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Epigenetic regulation by histone modification can activate or repress transcription through changes in chromatin dynamics and regulates development and the response to environmental signals in both animals and plants. Chromatin immunoprecipitation (ChIP) is an indispensable tool to identify histones with specific post-translational modifications. The lack of a ChIP technique for macroalgae has hindered understanding of the role of histone modification in the expression of genes in this organism. In this study, a ChIP method with several modifications, based on existing protocols for plant cells, has been developed for the red macroalga, Neopyropia yezoensis, that consists of a heterogeneous alternation of macroscopic leaf-like gametophytes and microscopic filamentous sporophytes. ChIP method coupled with qPCR enables the identification of a histone mark in generation-specific genes from N. yezoensis. The results indicate that acetylation of histone H3 at lysine 9 in the 5 ' flanking and coding regions from generation-specific genes was maintained at relatively high levels, even in generation-repressed gene expression. The use of this ChIP method will contribute significantly to identify the epigenetic regulatory mechanisms through histone modifications that control a variety of biological processes in red macroalgae.

Heat stress disrupts algal growth, development, and physiological processes, such as photosynthesis, and eventually decreases seaweed productivity. Previous studies of crop plants have revealed that exogenous application of phytohormones prior or parallel to stress can alleviate the negative effects of abiotic stressors, including heat stress. However, there is limited information on phytohormone-induced tolerance to abiotic stressors in seaweed. In the present study, the application of the major plant hormones abscisic acid and salicylic acid failed to mitigate the negative effects of heat stress on the marine red alga Neopyropia yezoensis, whereas 1-aminocyclopropane-1-carboxylic acid (ACC), the direct precursor of the plant hormone ethylene, regulates thermotolerance. In addition, the ACC analogs 1-aminocyclobutane-1-carboxylic acid and alpha-aminoisobutyric acid enhanced tolerance to heat stress. ACC increased the expression of genes involved in antioxidant defense systems to protect photosynthesis and respiration. These results suggest ACC acts as a phytohormone to mitigate the impact on heat stress independent of ethylene in N. yezoensis.

Heat stress disrupts algal growth, development, and physiological processes, such as photosynthesis, and eventually decreases seaweed productivity. Previous studies of crop plants have revealed that exogenous application of phytohormones prior or parallel to stress can alleviate the negative effects of abiotic stressors, including heat stress. However, there is limited information on phytohormone-induced tolerance to abiotic stressors in seaweed. In the present study, the application of the major plant hormones abscisic acid and salicylic acid failed to mitigate the negative effects of heat stress on the marine red alga Neopyropia yezoensis, whereas 1-aminocyclopropane-1-carboxylic acid (ACC), the direct precursor of the plant hormone ethylene, regulates thermotolerance. In addition, the ACC analogs 1-aminocyclobutane-1-carboxylic acid and alpha-aminoisobutyric acid enhanced tolerance to heat stress. ACC increased the expression of genes involved in antioxidant defense systems to protect photosynthesis and respiration. These results suggest ACC acts as a phytohormone to mitigate the impact on heat stress independent of ethylene in N. yezoensis.

Background 1-aminocyclopropane 1-carboxylic acid (ACC) is the immediate precursor of the plant hormone ethylene. However, recent studies have suggested that ACC also acts as a signaling molecule to regulate development and growth independently from ethylene biosynthesis. In red algae, ACC stimulates the switch from a vegetative to a sexual reproductive phase. However, despite evidence that ACC signaling in plants and algae is widespread, the mechanistic basis of the ACC signaling pathway remains unknown. Results We demonstrate that exogenous ACC increased the activity of phospholipase D (PLD) and induced the accumulation of PLD transcripts in the marine red alga Neopyropia yezoensis. The product of PLD, the lipid second messenger phosphatidic acid (PA), also increased in response to ACC. Furthermore, the pharmacological inhibition of PLD by 1-butanol blocked ACC-induced spermatangia and carpospore production, but the inactive isomer t-butanol did not. In addition, 1-butanol prevented ACC-induced growth inhibition and inhibited transcript accumulation of genes upregulated by ACC, including extracellular matrix (ECM)-related genes, and alleviated the transcriptional decrease of genes downregulated by ACC, including photosynthesis-related genes. Conclusions These results indicate that PLD is a positive regulator of sexual cell differentiation and a negative regulator of growth. This study demonstrates that PLD and its product, PA, are components of ACC signaling during sexual reproduction in N. yezoensis.

The process of biogenic silicon (Si) deposition in sporophytes of kelp (Saccharina japonica) was investigated by culturing sporophyte disks in the medium with and without hydrogen peroxide (H2O2). Culture with H2O2 remarkably promoted silicate-Si uptake by the disks. Silicate-Si uptake was completely inhibited by the treatment with potassium bromide (KBr), a competitive inhibitor of iodoperoxidase (IPO), even in the medium with H2O2. In addition, disk culture with phenol red in the medium with H2O2 resulted in the formation of bromophenol blue, indicating the presence of extracellular IPO. However, KBr treatment inhibited the formation of bromophenol blue in the medium with H2O2. These results showed that silicate-Si uptake is activated by extracellular IPO in the presence of radical oxygen species (ROS), such as H2O2. Interestingly, 1 mM salicylic acid also promoted silicate-Si uptake, even in the medium without H2O2. Furthermore, the allocation of silicate-Si to the soluble and cell wall-bound phlorotannin fractions using rhodamine 123 (R123), a tracer of biogenic Si, was estimated. Tracer fluorescence was detected in not only the soluble but also the cell wall-bound phlorotannin fraction. R123 fluorescence was histologically detected in the apoplasts of the epidermal and outer cortical layers of the disk, overlapping the histochemical distribution of IPO activity. These results suggested that silicate-Si uptake is activated by an oxidative burst induced by various stresses, and Si deposition is catalyzed by extracellular IPO in the presence of both ROS and phlorotannin in the extracellular matrix of sporophytes. Based on these findings, the hypothetical mechanisms of the extracellular silicate-Si uptake for defense responses in kelp were discussed.

The transition from the vegetative to sexually reproductive phase is the most dynamic change to occur during a plant's life cycle. In the present study, we showed that the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) induces sexual reproduction in the marine red alga Pyropia yezoensis independently from ethylene. Exogenous application of ACC, which contains a three membered carbocyclic ring, promoted the formation of spermatia and carporspores in gametophytes, whereas ethephon, an ethylene-releasing compound, did not stimulate sexual reproduction. In addition, an ACC analog, 1-aminocyclobutane-1-carboxylic acid (ACBC), which contains a four membered carbocyclic ring, promoted sexual reproduction and enhanced tolerance to oxidative stress in the same manner as ACC, but 1-aminocyclopentane-1-carboxylic acid (cycloleucine; which contains a cyclopentane ring) did not. The application of ACC increased the generation of reactive oxygen species (ROS) and induced the expression of PyRboh gene encoding NADPH oxidase. ACC also stimulated the synthesis of ascorbate (AsA) by inducing transcripts of PyGalLDH, which encodes galactono-1,4-lactone dehydrogenase, the catalyst for the final enzymatic step of the AsA biosynthetic pathway. Conversely, ACC caused a decrease in the synthesis of glutathione (GSH) by repressing transcripts of PyGCL, which encodes glutamate cysteine ligase, the catalyst for the rate-limiting step in the formation of GSH. These results suggest a possible role played by ACC as a signaling molecule independent from ethylene in the regulation of sexual reproduction through alterations to the redox state in P. yezoensis.

The transition from the vegetative to sexually reproductive phase is the most dynamic change to occur during a plant's life cycle. In the present study, we showed that the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) induces sexual reproduction in the marine red alga Pyropia yezoensis independently from ethylene. Exogenous application of ACC, which contains a three membered carbocyclic ring, promoted the formation of spermatia and carporspores in gametophytes, whereas ethephon, an ethylene-releasing compound, did not stimulate sexual reproduction. In addition, an ACC analog, 1-aminocyclobutane-1-carboxylic acid (ACBC), which contains a four membered carbocyclic ring, promoted sexual reproduction and enhanced tolerance to oxidative stress in the same manner as ACC, but 1-aminocyclopentane-1-carboxylic acid (cycloleucine; which contains a cyclopentane ring) did not. The application of ACC increased the generation of reactive oxygen species (ROS) and induced the expression of PyRboh gene encoding NADPH oxidase. ACC also stimulated the synthesis of ascorbate (AsA) by inducing transcripts of PyGalLDH, which encodes galactono-1,4-lactone dehydrogenase, the catalyst for the final enzymatic step of the AsA biosynthetic pathway. Conversely, ACC caused a decrease in the synthesis of glutathione (GSH) by repressing transcripts of PyGCL, which encodes glutamate cysteine ligase, the catalyst for the rate-limiting step in the formation of GSH. These results suggest a possible role played by ACC as a signaling molecule independent from ethylene in the regulation of sexual reproduction through alterations to the redox state in P. yezoensis.