Plants must cope with ever-changing and sometimes adverse environmental conditions. To deal with these circumstances, they have evolved a wealth of developmental solutions to shape their body plan and optimize their metabolism according to the given environmental demands. First and foremost, such responses are based on and also witness the remarkable developmental flexibility of plants, including permanent meristematic activity, de novo organogenesis, and enormous capacity for regeneration. However, coordinated plant growth and development, as well as appropriate plant stress responses, require efficient communication not only of single cells, but of whole tissues and plant organs. As in animals, plants use a limited number of hormonal compounds to accomplish this task. To date, we know that the plant life cycle, from germination until reproduction, is controlled by a highly complex network of low-abundance signaling molecules, called phytohormones. The existence of such compounds has already been proposed more than 125 years ago by the famous plant physiologists Charles Darwin (1880) and Julius von Sachs (1887). Already back then, they have been convinced that coordinated morphogenesis and functionality of multicellular organisms must rely on transmissible signals that are transported within the plant cormus.

The function of plant hormones has traditionally been categorized as growth-promoting or growth-inhibiting, although our current understanding is much more detailed and complex. Today, we know that plant hormones act in a combinatorial manner to trigger a multitude of different responses that depend not only on the perceived stimulus of a single plant hormone but also on the interplay and crosstalk of the different phytohormones and on the specific molecular properties of the responding tissue. Among growth-promoting phytohormones, the class of auxins, and here in particular indole-3-acetic acid (IAA), as the major naturally occurring auxin, has long been in the focus of our work. IAA is recognized to be involved in virtually all aspects of plant growth and differentiation, for example, in the promotion of shoot elongation, the induction of cambial cell division, the maintenance of apical dominance, and the induction of lateral and adventitious root formation. In recent decades, evidence has been accumulated that emphasizes the physiological importance of auxins in the context of coordinating plant development and describes the molecular mode of auxin action. IAA constitutes a rather simple molecule, sharing major structural features with the proteinogenic amino acid L-tryptophan. However, the biosynthesis of IAA remained elusive for a very long time, until the laboratories of Yunde Zhao and José Alonso disclosed the main auxin biosynthesis pathway, which proceeds from L-Trp through indole-3-pyruvic acid (IPyA) to IAA. However, further biochemical analysis provided evidence for the existence of a small number of alternative pathways, each of them designated for an intermediate that is a hallmark of the pathway (Fig. 1).

Figure 1. Proposed anabolic routes for IAA biosynthesis in Arabidopsis. Dashed lines represent assumed reaction steps for which the corresponding genes/enzymes have not yet been identified. AMI1, AMIDASE 1; CYP71A13, CYTOCHROME P450 MONOOXYGENASE 71A13; CYP79B2/B3, CYTOCHROME P450 MONOOXYGENASE 79B2/B3; IAA, indole-3-acetic acid; IAAld, indole-3-acetaldehyde; IAM, indole-3-acetamide; IAN, indole-3-acetonitrile; IAOx, indole-3-acetaldoxime; IGs, indole glucosinolates; IPyA, indole-3-pyruvic acid; L-Trp, L-tryptophan; MYR, MYROSINASE; NIT1-3, NITRILASE 1-3; PAD3 PHYTOALEXIN DEFICIENT 3 (CYTOCHROME P450 MONOOXYGENASE 71B15); SUR1, SUPERROOT 1 (S-ALKYLTHIOHYDROXYMATE LYASE); SUR2, SUPERROOT 2 (CYTOCHROME P450 MONOOXYGENASE 83B1); TAA1, TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1; TAR, TRYPTOPHAN AMINOTRANSFERASE RELATED; TDC, TRYPTOPHAN DECARBOXYLASE; TRA, tryptamine; UGT74B1, UDP-GLUCOSYL TRANSFERASE 74B1. (Pérez-Alonso et al., 2021 J. Exp. Bot.)

Currently, four Trp-dependent and one Trp-independent pathways have been proposed for auxin biosynthesis. The latter are the indole-3-acetaldoxime (IAOx) pathway, the indole-3-acetamide (IAM) pathway, the IPyA pathway, and the tryptamine (TAM) pathway. To date, only the IPyA pathway has been fully disclosed with respect to the catalyzed reaction steps and the enzymes involved. Due to obvious gaps in the other pathways, functional redundancy, and tissue and plant-specific variations in the expression patterns of the identified components, the relevance of each of these pathways is still difficult to assess.

The work of our laboratory provided evidence that led to the suggestion that one route of auxin formation takes its course through the intermediate IAM, a compound that has been proven to be endogenous to Arabidopsis and several other plant species. In the following, we succeeded in identifying and characterizing the first plant IAM hydrolase (AMI1) from Arabidopsis, capable of catalyzing the conversion of IAM to IAA (for review see: Pollmann et al., 2006 Plant Biol.; Lehmann et al., 2010 Eur. J. Cell Biol.).

AMI1 is located in the cytoplasm, assumed to be the main locus of IAA biosynthesis. Judging by its primary amino acid composition and homology to other well-characterized enzymes, AMI1 is considered a member of the amidase signature family that comprises enzymes that can be found widespread in nature, catalyzing a diverse range of different reactions (for a review, see: Moya-Cuevas et al., 2021 Biomolecules). To date, more than 20 AMI1-like proteins have been identified from monocot and dicot plant species (Mano et al., 2010 J. Exp. Bot.; Lehmann et al., 2010 Eur. J. Cell Biol.), suggesting a conserved and likely important function of AMI1-like enzymes. Just recently, our lab was able to demonstrate that IAM is capable of driving the expression of a subgroup of genes that differs from those that respond to IAA (Fig. 2).

Figure 2. Transcriptomics analysis of differentially expressed genes (DEGs) in IAM versus mock treated ami1 mutant seedlings compared to IAA versus mock treated wild-type Arabidopsis seedlings (GSE631) (Okushima et al., 2005 Plant J.). (A) Venn diagram of differentially regulated genes applying a significance threshold of q < 0.05 and a log2FC = ±1.75. The two compared datasets share only a minor number of common transcripts. (B) Bar plots of GO biological function enrichment analysis of the non-overlapping DEGs under the two compared conditions. Color and length of the bars indicate the significance of the identified GO terms. The color code used to differentiate the significance levels is given at the bottom of the figure. The bars only show terms with a significance score –log10(q-value). (Ortiz-García et al., 2022 Front. Plant Sci.)

Our focus is on elucidating the role of AMI1-mediated IAA formation and how AMI1 integrates into the already deciphered framework of auxin biosynthesis in plants. Furthermore, we are interested in the regulation of auxin biosynthesis. As shown in Figure 3, recent work of our lab identified IAM as a novel molecular hub that connects the biosynthesis pathways of IAA and abscisic acid (ABA) and triggers growth repression through the regulation of a number of transcription factors in Arabidopsis (Pérez-Alonso et al., 2021 J. Exp. Bot.; Sánchez-Parra et al., 2021 Int. J. Mol. Sci.; Ortiz-García et al., 2022).

Figure 3. A model summarizing the IAM accumulation-mediated transcriptional activation of MYB74. Abiotic stresses, including osmotic stress, suppress the expression of AMI1, which translates into an accumulation of IAM. The auxin precursor IAM triggers ABA biosynthesis. We demonstrated an ABA dependent and an ABA independent transcriptional activation of abiotic stress-related TF MYB74. IAM directly induces the expression of MYB74. The IAM-mediated accumulation of MYB74 results in the transcriptional reprogramming of many osmotic stress-related genes, including further MYB factors, such as the dehydration stress memory gene MYB47 and the abiotic stress-related genes MYB90 and MYB102, the ethylene response factor ERF53, the basic helix–loop–helix factor NIG1, and the ABA-responsive heat shock response factor HSFA6a. Consequently, MYB74 is assumed to integrate ABA dependent and independent signals and to be involved in plant responses to osmotic stress. At the time, the accumulation of MYB74 suppresses plant growth considerably, possibly through the transcriptional activation of MYB11, which is known to be capable of delaying plant development, and the repression of MYB77 expression. MYB77 is involved in the modulation of auxin signal transduction and the control of lateral root formation. (Ortiz-García et al., 2022 Front. Plant Sci.)

A second major research line of our group is concerned with the investigation of molecular mechanisms triggered by beneficial bacterial and fungal root-colonizing endophytes that promote plant biomass production and improve plant stress tolerance, paying special attention to the signaling networks involved (Pérez-Alonso et al., 2020 J. Exp. Bot.). So far, our focus has been on fungal endophytes that were collected under extreme environmental conditions, including the Antarctic, the Atacama Desert in Chile, and the Thar Desert in India. Over the years, we contributed to studies that demonstrated that the endophytes isolated from plants that learned to survive under those harsh environmental conditions are capable of promoting plant growth and productivity of a broad range of relevant crops under unfavorable environmental conditions (Ramos et al., 2018 Fungal Ecol.; Barrera et al., 2020 Front. Ecol. Evol.; Morales-Quintana et al., 2021 Plant Physiol. Biochem.; Morales-Quintana et al., 2022 Front. Microbiol.). However, we are not only working on the plant side, but also dedicate part of our work on the microbial partner, trying to understand how the microbial partners acts in the studied mutual interaction with its host plant and what role they play in conferring increased stress tolerance to the plant (Lanza et al., 2019 Environm. Microbiol.; Conchillo et al., 2021 Front. Ecol. Evol.). Just recently, our lab disclosed the Ca²⁺ sensor CBL7 as an important modulator of plant–microbe interactions in Arabidopsis, involved in controlling potassium fluxes in the host plant and in adjusting plant defense mechanisms to allow the endophyte to proliferate in the root to a certain extend (Fig. 4). 

Figure 4. Transcriptional analysis of Arabidopsis seedlings co-cultivated with Serendipita indica. (A) Venn diagram showing the numbers of differentially expressed genes in Arabidopsis plants 2‐ and 10‐days post infection with S. indica compared to control plants that were mock infected. (B) ClueGo analysis of induced DEGs. The figure shows the representative molecular function interaction among the targets. (C) qPCR analysis of transcriptional responses of identified target genes in plants that were co-cultivated for 2 and 10 days, respectively, with S. indica compared to mock treated plants. The data represent means ± SE (n = 3). DEGs, differentially expressed genes; dpi, days post infection. (Pérez-Alonso et al., 2022 Plant Cell Environ.)

To tackle our objectives, we use a combination of different complementing methodologies, including transcriptomics (RNAseq), metagenomics (ITS/16S amplicon sequencing), genetics (GWAS) and reverse genetics (mutant studies), general molecular biological, cell biological (confocal laser scanning microscopy), and mass spectrometric techniques (metabolomics).