8 conjugated oxIAA exclusively with Asp while PaGH3.17 preferred Glu but also Leu (Dataset S1; Fig. S4). Feeding spruce seedlings with IAA, IAA–Asp, IAA–Glu, and oxIAA showed that oxIAA–Asp and oxIAA–Glu accumulated predominantly after oxIAA treatment (Dataset S2; Fig. 2e,f) and to a minor extent after feeding with IAA–Asp and IAA–Glu, respectively (Dataset S2; Fig. 2e). As spruce mutant lines are unavailable, we chemically inhibited the GH3-catalyzed conjugation step with the GH3 inhibitor kakeimide (KKI; Hayashi et al., 2021; Fukui et al., 2022). KKI was demonstrated to inhibit the synthesis of IAA-amino acid conjugates by binding to the IAA binding site of the GH3·ATP complex to form a ternary complex, which is described to selectively target IAA-conjugating GH3s (Fukui et al., 2022). Brunoni et al. (2020) previously reported that members of the spruce Group II GH3s showed IAA-conjugating activity. This evidence suggested that KKI could inhibit Group II GH3 conjugating activity in spruce seedlings. Co-treatment with oxIAA and KKI of spruce seedlings blocked the formation of oxIAA–Asp and oxIAA–Glu (Dataset S2; Fig. 2f), confirming that oxIAA conjugation is mediated by GH3 enzymes. Our results suggest that, in moss, the oxidative pathway is the main contributor to the formation of the oxIAA-amino acid conjugates, whereas the conjugation of oxIAA to amino acids contributes the least. On the contrary, our findings on spruce confirm that diversification of the homeostatic control of IAA occurred in conifers, as conjugation is the favored mechanism for oxIAA-amino acid conjugate formation. At the same time, the oxidation of IAA-amino acid conjugates is a minor metabolic route. Many decades of research were required to identify the critical genes involved in auxin inactivation. Nonetheless, the mechanisms by which auxin-inactivating enzymes governed auxin metabolism remained fragmented, and these enzymes appear to participate in different catabolic pathways. Recent research discoveries placed the IAA-inactivating enzymes GH3, ILR1, and DAO into a single catabolic route and described it to occur in angiosperms (Hayashi et al., 2021). Here, we undertook a functional analysis of GH3 enzymes and in planta-feeding assays using auxin catabolic intermediates in different genetic backgrounds or upon chemical knockdown of the auxin inactivation pathway to further study the mechanisms regulating IAA homeostasis in the distantly related species, Arabidopsis, P. abies, and P. patens. Our results suggest that, unlike in angiosperms, the GH3-ILR1-DAO regulatory framework does not operate in nonflowering plants. The oxidative inactivation of IAA- and IAA-amino acid conjugates does not significantly maintain IAA homeostasis in spruce and is not mediated by DAO-like enzymes (Brunoni et al., 2020; this study). The GH3-mediated IAA- and oxIAA-amino acid conjugation appear to be this species' primary auxin inactivation strategy (Brunoni et al., 2020; this study). Distinctively, the metabolic fate of IAA and IAA metabolites is more similar between moss and Arabidopsis, as the oxidative pathway contributes the most to the metabolism of IAA-amino acid conjugates. The GH3-mediated conjugation of oxIAA to amino acids is a minor route in moss and Arabidopsis. Nonetheless, the GH3-ILR1-DAO pathway can unlikely regulate IAA metabolism in lower-land plants. DAO-like enzymes evolved specifically with angiosperms, ILL enzymes were not found in the moss P. patens, and the Marchantia polymorpha ILR1 is not employed in auxin conjugate hydrolysis in this liverwort (Campanella et al., 2018, 2019; Brunoni et al., 2020; Takehara et al., 2020), suggesting that yet-to-be-discovered enzymes could be responsible for oxidation and hydrolysis reactions in lower vascular plants. Altogether, the GH3-mediated oxIAA conjugation is a metabolic pathway that occurred early during plant evolution, and its contribution to IAA homeostasis is species-dependent. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (European Regional Development Fund-Project 'Plants as a tool for sustainable global development' no. CZ.02.1.01/0.0/0.0/16_019/0000827), by the Internal Grant of Palacký University Olomouc (IGA_PrF_2022_016) to ON, by the Czech Science Foundation (no. 21-07661S) to DK and by the Palacký University Olomouc Young Researcher grant (JG_2020_001) to MK, and (JG_2020_002) to AŽ. We thank Professor Jutta Ludwig-Müller for providing plasmids carrying PpGH3.1 and PpGH3.2 cds and Adam Klingberg (SkogForsk) for providing spruce seeds. None declared. FB and ON conceived the experiments. FB, AP, AŽ, SC and DK designed the experiments. FB, AP, AŽ, AA and MK performed the experiments. FB wrote the paper with input from all the authors. Dataset S1 Raw mass spectrometry data of indole-3-acetic acid (IAA)- and oxIAA-amino acid conjugates from the bacterial assay. Dataset S2 Raw mass spectrometry data of indole-3-acetic acid metabolites from in vitro enzymatic activity and in planta-feeding assays. Fig. S1 Bacterial assay of oxIAA conjugation by recombinant Arabidopsis GRETCHEN HAGEN 3s. Fig. S2 Levels of oxIAA–Glu in Arabidopsis Col-0 and gh3 sextuple mutant after feeding with indole-3-acetic acid (IAA) metabolites. Fig. S3 Bacterial assay of oxIAA conjugation by recombinant moss GRETCHEN HAGEN 3. Fig. S4 Bacterial assay of oxIAA conjugation by recombinant spruce GRETCHEN HAGEN 3s. Fig. S5 Levels of oxIAA–Glu in spruce seedlings after feeding with indole-3-acetic acid (IAA) metabolites and the GRETCHEN HAGEN 3 inhibitor KKI. Table S1 List of primers used for cloning in this work. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Auxin belongs among major phytohormones and governs multiple aspects of plant growth and development. The establishment of auxin concentration gradients, determines, among other processes, plant organ positioning and growth responses to environmental stimuli. Herein we report the synthesis of new NBD- or DNS-labelled IAA derivatives and the elucidation of their biological activity, fluorescence properties and subcellular accumulation patterns in planta . These novel compounds did not show auxin-like activity, but instead antagonized physiological auxin effects. The DNS-labelled derivatives FL5 and FL6 showed strong anti-auxin activity in roots and hypocotyls, which also occurred at the level of gene transcription as confirmed by quantitative PCR analysis. The auxin antagonism of our derivatives was further demonstrated in vitro using an SPR-based binding assay. The NBD-labelled compound FL4 with the best fluorescence properties proved to be unsuitable to study auxin accumulation patterns in planta . On the other hand, the strongest anti-auxin activity possessing compounds FL5 and FL6 could be useful to study binding mechanisms to auxin receptors and for manipulations of auxin-regulated processes.

Jasmonates (JAs) are a family of oxylipin phytohormones regulating plant development and growth and mediating ‘defense versus growth’ responses. The upstream JA biosynthetic precursor cis -(+)-12-oxo-phytodienoic acid ( cis -OPDA) has been reported to act independently of the COI1-mediated JA signaling in several stress-induced and developmental processes. However, its means of perception and metabolism are only partially understood. Furthermore, cis -OPDA, but not JA, occurs in non-vascular plant species, such as bryophytes, exhibiting specific functions in defense and development. A few years ago, a low abundant isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected in wounded leaves of flowering plants, opening up to the possibility that conjugation of cis -OPDA to amino acids might be a relevant mechanism for cis -OPDA regulation. Here, we extended the analysis of amino acid conjugates of cis -OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp in response to biotic and abiotic stress in Arabidopsis. The newly identified OPDA-amino acid conjugates show cis -OPDA-related plant responses in a JAR1-dependent manner. We also discovered that the synthesis and hydrolysis of cis -OPDA amino acid conjugates are regulated by members of the amidosynthetase GH3 and the amidohydrolase ILR1/ILL families. Finally, we found that the cis -OPDA conjugative pathway already functions in non-vascular plants and gymnosperms. Thus, one level of regulation by which plants modulate cis -OPDA homeostasis is the synthesis and hydrolysis of OPDA-amino acid conjugates, which temporarily store cis -OPDA in stress responses.### Competing Interest StatementThe authors have declared no competing interest.

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions, similar to other phytohormones. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic–lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels reached a maximum of pmol/g. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Current synthetic and analytical methodologies support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.

Trees are long-lived organisms with complex life cycles that provide enormous benefits both in natural and cultivated stands [...].

Recalcitrant adventitious root (AR) development is a major hurdle in propagating commercially important woody plants. Although significant progress has been made to identify genes involved in subsequent steps of AR development, the molecular basis of differences in apparent recalcitrance to form AR between easy-to-root and difficult-to-root genotypes remains unknown. To address this, we generated cambium tissue-specific transcriptomic data from stem cuttings of hybrid aspen, T89 (difficult-to-root) and hybrid poplar OP42 (easy-to-root) and used transgenic approaches to verify the role of several transcription factors (TF) in the control of adventitious rooting. Increased peroxidase activity is positively correlated with better rooting. We found differentially expressed genes encoding Reactive Oxygen Species (ROS) scavenging proteins to be enriched in OP42 compared to T89. A higher number of differentially expressed TF in OP42 compared to T89 cambium cells was revealed by a more intense transcriptional reprograming in the former. PtMYC2 , a potential negative regulator, was less expressed in OP42 compared to T89. Using transgenic approaches, we have demonstrated that PttARF17.1 and PttMYC2.1 negatively regulate adventitious rooting. Our results provide insights into the molecular basis of genotypic differences in AR and implicate differential expression of the master regulator MYC2 as a critical player in this process.

Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth.

Abstract Background Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. Results A high-throughput sample preparation method for liquid chromatography–tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. Conclusion The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.

During plant development, a precise balance of cytokinin is crucial for correct growth and patterning, but it remains unclear how this is achieved across different cell types and in the context of a growing organ. Here we show that in the root apical meristem, the TMO5/LHW complex increases active cytokinin levels via two cooperatively acting enzymes. By profiling the transcriptomic changes of increased cytokinin at single-cell level, we further show that this effect is counteracted by a tissue-specific increase in CYTOKININ OXIDASE 3 expression via direct activation of the mobile transcription factor SHORTROOT. In summary, we show that within the root meristem, xylem cells act as a local organizer of vascular development by non-autonomously regulating cytokinin levels in neighbouring procambium cells via sequential induction and repression modules.

The aim of this study was to investigate the action spectrum of two urea derivatives, the 1,3-di(benzo[ d ]oxazol-5-yl)urea (5-BDPU) and the 1,3-di(benzo[ d ]oxazol-6-yl)urea (6-BDPU). In order to evaluate a possible adjuvant activity on cytokinins the compounds alone or in the simultaneous presence of different cytokinins were assayed either on in vitro typical cytokinin-related bioassays, or on in planta interaction with cytokinin signal transduction pathway. The compounds ability to activate the cytokinin receptor CRE1/AHK4 was studied either by a heterologous bacterial assay or by a competitive binding assay and docking simulations were performed with the crystal structure of the same receptor. Then, owing to their chemical structure which resembles that of urea-type cytokinins, the ability of 5- and 6-BDPU to inhibit the activity of cytokinin oxidase/dehydrogenase of Zea mays (ZmCKX1) was investigated and docking simulations were performed as well. Accordingly to the experimental results, we speculate that BDPUs could show a dual activity: the blocking of the conformational re-adaption of CRE1/AHK4 receptor maintaining the cytokinin inside its binding pocket, thus possibly enhancing its kinase action; the inhibition of cytokinin oxidase/dehydrogenase activity thus possibly preventing its cleavage of natural cytokinins with isoprenoid side chain. Graphic abstract

Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

Vegetative propagation relies on the capacity of plants to regenerate de novo adventitious roots (ARs), a quantitative trait controlled by the interaction of endogenous factors, such as hormones and environmental cues among which light plays a central role. However, the physiological and molecular components mediating light cues during AR initiation (ARI) remain largely elusive. Here, we explored the role of red light (RL) on ARI in de-rooted Norway spruce seedlings. We combined investigation of hormone metabolism and gene expression analysis to identify potential signaling pathways. We also performed extensive anatomical characterization to investigate ARI at the cellular level. We showed that in contrast to white light, red light promoted ARI likely by reducing jasmonate (JA) and JA-isoleucine biosynthesis and repressing the accumulation of isopentyl-adenine-type cytokinins. We demonstrated that exogenously applied JA and/or CK inhibit ARI in a dose-dependent manner and found that they possibly act in the same pathway. The negative effect of JA on ARI was confirmed at the histological level. We showed that JA represses the early events of ARI. In conclusion, RL promotes ARI by repressing the accumulation of the wound-induced phytohormones JA and CK.

Vegetative propagation relies on the capacity of plants to regenerate adventitious roots (ARs) de novo. Light plays a central role in this process; however the physiological and molecular components mediating light cues remain largely elusive, especially in conifers. Here, we explore the effect of light spectral quality on AR initiation (ARI) in de-rooted Norway spruce seedlings. We used light emitting diodes (LEDs) to study the effects of different light spectra on ARI, hormone metabolism and gene expression. We used sensitive mass spectrometry-based methods, and, since the Norway spruce genome sequence recently became available, we coupled this to gene expression analysis. We performed extensive anatomical characterization to investigate ARI at the cellular level. We showed that in contrast to constant white light (WL) and constant blue light (cBL), constant red light (cRL) promoted ARI by reducing jasmonate (JA) and JA-isoleucine contents and repressing the accumulation of isopentyl-adenine-type cytokinins and abscisic acid, which are known to inhibit AR development. We found that JA and CKs possibly act in the same pathway to repress ARI. Anatomical analysis showed that JA represses the early events of ARI. In conclusion, cRL promotes ARI by repressing the accumulation of the wound-induced phytohormones JA and CK.

Background Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC-ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein. Conclusions This new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.