Roman Skokan

The evolution of indoleacetic acid (IAA) into the plant hormone auxin remains unclear. Here, we compared IAA response and metabolism in land plants and their closest living relatives, the streptophyte algae, using chemical treatments, phytohormone profiling, and targeted and untargeted metabolomics. Streptophyte algae showed neither plant-like auxin responses nor canonical IAA metabolism. Here, we demonstrate IAA side-chain decarboxylation as a novel pathway of cellular IAA metabolism, operating in streptophyte algae and land plants alike. Decarboxylation also underpinned IAA’s innate photolability and yielded cytotoxic intermediates, which also accumulated endogenously under simulated sunlight. Together, these findings establish IAA decarboxylation as a major, conserved metabolic pathway, and suggest that the metabolic constraints arising from IAA’s instability and indirect cytotoxicity contributed to its adoption as a plant hormone.

The exocyst is a conserved octameric vesicle-tethering complex essential for targeted secretion. It is organized into two modules (I: SEC3, SEC5, SEC6, SEC8; II: SEC10, SEC15, EXO70, EXO84). In plants, the module II subunits SEC15, EXO84, and especially EXO70 have diversified into multiple subfamilies, yet the evolutionary origins and functional consequences of this diversification remain unclear. Here we reconstruct exocyst evolution across streptophytes using phylogenomic, functional complementation, and structural modeling analyses. We show that the three major EXO70 subfamilies originated in anydrophytes - the common ancestor of Zygnematophyceae and land plants - indicating that EXO70 diversification had begun at the dawn of plant terrestrialization. Complementation of Arabidopsis exo70 mutants with EXO70 paralogs from the liverwort Marchantia polymorpha and the streptophyte alga Klebsormidium nitens demonstrates that the ancestral canonical function is retained in the EXO70.1 lineage, whereas other subfamilies have undergone substantial functional specialization. We further uncover an evolutionary shift in exocyst membrane targeting: while Klebsormidium SEC3 retains autonomous membrane-recruitment capacity, land-plant SEC3 subunits have lost this ability, rendering exocyst targeting increasingly dependent on EXO70. Together, these findings suggest that early EXO70 diversification, combined with the redistribution of membrane-targeting functions within the exocyst, enabled paralog-specific exocyst recruitment and facilitated the emergence of specialized secretion pathways during plant terrestrialization.

The resolution of the 3D structure of PIN proteins and the ability to universally predict protein structures using AlphaFold allowed us to reconsider the classification of Auxin efflux carriers within the BART superfamily, and we propose to merge members of this superfamily, possessing a characteristic fold with a helical crossover, with other families carrying this fold (BASS, NhaA, CPA and others) into a newly proposed superfamily named X-Helices Carriers (XHC). We further demonstrate a profound divergence between PIN and PILS proteins and monophyly of eukaryotic PINs with the animal cholesterol receptor GPR155/LYCHOS. We hypothesise that its signalling capacity is derived from a general feature of PIN proteins to interact with membrane lipids within the core membrane fold. Finally, we discuss the auxin-transport ability of PIN and PILS proteins in the context of the described properties of bacterial homologs that act as organic acid permeases. Disclaimer This version is a draft and it will be updated during the next few weeks. Highlights PINs are in plants and fragmentary in many algae and protists, but never in fungi Eukaryotic PINs are monophyletic together with animal cholesterol receptor GPR155 PILS are frequently present in protists and in all fungi, but not in animals PINs/PILS split is occurred very early in Prokaryota AECs belongs to proposed superfamily of transporter with helical crossover

The genomes of charophyte green algae, close relatives of land plants, typically do not show signs of developmental regulation by phytohormones. However, scattered reports of endogenous phytohormone production in these organisms exist. We performed a comprehensive analysis of multiple phytohormones in Viridiplantae, focusing mainly on charophytes. We show that auxin, salicylic acid, ethylene and tRNA-derived cytokinins including cis-zeatin are found ubiquitously in Viridiplantae. By contrast, land plants but not green algae contain the trans-zeatin type cytokinins as well as auxin and cytokinin conjugates. Charophytes occasionally produce jasmonates and abscisic acid, whereas the latter is detected consistently in land plants. Several phytohormones are excreted into the culture medium, including auxin by charophytes and cytokinins and salicylic acid by Viridiplantae in general. We note that the conservation of phytohormone biosynthesis and signaling pathways known from angiosperms does not match the capacity for phytohormone biosynthesis in Viridiplantae. Our phylogenetically guided analysis of established algal cultures provides an important insight into phytohormone biosynthesis and metabolism across Streptophyta.

Multiple phytohormones act as conserved developmental regulators in land plants. Although the closely related streptophyte green algae typically lack full complements of molecular pathways underlying these responses, scattered reports of endogenous phytohormone production in these organisms exist. In this study, we performed a detailed LC/MS-based analysis of several phytohormones, their precursors and metabolites in all lineages of streptophyte algae. We also included chlorophyte algae and early-diverging land plants as outgroups. Free auxin, tRNA-derived cytokinins and certain phenolics including salicylic acid were found ubiquitously. However, land plants differed from green algae by the consistent detection of abscisic acid and the presence of auxin and cytokinin conjugates and trans -zeatin, supporting the hypotheses that these three phytohormones likely came to regulate development in the ancestral land plant. By contrast, we observed a patchy distribution of jasmonates among streptophytes. We additionaly analyzed the corresponding culture and empty media to account for phytohormone excretion and environmental contamination. Extracellular auxins and cytokinins were frequently detected, while agar constituted a major external source of phenolic compounds. We provide a highly comprehensive evolution-directed screen of phytohormone compound occurrence and thoroughly discuss our data in the context of current plant hormonomics and phylogenomics. GRAPHICAL

Auxin, represented by indole-3-acetic acid (IAA), has for a long time been studied mainly with respect to the development of land plants, and recent evidence confirms that canonical nuclear auxin signaling is a land plant apomorphy. Increasing sequential and physiological data show that the presence of auxin transport machinery pre-dates the emergence of canonical signaling. In this review, we summarize the present state of knowledge regarding the origins of auxin transport in the green lineage (Viridiplantae), integrating both data from wet lab experiments and sequence evidence on the presence of PIN-FORMED (PIN), PIN-LIKES (PILS), and AUXIN RESISTANT 1/LIKE-AUX1 (AUX1/LAX) homologs. We discuss a high divergence of auxin carrier homologs among algal lineages and emphasize the urgent need for the establishment of good molecular biology models from within the streptophyte green algae. We further postulate and discuss two hypotheses for the ancestral role of auxin in the green lineage. First, auxin was present as a by-product of cell metabolism and the evolution of its transport was stimulated by the need for IAA sequestration and cell detoxification. Second, auxin was primarily a signaling compound, possibly of bacterial origin, and its activity in the pre-plant green algae was a consequence of long-term co-existence with bacteria in shared ecological consortia.

PIN-FORMED (PIN) transporters mediate directional, intercellular movement of the phytohormone auxin in land plants. To elucidate the evolutionary origins of this developmentally crucial mechanism, we analysed the single PIN homologue of a simple green alga Klebsormidium flaccidum. KfPIN functions as a plasma membrane-localized auxin exporter in land plants and heterologous models. While its role in algae remains unclear, PIN-driven auxin export is probably an ancient and conserved trait within streptophytes.

Coordination of plant development requires modulation of growth responses that are under control of the phytohormone auxin. PIN-FORMED plasma membrane proteins, involved in intercellular transport of the growth regulator, are key to the transmission of such auxin signals and subject to multilevel surveillance mechanisms, including reversible post-translational modifications. Apart from well-studied PIN protein modifications, namely phosphorylation and ubiquitylation, no further post-translational modifications have been described so far. Here, we focused on root-specific Arabidopsis PIN2 and explored functional implications of two evolutionary conserved cysteines, by a combination of in silico and molecular approaches. PIN2 sequence alignments and modeling predictions indicated that both cysteines are facing the cytoplasm and therefore would be accessible to redox status-controlled modifications. Notably, mutant pin2^C−A alleles retained functionality, demonstrated by their ability to almost completely rescue defects of a pin2 null allele, whereas high resolution analysis of pin2^C−A localization revealed increased intracellular accumulation, and altered protein distribution within plasma membrane micro-domains. The observed effects of cysteine replacements on root growth and PIN2 localization are consistent with a model in which redox status-dependent cysteine modifications participate in the regulation of PIN2 mobility, thereby fine-tuning polar auxin transport.