Biology subjects

Brunoni, F.

Publications and source records attributed to Brunoni, F..

11 recordsLinked to original sources

bioRxiv2026

Spatially-restricted metabolic delivery of auxin for selective adventitious root induction

Auxin impacts on nearly every aspect of plant growth and development. Its exogenous application therefore results in pleiotropic growth responses. Exploiting this activity for plant propagation requires avoiding or minimizing such off-target effects and is generally achieved as a trade-off between toxicity and organogenetic efficacity. We recently identified the compound HYSPARIN (HYS) with potent, and uniquely selective adventitious root inductive activity. Unlike other root-inducing compounds, HYS preferentially activates auxin responses in the shoot via an unknown mechanism. Here, we show that HYS acts as a shoot-specific proauxin. Rather than acting through auxin homeostasis, we found that HYS is hydrolysed in planta independently of ILR1/ILL amidohydrolases to release the synthetic auxin MCPA. Structure-activity relationship analysis confirmed a strong dependence on its MCPA moiety for activating auxin responses, and identified its promoiety as a determinant of shoot-specificity and activity. Selective application of MCPA also potently induces AR is consistent with a model in which HYS metabolism produces a spatially restricted, AR inductive auxin signal. The activation mechanism of HYS thus provides a conceptual framework for tissue-specific metabolic delivery of auxin and may enable the programmable delivery of other xenobiotics in plants.

plant biology↗

bioRxiv2026

Wild rice Oryza rufipogon outperforms cultivated rice in stimulating beneficial bacterial endophytes.

Beneficial interactions between plants and microorganisms strongly influence plant health and productivity, and root exudates play a central role in shaping these associations. This study analyzed the transcriptional responses of the bacterial endophytes Enterobacter asburiae RCA24 and Kosakonia sacchari RCA25 to root exudates from two commercial Italian rice accessions (Oryza sativa Baldo and Vialone Nano) and from an accession of the wild progenitor of tropical rice, Oryza rufipogon. Bacterial transcriptome analyses revealed that RCA24 responds differently to O. sativa varieties and that RCA25 was more stimulated by O. rufipogon. Changes in bacterial gene expression were mainly related to central metabolism, stress response, and signal transduction, highlighting a precise pattern of interaction. On the other hand, transcriptome analysis of inoculated rice revealed that RCA24 triggered broader transcriptional changes in plants than RCA25. Differentially expressed genes were related, especially in shoots, to defense responses, hormone-mediated signaling, and ribosome biogenesis, revealing that plants discriminate bacterial strains in a genotype-specific manner at the transcriptional level. Our findings suggest that traits beneficial to plant-soil microbiota interactions present in O. rufipogon and lost during domestication and diversification could be identified and reintroduced into modern rice varieties to improve sustainable field performance through beneficial microbial associations.

plant biology↗

bioRxiv2026

Comprehensive characterisation of IAA inactivation pathways reveals the impact of glycosylation on auxin metabolism and plant development

Together with biosynthesis and transport, inactivation regulates the concentration of indole-3-acetic acid (IAA), a key auxinic compound with a myriad of functions in plant development. Main inactive IAA metabolites are categorised into oxidised forms and ester- or amide-linked conjugates. DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) and DAO2, 2-oxoglutarate and iron-dependent dioxygenases, contribute to IAA oxidative inactivation in collaboration with group II GRETCHEN HAGEN3 (GH3) IAA-amido synthetases, while a group of UDP-glycosyltransferases (UGTs) conjugate IAA to sugars. To study the IAA inactivation routes, we generated combinatorial mutants between all group II GH3s (gh3oct) and DAO1 or DAO2, as well as between the DAOs and main UGTs. In vivo [13C6]IAA feeding experiments traced the metabolic fate of the exogenously applied IAA, supporting the main IAA inactivation pathway, in which DAO acts downstream of GH3s. They also indicated that UGT-mediated IAA glycosylation is more important than previously assumed for modulating IAA levels and plant development. Our metabolic and transcriptomic data further revealed that gh3oct may still produce some GH3 activity, explaining previous reported phenotypic inconsistencies. Our data additionally suggest that other not yet identified metabolic activities might play a role in IAA overproducing plants, and that the premature downregulation of flowering time integrators like FLOWERING LOCUS C (FLC) likely underlies the early flowering of gh3oct and gh3oct dao1 plants.

plant biology↗

bioRxiv2024

Phenylacetic acid metabolism in land plants: novel pathways and metabolites

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 (PAA-Leu), phenylacetyl-phenylalanine (PAA-Phe), phenylacetyl-valine (PAA-Val), and phenylacetyl-glucose (PAA-glc). 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.

plant biology↗

bioRxiv2023

Cytokinin Dehydrogenase in Xylem Sap Reveals A Direct Link Between Cytokinin Metabolism and Long-Distance Transport

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.

plant biology↗

bioRxiv2023

Conjugation of cis-OPDA with amino acids is a conserved pathway affecting cis-OPDA homeostasis upon stress responses

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.

plant biology↗

bioRxiv2022

IPT9, a cis-zeatin cytokinin biosynthesis gene, promotes root growth

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 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 on local signaling which does not relate to 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.

plant biology↗

bioRxiv2021

Molecular basis of differential adventitious rooting competence in poplar genotypes

O_LIRecalcitrant 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. C_LIO_LITo 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. C_LIO_LIIncreased 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. C_LIO_LIOur 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. C_LI

plant biology↗

bioRxiv2020

Light controls de novo adventitious root regeneration by modulating jasmonate and cytokinin homeostasis in Norway spruce hypocotyls

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. We explored the effect of light spectral quality on ARI in de-rooted Norway spruce seedlings as well as on hormone metabolism with sensitive mass spectrometry-based methods. We coupled this to gene expression analysis to identify potential signaling pathways and to extensive anatomical characterization to investigate ARI at the cellular level. We showed that in contrast to white light and blue light, red light promoted ARI likely by reducing jasmonate (JA) and JA-isoleucine biosynthesis and repressing the accumulation of isopentyl-adenine-type cytokinins and abscisic acid. We confirmed that exogenously applied JA and/or CK inhibit ARI, and found that they possibly act in the same pathway. The negative effect of JA 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. HighlightBlue and red light have an opposite effect on adventitious root initiation in Norway spruce hypocotyl, red light having a promoting effect by modulating hormone homeostasis.

plant biology↗

bioRxiv2019

Conifers exhibit a characteristic inactivation of auxin to maintain tissue homeostasis

O_LIDynamic regulation of the levels of the natural auxin, indol-3-acetic acid (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 Arabidopsis and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms are still poorly investigated in conifers. To reduce this gap of knowledge, we investigated the different contribution of the IAA inactivation pathways in conifers.\nC_LIO_LIMass spectrometry-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 by a comprehensive phylogenetic analysis including Arabidopsis and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored.\nC_LIO_LICompared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA levels in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. On the other hand, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways.\nC_LIO_LITaken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.\nC_LI

plant biology↗

bioRxiv2019

A bacterial assay for rapid screening of IAA catabolic enzymes

BackgroundPlants 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 OF 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 expressing 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.\n\nResultsIn 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 express 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.\n\nConclusionsThis 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.

plant biology↗