Katarina Kurtović

Endogenous biological rhythms are central to temporal organization in land plants, yet their evolutionary origins in streptophyte algae remain unresolved. We investigated whether rhythmic morpho‐physiological processes in Chara braunii are consistent with endogenous regulation and how continuous light intensity modulates these responses. In C. braunii , time‐lapse imaging, pigment quantification, chlorophyll fluorescence measurements, and metabolomic profiling were conducted under continuous low (dim) light and continuous higher (regular) light conditions. Circadian oscillations in branchlet angle persisted under continuous dim light but were attenuated under continuous regular light, whereas internodal elongation showed only limited temporal structure. Pigment and metabolite measurements, obtained once per day under two different continuous light conditions, revealed intensity‐dependent physiological adjustment, including changes in chlorophyll and carotenoid contents and altered accumulation of tricarboxylic acid cycle intermediates such as malate and fumarate. Chlorophyll fluorescence parameters showed no detectable temporal variation under the applied sampling scheme. Comparative genomic analyses identified putative homologs of several canonical land‐plant clock‐associated components, whereas canonical TOC1 and ZTL orthologs were not detected. Together, these results are consistent with the presence of endogenous regulatory components governing rhythmic outputs in C. braunii , while highlighting irradiance as a major modulator capable of suppressing these outputs. The parallel attenuation of rhythmic outputs and the shift toward a stress‐associated metabolic profile under higher irradiance suggest that higher irradiance affects both endogenous regulatory outputs and physiological acclimation responses, although whether these processes are causally linked remains to be determined.

Auxin, primarily indole‐3‐acetic acid (IAA), is a central regulator of growth and development in land plants, but its physiological role in chlorophyte algae remains unclear. Here, we show that exogenous IAA modulates growth in Chlorella sorokiniana , Chlorella variabilis , and Chlamydomonas reinhardtii in a concentration‐dependent manner. Low IAA concentrations promoted growth by accelerating the onset of cell division without affecting cell size, whereas higher concentrations inhibited proliferation. Radiotracer assays showed that all three species take up and release IAA across the plasma membrane through a combination of passive diffusion and energy‐dependent, saturable processes. Competition by excess unlabeled natural and synthetic auxins further supported the presence of carrier‐mediated transport with broad substrate recognition. Phylogenetic analyses identified potential PIN‐like auxin exporters in chlorophytes and other non‐plant eukaryotes, and structural modeling supported conservation of the overall PIN fold and predicted auxin‐binding residues. However, functional assays in Xenopus laevis oocytes, tobacco BY‐2 cultured cells, and Arabidopsis thaliana did not support a role for these proteins in directional auxin export. Instead, non‐plant PIN homologs localized predominantly to the endoplasmic reticulum and showed limited or no transport activity in heterologous systems. Together, these findings indicate that auxin responsiveness and basic cellular auxin transport predate canonical PIN‐mediated directional auxin export, which appears to be a later innovation of the streptophyte lineage.

Summary: Auxin, indole‐3‐acetic acid (IAA), is a key phytohormone with diverse morphogenic roles in land plants, but its function and transport mechanisms in algae remain poorly understood. We therefore aimed to explore the role of IAA in a complex, streptophyte algae Chara braunii. Here, we described novel responses of C. braunii to IAA and characterized two homologs of PIN auxin efflux carriers: CbPINa and CbPINc. We determined their localization in C. braunii using epitope‐specific antibodies and tested their function in heterologous land plant models. Further, using phosphoproteomic analysis, we identified IAA‐induced phosphorylation events. The thallus regeneration assay showed that IAA promotes thallus elongation and side branch development. Immunolocalization of CbPINa and CbPINc confirmed their presence on the plasma membrane of vegetative and generative cells of C. braunii. However, functional assays in tobacco BY‐2 cells demonstrated that CbPINa affects auxin transport, whereas CbPINc does not. The IAA is effective in the acceleration of cytoplasmic streaming and the phosphorylation of evolutionary conserved targets such as homolog of RAF‐like kinase. These findings suggest that, although canonical PIN‐mediated auxin transport mechanisms might not be fully conserved in Chara, IAA is involved in morphogenesis and fast signaling processes.

The role of auxin indole-3-acetic acid (IAA), a phytohormone with numerous morphogenic functions, is now well-established in land plants. Although the role of IAA and its transport in algae remains unclear, PIN-driven auxin export is probably an ancient and conserved trait within Streptophytes. Among streptophyte algae, Chara represents a genus with a considerable degree of complexity in body arrangement. This study investigates the auxin response and characterizes homologs of land plant PIN auxin efflux carriers in Chara braunii . Through regeneration experiments, we observed that IAA significantly promotes elongation of thallus and side branch development upon thallus tip decapitation, indicating an effect on morphogenesis. We show that IAA is actively uptaken and metabolized by thallus cells and that this process is influenced by N-1-naphthylphthalamic acid (NPA). To elucidate the underlying mechanisms, we cloned and sequenced the most expressed Chara braunii PINs, CbPINa and CbPINc. Using epitope-specific antibodies, we showed their presence in the plasma membrane (PM) of vegetative internodal cells and generative antheridial cells. Functional tests in tobacco BY-2 cells, supported by in silico docking of IAA and NPA to CbPINa and CbPINc, showed that PM-localized CbPINa interferes with auxin transport in contrast to ER-localized CbPINc. Finally, our phosphoproteome analysis indicated that IAA rapidly induces specific phosphorylation events, including RAF-like kinase phosphorylation, highlighting a potential role for IAA in fast signaling processes in Chara braunii. Altogether, we provide new insights into IAA role in Chara braunii morphogenesis and suggest that while the canonical auxin transport mechanism may not be conserved, auxin still may likely play a role in rapid signaling pathways in this close relative of land plants.

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.

Chara has been used as a model for decades in the field of plant physiology, enabling the investigation of fundamental physiological processes. In electrophysiological studies, Chara has been utilized thanks to its large internodal cells that can be easily manipulated. Additionally, Chara played a pioneering role in elucidating the presence and function of the cytoskeleton in cytoplasmic streaming, predating similar findings in terrestrial plants. Its representation considerably declined following the establishment and routine application of genetic transformation techniques in Arabidopsis. Nevertheless, the recent surge in evo-devo studies can be attributed to the whole genome sequencing of the Chara braunii, which has shed light on ancestral traits prevalent in land plants. Surprisingly, the Chara braunii genome encompasses numerous genes that were previously regarded as exclusive to land plants, suggesting their acquisition prior to the colonization of terrestrial habitats. This review summarizes the established methods used to study Chara, while incorporating recent molecular data, to showcase its renewed importance as a model organism in advancing plant evolutionary developmental biology.

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

The present study was conducted to establish a protocol for the regeneration of virus-free garlic plants through somatic embryogenesis of two Croatian garlic ecotypes. Basal parts of cloves from mother plants were cultured on a full Murashige and Skoog (MS) or modified MS medium (¼ of KNO₃ and NH₄NO₃ and 2xMgSO₄) containing 0.1 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) or 1 mg L⁻¹ 2,4-D + 0.5 mg L⁻¹ kinetin (Kin) and representing four different treatments. Plants were regenerated in MS medium containing 0.1 mg L⁻¹ 2,4-D and rooted in a medium containing 0.05 mg L⁻¹ 1-naphthaleneacetic acid (NAA) + 0.005 mg L⁻¹ 6-(γ,γ-dimethylallylamino)purine (2iP). The presence of viruses (i.e., sanitary status) of the mother plants and regenerants was checked by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The mother plants were infected with onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV). In addition, the presence of garlic common latent virus (GCLV) was confirmed in four mother plants. Embryogenic callus developed in all four treatments with success ranging from 55% to 81% depending on treatment and ecotype. Plant conversion was significantly higher in somatic embryos developed in media containing 0.1 mg L⁻¹ 2,4-D than those developed in media containing 1 mg L⁻¹ 2,4-D + 0.5 mg L⁻¹ Kin. Virus elimination success ranged from 13.3% up to 62.5% depending on garlic ecotype and treatment. The overall rate of virus elimination by somatic embryogenesis for both treatments and ecotypes were 20.7%, 22.9%, and 30.5% for OYDV, GCLV, and LYSV, respectively. Based on these results, somatic embryogenesis has been shown to be equally or more successful in eliminating garlic viruses compared to other in vitro methods.