Phytohormone profiles in macroalgae: A comprehensive review of biochemical diversity and plant growth-promoting effects

1. Introduction

The contemporary transition toward sustainable global development paradigms has established macroalgae as ontological pillars of the so-called Blue Bioeconomy, redefining these organisms not merely as fundamental ecological components of coastal habitats, but as highly complex molecular biorefineries (García‐Poza et al., 2022). In an era marked by the urgent need to mitigate climate change and ensure food security for an exponentially growing population, macroalgae represent a renewable biological resource with vast, yet largely untapped, potential in the fields of white and green biotechnology (Ruggeri et al., 2026). The global macroalgal market, valued at over US$10 billion with steady growth projections, reflects an industrial demand that transcends traditional food sectors to venture into the domains of pharmaceuticals, nutraceuticals, and, pre-eminently, precision agriculture (Deepika et al., 2022). However, beyond the richness of structural polysaccharides and high-molecular-weight secondary metabolites, the interest of the international scientific community has recently coalesced around a class of signaling molecules operating at nanomolar concentrations: phytohormones (Rathod et al., 2023). These compounds act as the conductors of algal phenotypic plasticity, coordinating sophisticated physiological responses to dynamic and often extreme environmental stimuli, such as fluctuations in ultraviolet radiation, salinity gradients, and the mechanical stresses of tidal regimes (Dildar et al., 2025; Kaur et al., 2022). In this context, it is worth noting that the term “macroalgae” serves as an ecological and functional descriptor rather than a natural taxonomic classification. Indeed, the three major macroalgal groups are: Phaeophyceae (Brown algae, belonging to the Stramenopiles/Heterokontophyta); Rhodophyta (Red algae) and Chlorophyta (Green algae), both belonging to the Archaeplastida lineage, representing phylogenetically distant evolutionary lineages that evolved independently across distinct eukaryotic kingdoms. This evolutionary divergence underlies the vast diversity in their biochemical profiles and phytohormone biosynthetic pathways. More specific on phylogenetic standpoint, macroalgae do not constitute a monophyletic taxonomic entity but are distributed across three deeply divergent evolutionary lineages Phaeophyceae (brown algae), Rhodophyta (red algae), and Chlorophyta (green algae) which exhibit equally heterogeneous biochemical architectures and hormonal signaling strategies (Liu & Han, 2025; McCoy et al., 2020). The Phaeophyceae, belonging to the Stramenopiles supergroup, represent the dominant segment in the agricultural biostimulant sector, thanks to a biochemical complexity that integrates polyphenols, alginates, and fucoidans into a unique metabolomic synergy (Shukla et al., 2025). Conversely, Rhodophyta offers hormonal profiles that remain partially cryptic, characterized by an extraordinary variety of halogenated metabolites and accessory pigments (La Barre et al., 2010). Finally, Chlorophyta holds critical interest as they are closely related to the progenitors of land plants (Embryophyta), providing an essential evolutionary model for deciphering the origins of hormonal biosynthetic pathways (Donoghue et al., 2021). The question of evolutionary pre-adaptation is central here: evidence that most classical plant hormones, including indole-3-acetic acid (IAA), cytokinins (CKs), and abscisic acid (ABA), are present in macroalgae suggests that the molecular machinery for hormonal synthesis and perception emerged in the aquatic environment long before the colonization of terrestrial land (Gu et al., 2025; Shanab & Shalaby, 2021). This ancestral chemical language allowed autotrophic organisms to orchestrate the evolutionary transition toward environments characterized by lower water availability, laying the groundwork for the structural complexity of vascular plants (Dadras et al., 2025). The nexus between algal biochemistry and modern agriculture is most vigorously manifested in the concept of sustainable intensification (Xie et al., 2019). The need to increase agricultural yields while minimizing the use of synthetic chemical inputs whose environmental costs, including eutrophication and soil degradation, have become unsustainable has driven research toward the use of algal-derived biostimulants (Ercan et al., 2026). These extracts do not operate as fertilizers in the traditional sense; instead, they act as exogenous elicitors capable of reprogramming the hormonal homeostasis and the transcriptome of the host plant (Razzaq et al., 2025). The application of extracts from species such as Ascophyllum nodosum or Ecklonia maxima induces a cascade of signaling events, including the remodeling of root system architecture (RSA), the optimization of nutrient use efficiency (NUE), and the activation of priming responses toward abiotic stresses (Kumari et al., 2023). However, despite empirical successes, the scientific community faces a molecular black box. The intrinsic complexity of the algal matrix, rich in viscous polymers and polyphenolic compounds that interfere with biochemical analyses, has historically hindered the precise quantification of hormonal profiles (Bermudez et al., 2024). Only recently, with the advent of high-resolution analytical techniques such as ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), has it been possible to rigorously map the endogenous hormonal content of macroalgae, revealing a seasonal and ontogenetic variability that challenges any attempt at industrial standardization (Wang et al., 2020a). Beyond classical growth regulators, the current frontier of research is exploring the role of emerging phytohormones, such as brassinosteroids (BR), jasmonates, salicylic acid (SA), and strigolactones (Ali et al., 2024). These molecules, often overlooked in past algal models, are proving crucial in modulating biotic interactions and acquired systemic resistance (Lang et al., 2024). Of particular relevance is the role of these compounds in cross-kingdom signaling the biochemical communication between macroalgae and their associated microbiome, both in the original marine environment and in the rhizosphere of the treated terrestrial plant (Prisa et al., 2026a). It is hypothesized that the efficacy of an algal extract does not reside in a single component but in a metabolic synergy where phytohormones operate in concert with low-molecular-weight polysaccharides and protective osmolytes (Gòrka et al., 2018). This systemic vision requires a shift in scientific focus: that is, from simple chemical identification to the understanding of signal transduction networks. Despite the abundance of phenotypic studies, significant knowledge gaps persist that prevent the full realization of macroalgal potential (Moreira et al., 2022). For instance, a detailed understanding of the mechanisms of polar transport of phytohormones in multicellular algae and the hormonal receptors, which, in many algal lineages, appear to differ structurally from angiosperm models, is still lacking (Schmidt et al., 2024). Furthermore, the impact of extraction processes (acid or alkaline hydrolysis, microwave-assisted extraction [MAE], or ultrasound-assisted extraction [UAE]) on the stability and bioactivity of hormonal profiles remains an area of intense technical debate (Walayat et al., 2024). The need for a critical synthesis that integrates metabolomic, transcriptomic, and applicative data has never been more pressing. This review, therefore, aims to provide an exhaustive and rigorously updated state-of-the-art analysis regarding phytohormone profiles in macroalgae. Through an examination ranging from the biochemical diversity of different lineages to the critical estimation of biosynthetic pathways comparing tryptophan-dependent and independent pathways for auxin and de novo pathways for CKs, this work intends to consolidate fragmented knowledge into a coherent theoretical framework. To ensure transparency and comprehensiveness, the literature compiled in this synthesis was selected through a systematic query of major scientific databases, including Scopus, Web of Science, and PubMed, primarily focusing on peer-reviewed articles, books, and conference proceedings published between 2000 and 2026. Search criteria targeted keywords, such as “macroalgae,” “phytohormones,” “auxins,” “cytokinins,” and “plant growth promotion,” prioritizing studies that provided rigorous analytical quantification or validated functional applications in agriculture. Methodological challenges in purification from complex matrices will be analyzed, and the molecular mechanisms through which marine signals are translated into growth responses in land plants will be discussed. Ultimately, this review does not merely aim to summarize existing literature; on the contrary, it serves as a strategic roadmap for the future development of the blue bioeconomy, identifying the research pathways necessary to transform macroalgal biochemistry into an operational tool for a new era of sustainable and resilient agriculture. The final goal is to demonstrate that a profound understanding of algal hormonal physiology represents the necessary bridge between the conservation of marine ecosystems and global technological innovation.

2. Phytohormone profiles: A comprehensive overview

3. Biosynthetic pathways of phytohormones

3.1. Tryptophan-dependent and tryptophan-independent pathways for auxin biosynthesis

The biosynthesis of IAA can occur through tryptophan-dependent and tryptophan-independent pathways (Benjamins & Scheres, 2008). Tryptophan-dependent auxin biosynthesis comprises multiple routes, including the indole-3-pyruvate pathway, considered the main one, and alternative pathways, such as the indole-3-acetaldoxime (IAOx), tryptamine (TAM), and indole-3-acetamide pathways (Figure 1) (Chandler, 2009; Kiseleva et al., 2012). Among tryptophan-dependent pathways, the indole-3-pyruvate pathway is widely considered predominant in land plants; however, its presence and relevance in macroalgae are less clearly defined. In this process, tryptophan is converted to indole-3-pyruvate by tryptophan aminotransferase. Indole-3-pyruvate is then converted to IAA through oxidative reactions catalyzed by flavin-dependent monooxygenases of the YUCCA (Yucca Uncharacterized Complementary Cloned Arabidopsis) family or functionally similar enzymes. The involvement of intermediates such as indole-3-acetaldehyde has been proposed in some systems, although their role remains secondary or less well-defined (Chandler, 2009). An alternative pathway is the IAOx pathway, mediated by cytochromes P450 (CYP79B2 and CYP79B3), which convert tryptophan into IAOx. This intermediate can be directed toward the biosynthesis of IAA (Delker et al., 2008) or toward secondary metabolites, such as indole glucosinolates, particularly in Brassicaceae (Ljung et al., 2002). However, the conservation and role of this pathway in macroalgae are limited and less well-characterized than in land plants. A related variant is the TAM pathway, in which tryptophan is decarboxylated to TAM by tryptophan decarboxylase. TAM can then be converted to oxidized intermediates by flavin-dependent monooxygenases of the YUCCA family or functionally similar proteins, such as FLOOZY (Kiseleva et al., 2012; Tobena-Santamaria et al., 2002; Yamamoto et al., 2007). While genomic surveys indicate the presence of homologous sequences to embryophyte YUCCA genes in macroalgae, it must be emphasized that these pathways remain largely inferred via bioinformatic homology. Direct, experimental biochemical validation of these enzymatic steps in macroalgal systems is still limited compared to land plants. In parallel, a tryptophan-independent pathway has been proposed, which does not use tryptophan directly but uses intermediates of its biosynthesis, such as indole or indole-3-glycerol phosphate (Zhang et al., 2008). These compounds can be converted to indole-3-acetonitrile (IAN) or directly to IAA through pathways that are not yet fully characterized (Ljung et al., 2002; Ostin et al., 1999). Overall, IAA biosynthesis involves several enzyme classes, including aminotransferases, flavin-dependent monooxygenases, cytochromes P450 and decarboxylases, whose distribution and relevance vary significantly between terrestrial plants and macroalgae, reflecting the evolutionary diversity of these organisms (Chandler, 2009; Delker et al., 2008). Furthermore, given that several alternative IAA biosynthetic routes remain a subject of active discussion even in higher plants, it must be emphasized that the precise relative contribution of each pathway within macroalgal lineages is still completely unresolved, representing a critical knowledge gap for future functional studies (Ma et al., 2026). From an evolutionary perspective, a critical distinction must be maintained between three non-equivalent concepts to avoid the overinterpretation of sequence homology: the conservation of metabolites (the chemical presence of the hormone), the conservation of biosynthetic enzymes (the catalytic machinery), and the conservation of downstream signaling pathways (the receptor and transcriptional networks). While the presence of auxin molecules is nearly universal across photosynthetic lineages and several biosynthetic enzyme homologs are shared, the complex signaling cascades characteristic of land plants are often absent or radically different in macroalgae, demonstrating that chemical conservation does not equal regulatory or functional conservation (Yu et al., 2026).

Figure 1

3.1.1. Comparative enzymatic landscape of auxin biosynthesis in terrestrial plants and macroalgae

Comparative bioinformatic analyses based on protein sequences suggest that the distribution of enzymes involved in IAA biosynthesis in macroalgae differs significantly from that of land plants. Proteins homologous to flavin-dependent monooxygenases of the YUCCA family have been identified in several algal species, indicating the possible presence of oxidative reactions like those of the IAA pathway. However, the absence or poor conservation of key enzymes such as tryptophan aminotransferases suggests that this pathway may not be fully functional or may operate differently in algae. Similarly, the limited evidence for functional homologs of cytochrome P450s involved in the IAOx pathway indicates that this pathway may be absent or marginal. Conversely, the identification of enzymes with nitrilase activity in several algal lineages supports the hypothesis that alternative pathways, such as that mediated by IAN, may contribute to IAA biosynthesis (Kiseleva et al., 2012). Overall, these data highlight a remarkable diversity in auxin biosynthetic mechanisms in macroalgae, suggesting the existence of partially conserved or evolutionary distinct pathways compared to land plants.

3.2. GA biosynthesis

GAs are diterpenes derived from isoprenoid biosynthesis, which can occur via the mevalonic acid and methylerythritol phosphate (MEP) pathways (Schwender et al., 1996). In land plants, and potentially also in several algal lineages, GA biosynthesis occurs predominantly in plastids via the MEP pathway (Figure 2) (Kasahara et al., 2002). The initial steps of the pathway involve the cyclization of geranylgeranyl pyrophosphate (GGPP) to copalyl pyrophosphate, catalyzed by copalyl pyrophosphate synthase (CPS), followed by conversion to ent-kaurene by ent-kaurene synthase (KS). These reactions occur in the plastids of growing tissues, while subsequent steps also involve the endoplasmic reticulum, highlighting a subcellular compartmentalization of the pathway (Helliwell et al., 2001a; Yamaguchi et al., 1996). Ent-kaurene is subsequently converted to GAs through a series of oxidative reactions catalyzed by cytochrome P450-dependent monooxygenases, leading to the formation of key intermediates such as GA₁₂ and its 13-hydroxylated analog GA₅₃. The final steps of GA metabolism are catalyzed by soluble 2-oxoglutarate-dependent dioxygenases, including GA20 oxidase, GA3 oxidase, and GA2 oxidase, which are involved in the biosynthesis and inactivation of the bioactive forms, respectively (Hedden & Kamiya, 1997; Hedden & Proebsting, 1999; Helliwell et al., 2001b). However, in macroalgae, the presence and organization of these pathways are less well characterized, and available evidence suggests a possible diversity in biosynthetic mechanisms compared to land plants.

Figure 2

3.2.1. Comparative enzymatic landscape of GAs biosynthesis in terrestrial plants and macroalgae

Comparative analyses suggest that enzymes involved in GA biosynthesis in macroalgae are heterogeneous along the pathway. No clear homologs have been identified for enzymes catalyzing the early stages of biosynthesis, such as CPS, ent-KS, and ent-kaurenoic acid oxidase, suggesting a possible divergence or limited representation of these steps in the available data. In contrast, enzymes involved in the later stages of GA metabolism, belonging to the 2-oxoglutarate-dependent dioxygenase family (such as GA20 oxidase, GA3 oxidase, and GA2 oxidase), are more frequently represented in algal genomes. Interestingly, an enzyme with GA20 oxidase activity has been characterized in the microalga Chlamydomonas reinhardtii, although it shows limited similarity to its homologs in land plants, suggesting possible structural and functional differences (Kiseleva et al., 2012). Overall, these data indicate that, while some late stages of GA biosynthesis may be conserved in algae, the early stages are less evident or potentially divergent. This evidence supports the hypothesis that GA biosynthetic pathways in macroalgae may differ, at least in part, from those of land plants, or reflect limitations in the currently available genomic resources.

3.3. CKs biosynthesis

The biosynthesis of CKs, conserved in land plants and several algal lineages, can occur through two main pathways: a direct (de novo) pathway and a tRNA-dependent pathway (Figure 3). The de novo pathway is catalyzed by isopentenyltransferase (IPT), which transfers an isopentenyl group from isopentenyl pyrophosphate (IPP) to AMP, leading to the formation of N6-isopentenyladenosine monophosphate (iPMP) (Mok et al., 2000). In the alternative pathway, the biosynthesis of isoprenoid CKs results from the modification of tRNA molecules containing isopentenylated bases, such as cis-zeatin (Lindner et al., 2014; Stirk et al., 2003). Marine macroalgae (chlorophytes, phaeophytes, and rhodophytes) exhibit both isoprenoid and aromatic CKs, suggesting the possible presence of both biosynthetic pathways (Stirk et al., 2003, 2009). Interestingly, forms such as iP and cis-zeatin were detected in higher concentrations than dihydrozeatin (DHZ) conjugates. Conversely, the absence of N-glucosides in several macroalgae suggests that the tRNA-dependent pathway may represent a significant contribution to CK biosynthesis in these organisms (Von Schwartzenberg et al., 2007). Comparative analyses of amino acid sequences have also identified, in some algae, proteins homologous to IPTs of higher plants, suggesting the possible conservation of the de novo pathway (Kiseleva et al., 2012). Overall, these findings suggest that macroalgae are able to synthesize CKs through pathways that are partially conserved compared to land plants, although their organization and regulation remain incompletely characterized. Crucially, it must be underscored that sequence similarity or bioinformatic homology alone does not necessarily imply the absolute conservation of enzymatic function. In the absence of direct biochemical characterization, high sequence identity cannot rule out divergent substrate specificities or alternative regulatory mechanisms in macroalgal lineages, and readers should avoid overinterpreting genomic data as definitive functional proof (Yu et al., 2026).

Figure 3

3.4. ABA biosynthesis

ABA is derived primarily from the oxidative degradation of carotenoids, while potentially independent alternative pathways have been proposed in some photosynthetic organisms (Cutler & Krochko, 1999) (Figure 4). ABA biosynthesis in land plants begins with the formation of carotenoids from IPP. Specifically, IPP is converted to GGPP, which is the direct precursor of carotenoid synthesis. GGPP is converted to phytoene-by-phytoene synthase (PSY), followed by a series of desaturation and isomerization reactions, catalyzed by enzymes such as phytoene desaturase (PDS), which lead to the formation of lycopene and subsequently β-carotene and zeaxanthin (Kiseleva et al., 2012). In some cases, alternative pathways for carotenoid formation have been hypothesized, but the main pathway remains that mediated by GGPP (Hartung, 2010; Taiz et al., 2015). Zeaxanthin is converted to trans-violaxanthin by zeaxanthin epoxidase (ZEP), which catalyzes an epoxidation reaction, and is subsequently transformed into 9-cis-violaxanthin or 9-cis-neoxanthin by neoxanthin synthase. These cis-carotenoids are then cleaved by the key enzyme 9-cis-epoxycarotenoid dioxygenase (NCED), producing xanthoxin, a direct intermediate in ABA biosynthesis (Maier, 1993; Taiz et al., 2015). Xanthoxin is subsequently converted to ABA through a series of cytosolic enzymatic reactions involving the intermediate formation of abscisic aldehyde and its final oxidation to ABA (Seo & Koshiba, 2002). In this metabolic framework, it is important to note that while the upstream enzymatic steps involved in carotenoid precursor production have been experimentally demonstrated in several macroalgae, the downstream cleavage and oxidation steps leading specifically to ABA biosynthesis remain largely inferred through genomic homologies with higher-plant models, requiring further direct biochemical validation in marine lineages. Furthermore, the presence of specific carotenoid intermediates in green algae has been reported, while other key compounds are less evident. However, the absence or poor evidence of some key intermediates still makes the complete conservation of this pathway uncertain (Baroli & Niyogi, 2000). 9′-cis-neoxanthin is known to be present in green algae, while other key intermediates of the carotenoid pathway are less evident or not always detected. This may indicate that, in some algae, ABA production may also occur through alternative pathways, potentially involving precursors such as farnesyl diphosphate (Hartung, 2010). In other groups, such as cyanobacteria, Dinophyta and Rhodophyta, inhibition of carotenoid biosynthesis does not appear to affect ABA accumulation, suggesting the existence of alternative biosynthetic pathways, potentially carotenoid-independent and based on PPI-derived precursors (Cutler & Krochko, 1999). Overall, while the ABA carotenoid pathway appears well conserved in land plants, available evidence in macroalgae suggests greater metabolic diversity, with possible alternative pathways still poorly characterized.

Figure 4

3.4.1. Comparative enzymatic landscape of ABA biosynthesis in terrestrial plants and macroalgae

Since the initial steps of ABA biosynthesis are closely linked to the carotenoid pathway, it is plausible to hypothesize that enzymes involved in these processes are also widespread among different algal groups. Bioinformatic analyses conducted using BlastP on the amino acid sequences of the PSY, PDS, and ZEP enzymes of Arabidopsis thaliana have highlighted the presence of homologous proteins in numerous algal species. Similarly, homologs of enzymes implicated in the more advanced steps of the pathway, such as NCED and proteins belonging to the short-chain dehydrogenase/reductase family, involved in the conversion of xanthoxin, have been identified in several algal taxa. Such homologs have been found, for example, in Ectocarpus siliculosus, C. reinhardtii, and Chlorella variabilis. However, the presence of sequence homology does not necessarily imply functional conservation of the pathway (Kiseleva et al., 2012). Therefore, although these results suggest a possible partial conservation of the enzymatic elements associated with ABA biosynthesis in algae, the actual operation and organization of this pathway require further experimental confirmation. Caution must be exercised when interpreting these physiological responses alongside genomic data; the functional annotation of these biosynthetic networks is predominantly based on in silico homology with higher plants, whereas experimental verification of active, functional enzymes in macroalgae represents an ongoing challenge.

3.5. Ethylene

Ethylene biosynthesis in macroalgae can originate from methionine (Met) through different pathways, some of which diverge from those described in terrestrial plants (Figure 5). In some marine macroalgae, Met is the precursor for the synthesis of dimethylsulfoniopropionate (DMSP), an organosulfur compound widely distributed in photosynthetic marine organisms. DMSP can then be cleaved into acrylate and dimethylsulfide, and it has been proposed that acrylate-derived intermediates may contribute to the formation of ethylene, although the enzymatic details of this process remain poorly characterized (Watanabe & Kondo, 1976). Ethylene production associated with this pathway has been reported in several algal species, including U. lactuca, Pyropia tenera, Codium fragile, and Fucus vesiculosus (Watanabe & Kondo, 1976). Conversely, some unicellular green algae, such as Haematococcus pluvialis, appear to possess an ethylene biosynthetic pathway more like that described in land plants (Maillard et al., 1993). In this pathway, Met is converted to S-adenosylmethionine, a direct precursor for the synthesis of ACC. ACC is subsequently oxidized to ethylene by the action of the enzyme ACC oxidase. In particular, the enzyme identified in unicellular green algae is activated by Co²⁺, Mn²⁺, and Ag⁺ and inhibited by Cu2+, SA, and darkness. In contrast, in land plants, ACC oxidase is generally stimulated by Fe²⁺ and inhibited by Co²⁺ (Maillard et al., 1993). Overall, the available evidence suggests that ethylene biosynthesis in algae comprises both ACC-dependent pathways like those in land plants and alternative pathways potentially specific to certain algal groups. This indicates a partial evolutionary conservation of ethylene metabolism, accompanied by significant biochemical diversification among different algal lineages (Baroli & Niyogi, 2000; Booker & DeLong, 2015).

Figure 5

4. Methodological approaches in algal phytohormone analysis

The study and analysis of phytohormones in macroalgae, due to their low concentrations, represents a difficult research challenge. In addition to low concentrations, a significant obstacle is the diversity of compounds and the complexity of algal matrices. As seen in the previous paragraphs, the different classes of phytohormones produced exhibit many chemical and physical differences, which complicate the development and/or optimization of extraction processes and analytical techniques for identifying all components. In recent years, research has moved toward green and eco-sustainable extraction methods aimed at reducing the consumption of solvents used in extraction processes and extraction times (Ben Hammouda et al., 2026). Among the main approaches used for the extraction and analysis of phytohormones in algae are liquid-liquid extraction, liquid microextraction, solid phase extraction (SPE) and molecularly imprinted polymer extraction (Górka & Wieczorek, 2017). Meanwhile, chromatographic techniques, particularly high-performance liquid chromatography (HPLC) and gas chromatography (GC), are the most widely used methods for the qualitative and quantitative determination of plant hormones due to their high sensitivity and reproducibility. HPLC, which generally uses an aqueous mobile phase, is one of the main tools for the accurate determination of hormones due to its high sensitivity and good repeatability. The coupling of liquid chromatography with mass spectrometry (LC-MS/MS) combines the high separation capacity of HPLC with the sensitivity and selectivity of MS, effectively reducing interference from ultraviolet-absorbing compounds and improving the detection of phytohormones (Hoyerová et al., 2006; Zhou et al., 2003). SPE-LC-MS/MS methods with selective reaction monitoring have been used for the simultaneous purification and quantification of phytohormones such as GA₃, IAA, and ABA, demonstrating high analytical efficiency (Hou et al., 2008). GC is another widely used approach due to its high separation performance and sensitivity. In recent years, the field of algal phytomonitoring has seen a significant shift toward absolute quantitative LC-MS/MS workflows. In particular, the integration of stable isotope-labeled internal standards (such as ²H- or ¹³C-labeled analogs) has become increasingly crucial, as it allows researchers to effectively correct for matrix effects and extraction losses, ensuring unparalleled accuracy and reproducibility when profiling complex macroalgal matrices (Pantami et al., 2020). However, because many phytohormones exhibit high polarity and low volatility, GC analysis frequently requires a preliminary derivatization step, except for ethylene. This process increases the complexity of sample pretreatment and the operational burden. In recent years, gas chromatography-mass spectrometry (GC-MS) has become a key tool for the quantitative analysis of endogenous plant hormones (Birkemeyer et al., 2003). Although the GC-MS method guarantees high accuracy and reliability, it is expensive in terms of both equipment and maintenance, as well as requiring more laborious preparation protocols. A frequently used extraction protocol is based on the use of algal sap. After filtration through nylon fabrics with mesh sizes of approximately 20–50 µm, the sap is treated with organic solvents such as diethyl ether, ethyl acetate, and n-butanol. Subsequently, the solvent is evaporated, and the resulting pellets are resolubilized in ethanol for MS/MS analysis to identify phytohormones (Prasad et al., 2010). In a study conducted by Yokoya et al. (2010) three different solid-liquid extraction conditions were described for IAA, ABA, and CKs. In detail, auxins were extracted using ice-cold phosphate buffer with 0.02% sodium diethyl dithiocarbamate, CKs using ice-cold 70% ethanol, and ABA using a methanol:water:acetic acid mixture (10:89:1, v/v) containing sodium diethyldithiocarbamate. The obtained samples were subsequently purified by immunoaffinity chromatography and characterized by LC-MS/MS (Yokoya et al., 2010). Other solvents used for phytohormone extraction include methanol:water:formic acid (15:4:1) mixtures, used for the simultaneous extraction of different hormone classes in Ulva and Monostroma species (Lu et al., 2010), and acetonitrile:acetic acid mixtures, used in the red algae Bangia fuscopurpurea and Pyropia yezoensis (Mikami et al., 2016). Górka and Wieczorek optimized a protocol for the simultaneous extraction of nine phytohormones in C. glomerata and Spirulina sp., using supercritical CO₂ extraction (SFE-CO₂) at 500 bar and 40◦C, followed by analysis by reversed-phase high-performance liquid chromatography with a photodiode array (PDA) detector. This approach is distinguished using a “green” high-pressure extraction technique combined with chromatographic separation with optical detection. A different protocol was applied to aqueous extracts of dried Padina durvillaei and U. lactuca (1:10 w/v). In this case, the material is freeze-dried and then resuspended in 80% methanol containing 1% acetic acid. After centrifugation and concentration of the supernatant, the residue is solubilized in 1% acetic acid and purified by a reversed-phase column. Compared to previous protocols, this method involves a more complex fractionation step: acidic hormones are eluted with methanol, while basic hormones are eluted with 60% methanol containing 5% aqueous ammonia. The final residues are then resuspended in a mixture of 5% acetonitrile, 1% methanol, and 1% acetic acid before quantification by UHPLC-MS (Benítez García et al., 2020). This approach is characterized by a highly complex purification process and the use of highly sensitive MS as the final detection system. Additional analytical techniques, such as GC/MS and UPLC-MS/MS, have also been used to identify phytohormones in microalgae such as Chlorella minutissima (Stirk et al., 2014a). GAs and BR can be extracted using 80% acetonitrile containing 5% formic acid and subsequently analyzed by UPLC-MS/MS (Stirk et al., 2013b), highlighting the use of highly acidic conditions and highly sensitive tandem techniques. Endogenous hormones from Chlorella minutissima can also be extracted in cold phosphate buffer (50 mM, pH 7.0) containing sodium diethyldithiocarbamate and analyzed by UPLC coupled to electrospray ionization (ESI) (Stirk et al., 2013). Compared to other approaches, this method stands out for the use of an aqueous buffer environment and for detection based on ESI without necessarily resorting to tandem fragmentation. In addition to chromatographic methods, alternative approaches such as electrochemical analysis, biological assays and immunoassays have been proposed. Electrochemical analysis is a relatively simple and inexpensive technique, although determination is strongly influenced by the nature and pH of the background solution, especially in complex biological samples, limiting its practical application (Chen et al., 2002). In recent years, innovative electrochemical biosensors have shown considerable potential thanks to their wide operating range, good selectivity, and high sensitivity (Li et al., 2002). Bioassays, on the other hand, represent one of the first approaches used for the determination of endogenous plant hormones. Although these methods allow the evaluation of the physiological activity of hormones, they have low specificity and require the elimination of interferents such as auxin analogs and antagonists, making the analytical process complex. For this reason, they are mainly used for qualitative analyses or in combination with quantitative physicochemical methods (Plettner et al., 2005). Finally, immunoassays exploit the specific antigen-antibody interaction for the qualitative and quantitative analysis of analytes. Despite their potential, these techniques still suffer from operational instability, high interference, and poor reproducibility, factors that limit their application in the analysis of plant phytohormones (Swaczynová et al., 2007). Overall, the various studies differ mainly in the extraction strategy adopted (acidic organic solvents, supercritical CO₂, or aqueous buffers), the degree of chromatographic purification, and the sensitivity of the detection techniques employed (PDA, single-dose MS, LC-MS/MS, or GC-MS). Despite significant advances in extraction methodologies and high-resolution analytical platforms, standardizing protocols for the analysis of phytohormones in macroalgae remains an open challenge, especially due to the high compositional variability of algal matrices.

5. Macroalgal extracts as plant growth promoters

The transition from analytical biochemistry to applied agronomy constitutes the fundamental translational step in the valorization of macroalgal phytohormones. Within the framework of the Blue Bioeconomy, the demonstration that seaweed-derived extracts exert profound morphophysiological effects on terrestrial crops has transformed these marine organisms from mere ecological curiosities into strategic biotechnological tools for sustainable intensification (Khan et al., 2009). Unlike synthetic agrochemicals that operate via acute toxicity or brute-force nutrient delivery, macroalgal biostimulants function as complex signal orchestrators, capable of reprogramming the plant transcriptome and restoring hormonal homeostasis under suboptimal growth conditions (Shukla et al., 2019). The mechanistic basis of this plant growth promotion (PGP) lies not in a single master regulator but in the metabolic synergy among auxins, CKs, GAs, and bioactive polysaccharides, which collectively mimic and amplify endogenous plant signaling cascades (du Jardin, 2015). This section dissects the sequential impact of these extracts across the plant life cycle, from the first imbibition of the seed to the physiological tolerance of terminal abiotic stressors, while critically evaluating the dose-dependent and species-specific responses that govern field efficacy (Battacharyya et al., 2015). Also, when evaluating the agricultural potential of macroalgal derivatives, a stringent terminological distinction must be maintained between the endogenous phytohormones baseline quantified within the algal biomass, the hormone-like elicitor activity exhibited by crude extracts in bioassays, and the comprehensive plant biostimulant effects observed in crops, which often result from a synergistic network of multiple bioactive compounds rather than hormones alone.

6. Factors influencing phytohormone composition

The endogenous phytohormone profile of a given macroalga is not a static taxonomic fingerprint but a dynamic, plastic phenotype that responds to a multitude of interacting biotic and abiotic factors (Rathod et al., 2023). The reproducibility of biostimulant formulations, a critical requirement for industrial standardization and agricultural efficacy, is thus continuously challenged by natural variability (Khoulati et al., 2025). Understanding the drivers of this chemodiversity is not merely an academic pursuit; it is a prerequisite for the predictable and sustainable exploitation of macroalgal biomass. This section systematically deconstructs the primary sources of hormonal variability, ranging from predictable seasonal rhythms and ontogenetic programming to acute environmental stressors and post-harvest processing artifacts.

7. Concluding remarks and future perspectives

The systematic mapping of phytohormone profiles across the three major macroalgal lineages (Phaeophyceae, Rhodophyta, and Chlorophyta) definitively consolidates the role of these organisms as complex molecular biorefineries capable of bridging the evolutionary divide between marine ecosystems and terrestrial agriculture through an ancestral chemical language. As highlighted throughout this review, elucidating the biochemical diversity of both classical growth regulators (auxins, CKs, GAs, ABA, and ethylene) and emerging signaling molecules (BR, jasmonates, and SA) not only sheds light on the mechanisms of evolutionary pre-adaptation to land colonization but also offers a strategic roadmap for sustainable agricultural intensification within the framework of the blue bioeconomy. However, transitioning from empirical field applications to predictable, standardized molecular tools requires overcoming the critical analytical and enzymatic bottlenecks discussed throughout this work. At the methodological level, priority must be given to optimizing UHPLC-MS/MS workflows by mitigating the severe ion suppression phenomena caused by recalcitrant matrices rich in polyphenols and viscous polysaccharides; this entails shifting away from harsh chemical extraction treatments (such as acid or alkaline hydrolysis) toward green, targeted technologies like MAE or UAE, which are capable of preserving highly labile hormonal fractions, including aromatic CKs and free IAA pools. At the biosynthetic level, the heterogeneity of the enzymatic landscape evidenced by the coexistence of tryptophan-dependent and independent pathways for auxin, the prominence of the tRNA-dependent pathway for CKs, and the genomic divergence in the early stages of GA synthesis despite the conservation of terminal dioxygenases represents a bioinformatic “dark matter” that demands targeted functional characterization, aiming also to identify algal receptors and polar transport systems structurally distinct from established angiosperm models. Furthermore, it is essential to unravel cross-kingdom signaling dynamics by disentangling the endogenous metabolic contributions of macroalgae from those of the epibiotic bacterial consortia within the holobiont, while concurrently investigating the role of jasmonates in driving systemic defense responses against marine herbivores. For future prospecting, integrating comparative genomics with the rigorous evaluation of metabolic stability will be vital to deciphering the exact signaling cascades through which these marine extracts reprogram the transcriptome and hormonal homeostasis of terrestrial crops, leading to RSA remodeling, enhanced NUE, and the induction of priming against osmotic and thermal stresses, thereby transforming marine chemical ecology into a robust technology for global agricultural resilience.