External Stress Signals Research Articles

Emerging insights into the pathogenesis and therapeutic strategies for vascular endothelial injury-associated diseases: focus on mitochondrial dysfunction.

As a vital component of blood vessels, endothelial cells play a key role in maintaining overall physiological function by residing between circulating blood and semi-solid tissue. Various stress stimuli can induce endothelial injury, leading to the onset of corresponding diseases in the body. In recent years, the importance of mitochondria in vascular endothelial injury has become increasingly apparent. Mitochondria, as the primary site of cellular aerobic respiration and the organelle for "energy information transfer," can detect endothelial cell damage by integrating and receiving various external stress signals. The generation of reactive oxygen species (ROS) and mitochondrial dysfunction often determine the evolution of endothelial cell injury towards necrosis or apoptosis. Therefore, mitochondria are closely associated with endothelial cell function, helping to determine the progression of clinical diseases. This article comprehensively reviews the interconnection and pathogenesis of mitochondrial-induced vascular endothelial cell injury in cardiovascular diseases, renal diseases, pulmonary-related diseases, cerebrovascular diseases, and microvascular diseases associated with diabetes. Corresponding therapeutic approaches are also provided. Additionally, strategies for using clinical drugs to treat vascular endothelial injury-based diseases are discussed, aiming to offer new insights and treatment options for the clinical diagnosis of related vascular injuries.

Relevance and regulation of alternative splicing in plant secondary metabolism: current understanding and future directions.

The secondary metabolism of plants is an essential life process enabling organisms to navigate various stages of plant development and cope with ever-changing environmental stresses. Secondary metabolites, abundantly found in nature, possess significant medicinal value. Among the regulatory mechanisms governing these metabolic processes, alternative splicing stands out as a widely observed post-transcriptional mechanism present in multicellular organisms. It facilitates the generation of multiple mRNA transcripts from a single gene by selecting different splicing sites. Selective splicing events in plants are widely induced by various signals, including external environmental stress and hormone signals. These events ultimately regulate the secondary metabolic processes and the accumulation of essential secondary metabolites in plants by influencing the synthesis of primary metabolites, hormone metabolism, biomass accumulation, and capillary density. Simultaneously, alternative splicing plays a crucial role in enhancing protein diversity and the abundance of the transcriptome. This paper provides a summary of the factors inducing alternative splicing events in plants and systematically describes the progress in regulating alternative splicing with respect to different secondary metabolites, including terpenoid, phenolic compounds, and nitrogen-containing compounds. Such elucidation offers critical foundational insights for understanding the role of alternative splicing in regulating plant metabolism and presents novel avenues and perspectives for bioengineering.

HigA2 (Rv2021c) Is a Transcriptional Regulator with Multiple Regulatory Targets in Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems are the major mechanism for persister formation in Mycobacterium tuberculosis (Mtb). Previous studies found that HigBA2 (Rv2022c-Rv2021c), a predicted type II TA system of Mtb, could be activated for transcription in response to multiple stresses such as anti-tuberculosis drugs, nutrient starvation, endure hypoxia, acidic pH, etc. In this study, we determined the binding site of HigA2 (Rv2021c), which is located in the coding region of the upstream gene higB2 (Rv2022c), and the conserved recognition motif of HigA2 was characterized via oligonucleotide mutation. Eight binding sites of HigA2 were further found in the Mtb genome according to the conserved motif. RT-PCR showed that HigA2 can regulate the transcription level of all eight of these genes and three adjacent downstream genes. DNA pull-down experiments showed that twelve functional regulators sense external regulatory signals and may regulate the transcription of the HigBA2 system. Of these, Rv0903c, Rv0744c, Rv0474, Rv3124, Rv2603c, and Rv3583c may be involved in the regulation of external stress signals. In general, we identified the downstream target genes and possible upstream regulatory genes of HigA2, which paved the way for the illustration of the persistence establishment mechanism in Mtb.

Appropriate induction of TOC1 ensures optimal MYB44 expression in ABA signaling and stress response in Arabidopsis.

Plants possess the remarkable ability to integrate the circadian clock with various signalling pathways, enabling them to quickly detect and react to both external and internal stress signals. However, the interplay between the circadian clock and biological processes in orchestrating responses to environmental stresses remains poorly understood. TOC1, a core component of the plant circadian clock, plays a vital role in maintaining circadian rhythmicity and participating in plant defences. Here, our study reveals a direct interaction between TOC1 and the promoter region of MYB44, a key gene involved in plant defence. TOC1 rhythmically represses MYB44 expression, thereby ensuring elevated MYB44 expression at dawn to help the plant in coping with lowest temperatures during diurnal cycles. Additionally, both TOC1 and MYB44 can be induced by cold stress in an Abscisic acid (ABA)-dependent and independent manner. TOC1 demonstrates a rapid induction in response to lower temperatures compared to ABA treatment, suggesting timely flexible regulation of TOC1-MYB44 regulatory module by the circadian clock in ensuring a proper response to diverse stresses and maintaining a balance between normal physiological processes and energy-consuming stress responses. Our study elucidates the role of TOC1 in effectively modulating expression of MYB44, providing insights into the regulatory network connecting the circadian clock, ABA signalling, and stress-responsive genes.

The Role of Endogenous Brassinosteroids in the Mechanisms Regulating Plant Reactions to Various Abiotic Stresses

Plants are vulnerable to many abiotic stresses, resulting in reduced plant productivity. Its adaptation to unfavorable environments relies on transmitting external stress signals into internal signaling pathways. A series of stress response mechanisms have been developed. Among them, brassinosteroids (BRs) are a class of steroid hormones that are widely involved in plant growth, development, and stress response. Via genetics, proteomics, and genomics studies, the major components of signaling and signaling pathways through a series of phosphorylation cascade reactions have been identified in model plants such as Arabidopsis. Numerous studies have shown that BRs play important roles in plant responses to drought, temperature, salt, heavy metals, and other environmental stresses. The application of BRs to improve plant stress resistance has become the focus of research in recent years, especially the regulation of stress via endogenous BRs. Therefore, this paper systematically summarizes the research progress related to endogenous BR levels and provides an overview of BR biosynthesis and the signaling pathway, as well as the function of endogenous BRs in the response to abiotic stresses.

LsrR-like protein responds to stress tolerance by regulating polysaccharide biosynthesis in Lactiplantibacillus plantarum

In addition to their biological functions, polysaccharides assist Lactiplantibacillus plantarum in resisting harsh conditions. To enhance the polysaccharide biosynthesis and increase the survival of L. plantarum in gut environment. We analyzed the transcriptional regulators that regulated the polysaccharide biosynthesis. A new transcriptional inhibitor, LsrR (UniProtKB: Q88YH7), had been identified, which repressed polysaccharide synthesis by binding to the polysaccharide synthesis promoter cps4A-J (Pcps4A-J). The EPSs and CPSs production of L. plantarum 163 was reduced by 42 % and 36 % (p < 0.05), respectively, when lsrR was overexpressed. Furthermore, alkaline shock proteins Asp2 and Asp1, heat shock protein Hsp3, and an autoinducer-2 (AI-2) related quorum-sensing regulator Rrp6 recovered the synthesis of polysaccharides to 50, 33, 55, and 60 %, respectively, by inhibiting the LsrR activity. This suggested that LsrR regulates polysaccharide synthesis in response to external stress signals such as pH, temperature, and AI-2 concentration. Finally, we showed that polysaccharides increased the survival rate of L. plantarum (Lp163-ΔlsrR) by 2.1 times during lyophilization and enhanced its tolerance to pH 2.0 and 0.2 % bile salts by 15.3 and 60 times due to increased capsular thickness and enhanced the autoaggregation. We provide critical data regarding Lactobacillus survival during preservative lyophilization and under gastrointestinal conditions.

Regulation of crop agronomic traits and abiotic stress responses by brassinosteroids: a review

Plant adaptation to adverse environment depends on transmitting the external stress signals into internal signaling pathways, and thus forming a variety of stress response mechanisms during evolution. Brassinosteroids (BRs) is a steroid hormone and widely involved in plant growth, development and stress response. BR is perceived by cell surface receptors, including the receptor brassinosteroid-insensitive 1 (BRI1) and the co-receptor BRI1-associated-kinase 1 (BAK1), which in turn trigger a signaling cascade that leads to the inhibition of BIN2 and activation of BES1/BZR1 transcription factors. BES1/BZR1 can directly regulate the expression of thousands of downstream responsive genes. Studies in the model plant Arabidopsis thaliana have shown that members of BR biosynthesis and signal transduction pathways, particularly protein kinase BIN2 and its downstream transcription factors BES1/BZR1, can be extensively regulated by a variety of environmental factors. In this paper, we summarize recent progresses on how BR biosynthesis and signal transduction are regulated by complex environmental factors, as well as how BR and environmental factors co-regulate crop agronomic traits, cold and salt stress responses.

Evolutionary Analysis of the YABBY Gene Family in Brassicaceae.

The YABBY gene family is one of the plant transcription factors present in all seed plants. The family members were extensively studied in various plants and shown to play important roles in plant growth and development, such as the polarity establishment in lateral organs, the formation and development of leaves and flowers, and the response to internal plant hormone and external environmental stress signals. In this study, a total of 364 YABBY genes were identified from 37 Brassicaceae genomes, of which 15 were incomplete due to sequence gaps, and nine were imperfect (missing C2C2 zinc-finger or YABBY domain) due to sequence mutations. Phylogenetic analyses resolved these YABBY genes into six compact clades except for a YAB3-like gene identified in Aethionema arabicum. Seventeen Brassicaceae species each contained a complete set of six basic YABBY genes (i.e., 1 FIL, 1 YAB2, 1 YAB3, 1 YAB5, 1 INO and 1 CRC), while 20 others each contained a variable number of YABBY genes (5–25) caused mainly by whole-genome duplication/triplication followed by gene losses, and occasionally by tandem duplications. The fate of duplicate YABBY genes changed considerably according to plant species, as well as to YABBY gene type. These YABBY genes were shown to be syntenically conserved across most of the Brassicaceae species, but their functions might be considerably diverged between species, as well as between paralogous copies, as demonstrated by the promoter and expression analysis of YABBY genes in two Brassica species (B. rapa and B. oleracea). Our study provides valuable insights for understanding the evolutionary story of YABBY genes in Brassicaceae and for further functional characterization of each YABBY gene across the Brassicaceae species.

Glucocorticoids as Regulators of Macrophage-Mediated Tissue Homeostasis.

Our immune system has evolved as a complex network of cells and tissues tasked with maintaining host homeostasis. This is evident during the inflammatory responses elicited during a microbial infection or traumatic tissue damage. These responses seek to eliminate foreign material or restore tissue integrity. Even during periods without explicit disturbances, the immune system plays prominent roles in tissue homeostasis. Perhaps one of the most studied cells in this regard is the macrophage. Tissue-resident macrophages are a heterogenous group of sensory cells that respond to a variety of environmental cues and are essential for organ function. Endogenously produced glucocorticoid hormones connect external environmental stress signals with the function of many cell types, producing profound changes in immune cells, including macrophages. Here, we review the current literature which demonstrates specific effects of glucocorticoids in several organ systems. We propose that tissue-resident macrophages, through glucocorticoid signaling, may play an underappreciated role as regulators of organ homeostasis.

JNK Pathway in CNS Pathologies.

The c-Jun N-terminal kinase (JNK) signalling pathway is a conserved response to a wide range of internal and external cellular stress signals. Beside the stress response, the JNK pathway is involved in a series of vital regulatory mechanisms during development and adulthood that are critical to maintain tissue homeostasis. These mechanisms include the regulation of apoptosis, growth, proliferation, differentiation, migration and invasion. The JNK pathway has a diverse functionality and cell-tissue specificity, and has emerged as a key player in regeneration, tumorigenesis and other pathologies. The JNK pathway is highly active in the central nervous system (CNS), and plays a central role when cells need to cope with pathophysiological insults during development and adulthood. Here, we review the implications of the JNK pathway in pathologies of the CNS. More specifically, we discuss some newly identified examples and mechanisms of JNK-driven tumor progression in glioblastoma, regeneration/repair after an injury, neurodegeneration and neuronal cell death. All these new discoveries support the central role of JNK in CNS pathologies and reinforce the idea of JNK as potential target to reduce their detrimental effects.

Dual Role of Autophagy in Regulation of Mesenchymal Stem Cell Senescence.

During their development and overall life, mesenchymal stem cells (MSCs) encounter a plethora of internal and external stress signals and therefore, they need to put in action homeostatic changes in order to face these stresses. To this aim, similar to other mammalian cells, MSCs are endowed with two crucial biological responses, autophagy and senescence. Sharing of a number of stimuli like shrinkage of telomeres, oncogenic and oxidative stress, and DNA damage, suggest an intriguingly close relationship between autophagy and senescence. Autophagy is at first reported to suppress MSC senescence by clearing injured cytoplasmic organelles and impaired macromolecules, yet recent investigations also showed that autophagy can promote MSC senescence by inducing the production of senescence-associated secretory proteins (SASP). These apparently contrary contributions of autophagy may mirror an intricate image of autophagic regulation on MSC senescence. We here tackle the pro-senescence and anti-senescence roles of autophagy in MSCs while concentrating on some possible mechanistic explanations of such an intricate liaison. Clarifying the autophagy/senescence relationship in MSCs will help the development of more effective and safer therapeutic strategies.

Physio-biochemical insights into sugarcane genotypes under water stress

ABSTRACT Sugarcane (Saccharum officinarum L.) is an economic crop cultivated in the tropical and subtropical zones. It is the world’s major crop according to production quantity. Sugarcane crop faces all types of environmental constraints due to its long life cycle period. Among various stresses, water stress creates a major obstacle to sugarcane production. Water deficit during formative stage in sugarcane causes more deleterious effect on crop growth. Plants develop mechanism to perceive external stress signals and adapt by changing in their physiological and biochemical responses. Therefore, this experiment was undertaken to elucidate the changes occur in to physiological and biochemical parameters under water stress in ten sugarcane genotypes. The water stress was applied by water suppression during formative stage (120–150 days). The third fully expanded leaf from random plant of each biological replication was collected on 120th day (irrigated condition), 135th day and 150th day after applying water stress. The obtained results showed significant differences among physio-biochemical responses in all genotypes. Minor fluctuations in physio-biochemical observations were found to be in 15 days of water stress, whereas higher level of percent changes were to be noted in samples of 30 days water stress. A minimum reduction in RWC, MSI, SCMR value, NR activity and higher increase in proline accumulation, catalase, peroxidase activities were found to be in sugarcane genotypes Co 98014, Co 0118, CoPk 05191, Co 0238 and Co 05011. Whereas the genotype Co 89003 and CoJ 64 were more affected in both stress treatment.

OsLRR-RLK1, an early responsive leucine-rich repeat receptor-like kinase, initiates rice defense responses against a chewing herbivore.

Plants are constantly exposed to a variety of environmental stresses, including herbivory. How plants perceive herbivores on a molecular level is poorly understood. Leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest subfamily of RLKs, are essential for plants to detect external stress signals, and may therefore also be involved in herbivore perception. Here, we employed RNA interference silencing, phytohormone profiling and complementation, as well as herbivore resistance assays, to investigate the requirement of an LRR-RLK for the initiation of rice (Oryza sativa) defenses against the chewing herbivore striped stem borer (SSB) Chilo suppressalis. We discovered a plasma membrane-localized LRR-RLK, OsLRR-RLK1, whose transcription is strongly up-regulated by SSB attack and treatment with oral secretions of Spodoptera frugiperda. OsLRR-RLK1 acts upstream of mitogen-activated protein kinase (MPK) cascades, and positively regulates defense-related MPKs and WRKY transcription factors. Moreover, OsLRR-RLK1 is a positive regulator of SSB-elicited, but not wound-elicited, levels of jasmonic acid and ethylene, trypsin protease inhibitor activity and plant resistance towards SSB. OsLRR-RLK1 therefore plays an important role in herbivory-induced defenses of rice. Given the well-documented role of LRR-RLKs in the perception of stress-related molecules, we speculate that OsLRR-RLK1 may be involved in the perception of herbivory-associated molecular patterns.

Mir-34a-5p and mir-34a-3p contribute to the signaling pathway of p53 by targeting overlapping sets of genes

In contrary to the common belief that only one strand of the pre-miRNA is active (usually the 5p one that is the more abundant) while the second one (miRNA*) is discarded, functional 5p and 3p have been observed for many miRNAs. Among those miRNAs is miR-34a which is a target gene of the tumor suppressor p53. In this paper we have re-examined the role of miR-34a-5p and miR-34a-3p in the signaling pathway of p53. We found that they target overlapping sets of genes (MDM2 and THBS1). By a GO enrichment analysis we found that THBS1 is involved in cancer and metastasis relevant processes. We have also deduce that p53, MDM2 and miR-34a are linked by a type 1 incoherent FFL that represent a novel mechanism for accelerating the response of p53 to external stress signals.

Ubiquitin ligases in oncogenic transformation and cancer therapy.

The cellular response to external stress signals and DNA damage depends on the activity of ubiquitin ligases (E3s), which regulate numerous cellular processes, including homeostasis, metabolism and cell cycle progression. E3s recognize, interact with and ubiquitylate protein substrates in a temporally and spatially regulated manner. The topology of the ubiquitin chains dictates the fate of the substrates, marking them for recognition and degradation by the proteasome or altering their subcellular localization or assembly into functional complexes. Both genetic and epigenetic alterations account for the deregulation of E3s in cancer. Consequently, the stability and/or activity of E3 substrates are also altered, in some cases leading to downregulation of tumour-suppressor activities and upregulation of oncogenic activities. A better understanding of the mechanisms underlying E3 regulation and function in tumorigenesis is expected to identify novel prognostic markers and to enable the development of the next generation of anticancer therapies. This Review summarizes the oncogenic and tumour-suppressor roles of selected E3s and highlights novel opportunities for therapeutic intervention.

Genome-wide identification, annotation and expression profile analysis of SnRK2 gene family in grapevine

Background and Aims Protein kinases are widely involved in the response in plants to external environment stress signals. Sucrose non-fermenting 1-related protein kinase 2 (SnRK2) is an important class of plant-specific protein kinases that plays a crucial role in abscisic acid responses and abiotic stresses across some model plant species. Genome-wide annotation, however, and specific expression patterns of SnRK2s in grapevine remain unclear. Methods and Results From a genome-wide survey of grapevine, we identified eight VviSnRK2 genes through phylogenetic and local BLAST methods and various bioinformatics analyses. Phylogenetic analysis indicated that these VviSnRK2s could be classified into three subgroups as found in Arabidopsis. Comparison of the structure of the genes with those of Arabidopsis suggests that intron length may be a special evolutionary imprint of the SnRK2 gene family in grapevine. Sequence alignment and structural annotation provided an overview of variations that may contribute to functional divergence from Arabidopsis SnRK2s. Expressional analyses based on public microarray data and quantitative real-time polymerase chain reaction and co-expression network analyses suggest both conserved and variant biological functions exist in SnRK2s across different species. Conclusions Our thorough analysis of the SnRK2 gene family in the grapevine genome revealed functional conservation and diversity synchronously existed in grapevine SnRK2 gene family. Significance of the Study Our research provides a comprehensive overview of both the expression pattern and functional diversity of the grapevine SnRK2 gene family, which will facilitate the future functional characterisation of individual VviSnRK2 genes.

NFATc4 Regulates Sox9 Gene Expression in Acinar Cell Plasticity and Pancreatic Cancer Initiation.

Acinar transdifferentiation toward a duct-like phenotype constitutes the defining response of acinar cells to external stress signals and is considered to be the initial step in pancreatic carcinogenesis. Despite the requirement for oncogenic Kras in pancreatic cancer (PDAC) development, oncogenic Kras is not sufficient to drive pancreatic carcinogenesis beyond the level of premalignancy. Instead, secondary events, such as inflammation-induced signaling activation of the epidermal growth factor (EGFR) or induction of Sox9 expression, are required for tumor formation. Herein, we aimed to dissect the mechanism that links EGFR signaling to Sox9 gene expression during acinar-to-ductal metaplasia in pancreatic tissue adaptation and PDAC initiation. We show that the inflammatory transcription factor NFATc4 is highly induced and localizes in the nucleus in response to inflammation-induced EGFR signaling. Moreover, we demonstrate that NFATc4 drives acinar-to-ductal conversion and PDAC initiation through direct transcriptional induction of Sox9. Therefore, strategies designed to disrupt NFATc4 induction might be beneficial in the prevention or therapy of PDAC.

Dysregulation of ubiquitin ligases in cancer.

Ubiquitin ligases (UBLs) are critical components of the ubiquitin proteasome system (UPS), which governs fundamental processes regulating normal cellular homeostasis, metabolism, and cell cycle in response to external stress signals and DNA damage. Among multiple steps of the UPS system required to regulate protein ubiquitination and stability, UBLs define specificity, as they recognize and interact with substrates in a temporally- and spatially-regulated manner. Such interactions are required for substrate modification by ubiquitin chains, which marks proteins for recognition and degradation by the proteasome or alters their subcellular localization or assembly into functional complexes. UBLs are often deregulated in cancer, altering substrate availability or activity in a manner that can promote cellular transformation. Such deregulation can occur at the epigenetic, genomic, or post-translational levels. Alterations in UBL can be used to predict their contributions, affecting tumor suppressors or oncogenes in select tumors. Better understanding of mechanisms underlying UBL expression and activities is expected to drive the development of next generation modulators that can serve as novel therapeutic modalities. This review summarizes our current understanding of UBL deregulation in cancer and highlights novel opportunities for therapeutic interventions.

Quercetin conjugated poly(β-amino esters) nanogels for the treatment of cellular oxidative stress

PβAE polymers have emerged as highly promising candidates for biomedical and drug delivery applications owing to their tunable, degradable and pH sensitive properties. These polymeric systems can serve as prodrug carriers for the delivery of bioactive compounds which suffer from poor aqueous solubility, low bioavailability and are biologically unstable, such as the antioxidant, quercetin. Using acrylate functionalized quercetin, it is possible to incorporate the polyphenol into the backbone of the polymer matrix, permitting slow release of the intact molecule which is perfectly timed with the polymer degradation. While formulating these quercetin conjugated PβAE matrix into nanocarriers would allow for multiple delivery routes (oral, intravenous, inhalation etc.), well known oil-water nano-emulsion formulation methods are not amenable to the crosslinked hydrolytically sensitive nanoparticle/nanogel. In this work, a single-phase reaction–precipitation method was developed to formulate quercetin conjugated PβAE nanogels (QNG) via reaction of acrylated quercetin (4–5 acrylate groups) with a secondary diamine under dilute conditions using acetonitrile as the reaction medium, resulting in a self-stabilized suspension. The proposed approach permits the post synthesis modification of the spherical nanogels with a PEGylated coating, enhancing their aqueous stability and stealth characteristics. Nanogel size was controlled by varying feed reactant concentrations, achieving drug loadings of 25–38wt%. Uniform release of quercetin over 45–48h was observed upon PβAE ester hydrolysis under physiological conditions with its retained antioxidant activity over the extended times. Statement of SignificanceHere we present the first demonstration of using poly(beta amino ester) chemistry to form nanogels composed of a bioactive polyphenol for the control of cellular oxidative stress. Previous nanogel and nanoparticle approaches, which use a water phase, are not readily amenable to PBAE chemistry due to their hydrolytic sensitivity. Here we demonstrate a simple approach to control particle size, modify surface chemistry and achieve highly regulated controlled release of active antioxidants, which can protect cells against external oxidative stress signals. This work has importance in the area of controlling material biocompatibility through augmenting the antioxidant status of cells.

Extraction and Analysis of Oxylipins from Macroalgae Illustrated on the Example Gracilaria vermiculophylla.

Oxylipins are natural products that are derived by oxidative transformations of unsaturated fatty acids. These metabolites are found in a wide range of organisms from the animal kingdom to plants and algae. They represent an important class of signaling molecules, mediating intra- and intercellular processes such as development, inflammation, and other stress responses. In addition, these metabolites directly function as chemical defense against grazers and pathogens. In the red alga Gracilaria vermiculophylla, oxylipin production is initiated by mechanical tissue disruption and can also be induced in intact algae in response to external stress signals. The defense metabolites mostly result from the lipase- and lipoxygenase-mediated conversion of phospho- and galactolipids. Oxylipins can vary greatly in their size, degree of unsaturation, oxidation state, and functional groups. But also isomers with only subtle chemical differences are found. A variety of methods have been developed for separation, detection, and identification of oxylipins. This chapter focuses on the analysis of oxylipins in macroalgae and covers all aspects from sample preparation (including protocols for the investigation of oxylipins in wounded and intact algal tissue), extraction, purification, and subsequent analysis using liquid chromatography coupled to a UV detector or a mass spectrometer. The protocols developed for G. vermiculophylla can be readily adapted to the investigation of other macroalgae.