Next Article in Journal
A High-Precision Plant Disease Detection Method Based on a Dynamic Pruning Gate Friendly to Low-Computing Platforms
Next Article in Special Issue
The Cell Wall Characterization of Brown Alga Cladosiphon okamuranus during Growth
Previous Article in Journal
Variation in Vegetation Phenology and Its Response to Climate Change in Marshes of Inner Mongolian
Previous Article in Special Issue
Characterization of Chlorophyll Fluorescence and Antioxidant Defense Parameters of Two Gracilariopsis lemaneiformis Strains under Different Temperatures
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 12
Issue 11
10.3390/plants12112070
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Membrane Fluidization Governs the Coordinated Heat-Inducible Expression of Nucleus- and Plastid Genome-Encoded Heat Shock Protein 70 Genes in the Marine Red Alga Neopyropia yezoensis

by
Koji Mikami
1,* and
Ho Viet Khoa
2,†
1
School of Food Industrial Sciences, Miyagi University, Hatatate 2-2-1, Sendai 982-0215, Japan
2
Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-Cho, Hakodate 041-8611, Japan
*
Author to whom correspondence should be addressed.
†
Current address: Training Department, Ca Mau Community College, 126, 3/2 Street, Ward 6, Ca Mau City 98116, Vietnam.
Plants 2023, 12(11), 2070; https://doi.org/10.3390/plants12112070
Submission received: 6 May 2023 / Revised: 18 May 2023 / Accepted: 19 May 2023 / Published: 23 May 2023
(This article belongs to the Special Issue Current Topics in Macroalgal Research)
Browse Figures
Review Reports Versions Notes

Abstract

:
Heat shock protein 70 (HSP70) is an evolutionarily conserved protein chaperone in prokaryotic and eukaryotic organisms. This family is involved in the maintenance of physiological homeostasis by ensuring the proper folding and refolding of proteins. The HSP70 family in terrestrial plants can be divided into cytoplasm, endoplasmic reticulum (ER)-, mitochondrion (MT)-, and chloroplast (CP)-localized HSP70 subfamilies. In the marine red alga Neopyropia yezoensis, the heat-inducible expression of two cytoplasmic HSP70 genes has been characterized; however, little is known about the presence of other HSP70 subfamilies and their expression profiles under heat stress conditions. Here, we identified genes encoding one MT and two ER HSP70 proteins and confirmed their heat-inducible expression at 25 °C. In addition, we determined that membrane fluidization directs gene expression for the ER-, MT-, and CP-localized HSP70 proteins as with cytoplasmic HSP70s. The gene for the CP-localized HSP70 is carried by the chloroplast genome; thus, our results indicate that membrane fluidization is a trigger for the coordinated heat-driven induction of HSP70 genes harbored by the nuclear and plastid genomes in N. yezoensis. We propose this mechanism as a unique regulatory system common in the Bangiales, in which the CP-localized HSP70 is usually encoded in the chloroplast genome.

1. Introduction

Heat shock proteins (HSPs) were first discovered by the observation of rapid heat-inducible transcription during chromosome puffing in the fruit fly Drosophila melanogaster [1,2]. HSPs are ubiquitous, conserved in prokaryotes and eukaryotes, and accumulate under not only heat stress conditions, but also normal conditions and in response to other abiotic and biotic stresses [3,4,5]. The major function of HSPs is the maintenance of protein homeostasis by their chaperone activity, which assists with the folding of three-dimensional proteins during translation and the refolding of denatured proteins under stress conditions [6,7]. Thus, HSPs contribute to the stabilization of proteins, enabling cells to cope with the misfolding, unfolding, degradation, and aggregation of proteins under both optimum and adverse growth conditions.
HSPs are classified into subgroups based on their molecular weight, such as HSP of about 100 kDa (HSP100, also named Clp), HSP90 (HtpG), HSP70 (DnaK), HSP60 (GroEL), HSP40 (DnaJ), HSP10 (GroES), and small HSPs [6,7]. Of these, HSP70 (DnaK) proteins are the most abundant chaperones with high evolutionary conservation [8,9,10], and in collaboration with its co-chaperones HSP40 (DnaJ) and GrpE constitutes a functionally active chaperone [6,11]. Photosynthetic organisms possess four types of HSP70s, which localize to different cellular compartments: the cytoplasm, endoplasmic reticulum (ER), mitochondrion (MT), and chloroplast (CP) [12,13]. Gene duplication is proposed to have occurred following the divergence of the cytoplasm- and ER-localized HSP70 genes, whereas lateral gene transfer by endosymbiosis established the genes encoding MT- and CP-localized HSP70s in eukaryotic nuclei [14,15]. Based on their distinct localizations, HSP70s play important chaperone roles in a variety of cellular responses.
Much attention has been paid to resolving the transcriptional regulation of HSP genes under heat stress conditions. Studies into the underlying signal transduction pathways have identified several transcription factors directing the heat-inducible transcription of target genes, for instance, heat shock factor (HSF) [16,17,18], multiprotein bridging factor 1 (MBF1) [19,20], and DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) [21,22]. Other studies have focused on the trigger of the heat-inducible expression of HSP genes, which is related to the elucidation of how photosynthetic organisms sense heat stress [23,24,25]. This approach revealed the induction of HSP gene expression by heat-induced membrane fluidization in prokaryotes and eukaryotes [23,24,25,26,27,28,29,30,31,32], thus raising the possibility that organisms sense membrane fluidization as a signal of heat stress [33,34,35]. Ca2+ channels on cellular membranes have been proposed to act as heat sensors in terrestrial plants, such as the moss Physcomitrium patens and Arabidopsis (Arabidopsis thaliana) [36,37,38]. By contrast, HSP expression does not depend on membrane fluidization in the unicellular green alga Chlamydomonas reinhardtii [39]; thus, it is unclear whether the heat-inducible expression of HSP genes completely depends on membrane fluidization in other eukaryotes.
HSP70 genes have been identified in marine multicellular algae called seaweeds, including the green alga Ulva porfiera [40]; the brown algae Fucus spp. [41] and Fucus distichus [42]; and the Bangiales order of red algae that includes Porphyra umbilicalis [43,44], Neopyropia yezoensis [45,46], and Neoporphyra haitanensis [47]. Most of these HSP genes were found to be heat inducible. A distinctive feature of CP-localized HSP70s in the Bangiales is that they are encoded by DnaK genes in the chloroplast genome [43,48,49]; however, the expression profiles of these genes are unknown. A comparison of the mechanisms regulating the transcription of the HSP70 genes in nuclear and chloroplast genomes is necessary to understand the unique heat stress response in the Bangiales.
We recently analyzed the importance of the membrane physical state for the induction of heat-inducible gene expression in N. yezoensis [50], revealing that membrane fluidization by heat stress induced the expression of two cytoplasmic HSP70s and MBF1 but not the newly identified HIGH TEMPERATURE RESPONSE 2 (HTR2) or HTR2 LIKE (HTR2L). This observation indicates that N. yezoensis possesses membrane fluidization-dependent and -independent signal transduction pathways in its heat stress response, suggesting the presence of an additional unknown trigger for heat-inducible gene expression. These findings prompted us to question whether the nuclear genes for ER- and MT-localized HSP70 proteins and the chloroplast gene for CP-localized HSP70 are heat inducible and, if so, whether the trigger(s) for the heat-inducible expression of these nuclear and plastid genes is common or distinct in N. yezoensis.
In the present study, we identified the genes for ER- and MT-localized HSP70s in N. yezoensis and confirmed the heat inducibility of these nuclear genes and the previously identified cytoplasmic and plastid HSP70 genes. In addition, we determined the contribution of membrane fluidization in the heat-inducible expression of all HSP genes. Our findings reveal the trigger of the heat-induced expression of the HSP70 genes in N. yezoensis and provide novel and unique insights into the coordinated expression of the nuclear and plastid genomes, as well as the diversification of the regulatory mechanisms of the heat stress response in photosynthetic organisms.

2. Results

2.1. Identification of the Genes for ER and MT HSP70 in N. yezoensis

Using a functional annotation of our previous transcriptome data [51], we identified five contigs and unigenes (CL1648.Contig2, CL2684, Unigene14751, Unigene14752, and Unigene2869) representing candidate HSP70 genes. To classify these genes, we performed a phylogenetic analysis using the amino acid sequences encoded by these genes and HSP70s from unicellular red algae and the Bangiales. We included HSP110 members, which are related to cytosolic HSP70s but exhibit unique structural characteristics in the ATPase- and C-terminal domains [52,53,54], as the outgroup. We also included the previously identified CP HSP70, designated NycpDnaK, encoded by the chloroplast genome of N. yezoensis (GenBank accession number: DQ497595).
The translational products of all candidate genes contained three HSP70 family signatures [55] in their N-terminal ATPase domains (Figure S1); thus, we concluded that these are bona fide HSP70 genes in N. yezoensis. As shown in Figure 1, these proteins clustered into two major clades, each consisting of two subclades. The previously identified genes NyHSP70-1 and NyHSP70-2 [45,50], which correspond to Unigene14751 and CL1648.Contig2, respectively, grouped in the same subclade. Members of this subclade contain the cytoplasmic HSP70-specific motif EDID or SEVD (Figure S1), taking on the typical sequence of EEVD in the HSP70s of other eukaryotic organisms [54,55]. This subclade is therefore a distinct class of cytoplasmic HSP70s. The other subclade in the same major clade contained the proteins encoded by Unigene2869 and CL2684. These proteins contain a putative ER-retention tetrapeptide, K/HEEL (typically K/HDEL) [54,55]; therefore, we conclude that this subclade is for ER-localized HSP70 (also named binding protein [BiP]), and we designated them NyBiP1 and NyBiP2, respectively (Figure 1). We detected a 43-amino acid signal peptide (MAATPGSPARRPRPAAAKRRSVWTAVLVAAASIATATVAVSAA) in NyBiP1, while BiP2 did not appear to harbor a signal peptide.
Chloroplast genome-encoded NycpDnaK and proteins included in the same subclade possessed the motif TVIDTDFSEAK (Figure S1), which is related to the DVIDADFTDSK motif conserved in chloroplast HSP70s [54]. This subclade therefore contains the CP-localized HSP70s. The proteins in the rest of the subclade had characteristic motifs, such as GDAWVS and YSPSQV (Figure S1), which are conserved signature sequences for MT-localized HSP70s [54]. We thus designated Unigene14751 in this subclade NymtDnaK. Its encoded protein had a 33-amino acid signal peptide (MASAVSARAALARVGACRPGLLGLAASGGGGLA).
These findings indicate that the Bangiales, including N. yezoensis and unicellular red algae, possess all four classes of HSP70s, as well as HSP110, found in terrestrial plants [14,54,56,57]. The ER and MT HSP70s are newly identified; we deposited the sequence information of NyBiP1, NyBiP2, and NymtDnaK to DDBJ/EMBL/GenBank (accession numbers LC764584, LC764585 and LC764586, respectively). The NyHSP110 sequence was also deposited (LC764587).

2.2. Heat-Inducible Expression of the ER, CP, and MT HSP70 Genes

Our previous study demonstrated that when algal samples were cultured under a 10 h light and 14 h dark photoperiod at 15 °C, the expression of NyHSP70-1 and NyHSP70-2 was induced transiently by a treatment of 25 °C, peaking 2 h after the onset of temperature increase and again after 1 h of darkness (8 h after the beginning of the higher temperature treatment) [50]. We therefore analyzed the expression profiles of genes for the ER-, CP-, and MT-localized HSP70s in the three life stages—the gametophyte, sporophyte, and conchosporophyte [51]—under these conditions using the gene-specific primer sets indicated in Table S1.
As shown in Figure 2, NyBiP1 and NyBiP2 expression was transiently induced twice as much as observed for cytoplasmic HSP70 genes when algae were grown at 25 °C but not at 5 °C in all three life stages, indicating the transient heat-inducible and dark-stimulated expression of these genes. However, we observed differences in the expression profiles of the genes: NyBiP1 was mainly induced by 25 °C with a peak at 2 h into the higher temperature in the sporophyte and conchosporophyte, whereas NyBiP2 was mainly induced by darkness under heat stress conditions in all three life stages (8 h after onset of higher temperature and 1 h of darkness). In addition, the expression of two organellar HSP70 genes, NycpDnaK and NymtDnaK, was induced at 25 °C in the light and darkness at 25 °C, with expression peaks at 2 and 8 h into the temperature increase, although NycpDnaK was mainly induced by darkness and its expression level in the conchosporophyte remained low (Figure 3). Furthermore, when the algal samples were cultured under continuous light to explore the factor required for the second expression peak, we noticed that the dark-stimulated expression of the genes for ER- and organelle-localized HSP70 proteins is commonly inhibited by light in all three life stages (Figure S2). This result indicates that darkness, and not the duration since onset of heat treatment, is the factor required for the expression of the organellar HSP70-encoding genes under heat stress conditions, as was also shown previously for the genes encoding two cytoplasmic HSP70s [50].

2.3. Contribution of Membrane Fluidization to the Heat-Inducible and Dark-Stimulated Expression of HSP70 Genes

Our previous findings demonstrated the presence of two different heat stress signal transduction cascades, the membrane fluidization-dependent and -independent pathways, and the involvement of the former in NyHSP70-1, NyHSP70-2, and NyMBF1 expression [50]. To determine which pathway regulates the heat-inducible expression of the genes for ER- and organelle-localized HSP70 proteins, we examined the effects of physiological membrane changes on the expression of these genes using a membrane fluidizer (benzyl alcohol; BA) and a membrane rigidizer (dimethyl sulfoxide; DMSO). The treatment with 2.5 mM BA, but not 4% (v/v) DMSO, induced the expression of all four genes at 15 °C with similar expression kinetics, with an expression peak observed 15 min after the onset of pharmacological treatment (Figure 4 and Figure 5). The heat-induced gene expression for ER- and organelle-localized HSP70 is thus regulated by the membrane fluidization-dependent pathway.
We asked whether the second peak seen under darkness and heat conditions requires membrane fluidization. Accordingly, we examined the effects of 2.5 mM BA on the expression of the genes for ER- and organelle-localized HSP70 under continuous light in the three life stages at 15 °C. We determined that BA induces the expression of all genes at 1 and 5 h into treatment in the three life stages (Figure 6). We also subjected the three life stages of the alga to heat stress, either with or without 4% DMSO, under dark conditions. This DMSO treatment decreased the dark-stimulated expression of all genes (Figure 7). Thus, darkness under heat stress conditions further fluidizes the membranes whose state of the fluidity has already adapted to 25 °C under the light, resulting in the promotion of the membrane fluidization-dependent induction of the HSP70 genes. These findings indicate that the dark-stimulated expression of the genes encoding ER and organelle-localized HSP70 is regulated by a pathway activated by membrane fluidization in N. yezoensis.

2.4. Effects of Temperature Changes on the Expression of Plastid Genes for the Two-Component System

Red algal plastid genomes contain genes encoding one histidine kinase (Ycf26) and four response regulators (Ycf27, Ycf28, Ycf29, and Ycf30) [58]. Of these, Ycf26 is proposed to regulate the transcription of the chloroplast gene NycpDnaK, as its homolog in the photosynthetic cyanobacterium Synechocystis sp. PCC 68803, Hik33, contributes to inducing gene expression in an environmental stress-dependent manner [59,60].
To explore this possibility, we examined the effects of temperature changes on the expression of genes encoding Ycf26 and Ycf27, a response regulator proposed to work together with Ycf26 as a two-component system. We detected no response for Ycf26 or Ycf27 expression to either 5 °C (cold stress) or 25 °C (heat stress) in the three life stages (Figure S3). Thus, the transcriptional induction of these genes might not be involved in the heat-inducible expression of NycpDnaK.

3. Discussion

HSP70 is conserved in a wide variety of organisms [8,9,10], and the regulatory mechanisms underlying the heat-inducible expression of the encoding genes have been extensively studied [13,14,15,16,17,18]. Despite these insights, the analysis of heat-inducible transcription of the HSP70 genes in the Bangiales has been limited to genes encoding cytoplasmic HSP70 in N. yezoensis [45,50]. Here, we identified new members of the HSP70 family and analyzed their expression profiles under heat stress conditions in the marine red alga N. yezoensis. A phylogenetic analysis indicated that the family members were divided into four classes, cytoplasmic HSP70, ER-localized HSP70 (BiP), CP-localized HSP70, and MT-localized HSP70, as observed in other eukaryotes [12,13]. In addition, genes for ER- and organelle-localized HSP70s display heat-induced and dark-stimulated expression in the three studied life stages. Genes for cytoplasmic HSP70 also displayed these characteristics [50], leading us to conclude that the expression of all HSP70 genes in N. yezoensis is heat inducible in the three life stages studied, as previously reported for NyMBF1 [50]. Therefore, our study demonstrates for the first time that ER- and organelle-localized HSP70 proteins are encoded by heat-inducible genes whose expression is regulated by the membrane fluidization-dependent signal transduction pathway in the red alga N. yezoensis. Together with our previous data regarding cytoplasmic HSP70 [50], we conclude that genes of all four HSP70 classes are commonly and coordinately induced by heat stress in a membrane fluidization-dependent manner.
Recently, a genome-wide survey of the HSP70 genes in N. yezoensis led to the identification of 15 genes [46], although these included HSP110 genes, the number may not be accurate. In their study, four classes of HSP70s were proposed, as in our present study; however, their phylogenetic tree was different from the one presented in Figure 1. Our tree clearly demonstrated the separation of cytoplasm- and ER-localized HSP70s from organellar HSP70s, revealing differences in the origins of the organellar HSP70s and the other HSP70s [14,15]. By contrast, the phylogenetic tree produced by Yu et al. [46] included ER-localized HSP70s in the clade containing organellar HSP70s, and one of the CP-localized HSP70s was located in the cytoplasmic HSP70s clade. It is not clear why such a difference occurred; thus, we must identify other HSP70 genes in N. yezoensis and revise the phylogenetic trees to validate their true relationships and origins. Moreover, Yu et al. [46] reported the presence of two nuclear genes encoding chloroplast-localized HSP70 in N. yezoensis. They used established analysis tools to predict the subcellular localization of HSP70s; however, it remains necessary to identify the conserved amino acid sequence motifs and putative chloroplast signal peptides to resolve the uncertain classification and characterization of the N. yezoensis HSP70s.
NycpDnaK is characteristically a chloroplast gene in red algae [43,48,49], while this chloroplast-localized HSP70 is usually a nuclear gene in other eukaryotes [61,62,63]. The red algae genes encoding the chaperonin GroES and two-component histidine kinase and response regulators have also been retained in the chloroplast genome, while their homologs in terrestrial plants are in the nuclear genome. The red algal chloroplast genome thus appears to be in an evolutionarily intermediate state, with an incomplete transfer of genes from the ancient symbiotic chloroplast genome to the nuclear genome. The maintenance of these genes in the organelle may provide functional benefits in red algae, however, enabling them to survive in the ever-changing conditions of the intertidal region. In the presence of chloroplast-targeted DnaJ proteins, for example, the heat-inducible expression of NycpDnaK might be faster from the chloroplast than the nuclear genome, rapidly producing HSP70s (and the HSP70/HSP40 chaperone complex) to protect and fine-tune various organellar proteins. This hypothesis might be supported by the presence of GroEL and GroES as an operon in the chloroplast genomes of the Bangiales, facilitating the rapid supply of the HSP60/HSP10 chaperonin complex in this organelle, although it is uncertain whether the expression of the GroEL-GroES operon is heat inducible. To understand why NycpDnaK and GroES are located in the chloroplast genome of the Bangiales, the regulatory mechanisms of their transcription should be analyzed under various environmental stress conditions.
Although membrane fluidization is a common trigger of the heat-inducible expression of N. yezoensis HSP70 genes, the regulatory mechanisms of heat-inducible transcription are proposed to be different between nuclear and chloroplast genes. The involvement of HSF, a transcription factor responsible for controlling the heat-inducible expression of HSP genes, has been confirmed in many eukaryotes [64,65]. Red algae also have HSF [66,67], but the identification and expression analysis of the HSF gene(s) have yet to be conducted in N. yezoensis. In addition, the expression of chloroplast genes is regulated by nucleus-encoded transcription factors called sigma factors, which contribute to the differential expression of chloroplast genes under various environmental stress conditions. In this study, we discovered that the expression of genes encoding histidine kinase and the response regulator were not induced by heat stress (Figure S3); however, it is highly possible that the heat-dependent activation of Ycf26 by histidine phosphorylation and the phosphorylation of Ycf27 by activated Ycf26 are involved in NycpDnaK expression. The identification of transcription factors and the elucidation of the functional mode of the chloroplast two-component system are therefore essential for understanding the regulatory mechanisms underlying the coordinated heat-inducible gene expression for nuclear and chloroplast HSP70 genes in N. yezoensis.
The involvement of membrane fluidization in heat-inducible gene expression suggests the presence of a heat sensor that perceives heat-dependent changes in the membrane physical state and acts as an initiator to activate heat signal transduction pathways that govern the expression of all HSP genes in N. yezoensis. It has been proposed that membrane-localized calcium ion (Ca2+) channels are heat sensors in terrestrial plants [36,37,38,68]. Indeed, many lines of evidence indicate the contribution of Ca2+ influxes in the early phase of the heat stress response in terrestrial plants [36,37,38,68,69,70]; in moss, the activation of plasma membrane-embedded cyclic nucleotide-gated Ca2+ channels depends on membrane fluidization [36,37,38,68]. Thus, it is plausible that Ca2+ channels might be heat sensors that become activated by the heat-induced membrane fluidization in N. yezoensis; however, little is currently known about the heat sensor and heat stress signal transduction pathways. Understanding how membrane fluidization activates the signal transduction pathways that target the expression of nuclear and chloroplast HSP70 genes will therefore require the elucidation of whether Ca2+ influx is involved in either or both signal transduction pathways. These experiments could enable us to examine the relationship between Ca2+ influx and the expression and activation of transcription factors such as HSFs and sigma factors in red algae. The resulting insights may permit the identification of the heat sensor and the elucidation of the regulatory mechanisms involved in the recognition and transmission of the membrane fluidization signal to different signaling pathways to regulate the heat-inducible expression of the N. yezoensis HSP70 genes in a coordinated manner.
Together with our previous results [50], our findings demonstrate the presence of two different cascades for regulating heat-inducible gene expression in N. yezoensis. One is the membrane fluidization-dependent pathway that regulates multiple HSP70 and NyMBF1 genes, which are part of different sub-pathways for the regulation of heat-inducible nuclear and chloroplast genes as a unique system in the Bangiales. The other is the membrane fluidization-independent pathway for the expression of genes encoding the uncharacterized proteins NyHTR2 and NyHTR2L [50]; the trigger for the activation of this pathway is unknown. Signaling factors such as Ca2+ and the transcription factors involved in each pathway must be elucidated to identify the heat sensors and illuminate the entire regulatory system of the heat stress response mediated by multiple heat signal transduction pathways with their own heat-inducible target genes in N. yezoensis.

4. Materials and Methods

4.1. Algal Materials and Culture Conditions

Artificial sterilized seawater (SEALIFE, Marinetech, Tokyo, Japan) enriched with ESS2 [71] was used for the maintenance and examination of the three life stages of N. yezoensis (strain U-51)—gametophytes, sporophytes, and conchosporophytes [51]—under 60–70 μmol m–2 s–1 of light with a short-day photoperiod (10 h light/14 h dark) at 15 °C. Air was filtered through a 0.22 μm filter (Whatman, Maidstone, UK). The culture medium was changed weekly.
Heat stress and pharmacological treatments were performed as described by Khoa and Mikami [50]. In brief, the three life stages were incubated at 5 °C, 15 °C, or 25 °C for 0.5, 1, 2, 4, 6, 8, and 12 h. The experiments started at 12:00, 3 h after the start of light irradiation (9:00). In the short-day photoperiod with 10 h light, the 8 and 12 h time points comprised 1 and 5 h of darkness, respectively. Continuous 24 h lighting was also employed as a control condition. Treatments with 2.5 mM of benzyl alcohol (BA) and 4% dimethyl sulfoxide (DMSO) were performed by incubating algae at 15 °C for 5, 15, or 30 min. Algal cells were also treated with BA at 25 °C in the light or with DMSO under dark conditions for 8 or 12 h. After these treatments, algae were frozen in liquid nitrogen and stored at −80 °C prior to their use for gene expression analysis.

4.2. Identification and Phylogenetic Analysis of HSP70 Genes

Contigs and unigenes annotated as HSP70 genes in our N. yezoensis transcriptome data [51] were examined with open reading frame (ORF) finder (https://www.ncbi.nlm.nih.gov/orffinder/ accessed on 10 March 2023) to identify full-length coding sequences. A BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the predicted amino acid sequences for their translated products was performed to validate their HSP70 identity. The presence of a signal peptide was examined using the SOSUIsignal program (https://harrier.nagahama-i-bio.ac.jp/sosui/sosuisignal/sosuisignal_submit.html accessed on 15 April 2023). The amino acid sequences of all obtained HSP70 homologs were combined with previously known red algal HSP70s to reconstruct a neighbor-joining phylogenetic tree in MEGA 7 (https://www.megasoftware.net), using ClustalW to align the sequences.

4.3. Total RNA Extraction, DNA Removal, cDNA Synthesis, and Quantitative Gene Expression Analysis

All procedures were performed as described by Khoa and Mikami [50], except that primers for the HSP70 genes were used (Table S1) for reverse transcription quantitative PCR (RT-qPCR). Total RNAs from the three stages were separately extracted using a FavorPrep Plant Total RNA Mini Kit (FAVORGEN, Ping Tung, Taiwan) and subjected to treatment with DNase I (TURBO DNA-free TM kit; Thermo Fisher Scientific, Waltham, MA, USA) to remove any genomic DNA contamination. Total RNA samples (300 ng) with A260/A280 ratios ranging from 1.9 to 2.1 were used to synthesize first-strand complementary DNA (cDNA) using a PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio, Kusatsu, Japan). The thermal cycling parameters consisted of an initial denaturation step at 98 °C for 30 s, followed by 30 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 20 s and a final extension step at 72 °C for 5 min.
The primers for the HSP70 genes were designed using Primer Premier 5 (http://www.premierbiosoft.com accessed on 11 December 2021). The sizes of the amplified products and the specificity of the primers were confirmed using PCR reactions with a mixture of three cDNA samples and each primer set, using a Phusion high-fidelity DNA polymerase and GC buffer (Biolabs, Boston, MA, USA), according to the manufacturer’s instructions. The PCR products were checked by agarose gel electrophoresis. Primer sets generating amplified DNA bands with the expected sizes were employed for the qPCR, which was carried out in a total volume of 20 μL, containing 10 μL of 2× SYBR Premix Ex Taq GC, 0.4 μL of ROX Reference Dye, 2 μL of cDNA template, and 0.4 μL (10 μM) of each primer, using a SYBR Premix Ex Taq GC kit (Takara Bio). The thermal cycling parameters for the AriaMX (3000P) real-time PCR system (Agilent Technologies, Santa Clara, Hercules, CA, USA) consisted of 95 °C for 3 min and 40 cycles of 95 °C for 5 s and 60 °C for 20 s. A dissociation curve was generated to check for the specificity of amplification using the following conditions: 95 °C for 1 min, 55 °C for 30 s, and 95 °C for 30 s. The translation initiation factor 4A (eIF4A) gene was employed as a reference gene [50].

4.4. Statistical Analysis

Values are presented as means ± standard deviation (SD) from triplicate experiments. A one-way ANOVA followed by a Tukey–Kramer test was used for multiple comparisons, and significant differences were determined using a cutoff value of p < 0.05.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12112070/s1, Table S1: Primer sequences used for RT-qPCR analyses; Figure S1: The presence of subcellular localization signature motifs in four classes of HSP70 proteins from the Bangiales and unicellular red algae; Figure S2: Requirement of dark treatment for the second expression peak of genes for ER and organellar HSP70 proteins in the three life stages of Neopyropia yezoensis at 25 °C; Figure S3: Effects of temperature changes on the expression of NyYcf26 and NyYcf27 in the three life stages of Neopyropia yezoensis.

Author Contributions

K.M. conceived and designed the study, made the figures and table, and wrote the manuscript. H.V.K. collected and analyzed the data and made the figures and table. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported in part by a grant for Academic General Research from the Toyo Suisan Foundation, Tokyo, Japan.

Data Availability Statement

Data are contained within the article.

Acknowledgments

We thank Hiroyuki Mizuta, Hokkaido University, for permitting Ho Viet Khoa to study in Miyagi University. Ho Viet Khoa was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Ritossa, F. A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 1962, 18, 571–573. [Google Scholar] [CrossRef]
  2. Ritossa, F. Discovery of the heat shock response. Cell Stress Chaperones 1996, 1, 97–98. [Google Scholar] [CrossRef] [PubMed]
  3. AI-Whaibi, M.H. Plant heat-shock proteins: A mini review. J. King Saud Univ. Sci. 2011, 23, 139–150. [Google Scholar] [CrossRef]
  4. Jacob, P.; Hirt, H.; Bendahmane, A. The heat-shock protein/chaperone network and multiple stress resistance. Plant Biotechnol. J. 2017, 15, 405–414. [Google Scholar] [CrossRef]
  5. Khan, S.; Jabeen, R.; Deeba, F.; Waheed, U.; Khanum, P.; Iqbal, N. Heat shock proteins: Classification, functions and expressions in plants during environmental stresses. J. Biores. Manag. 2021, 8, 85–97. [Google Scholar] [CrossRef]
  6. Wang, W.; Vinocur, B.; Shoseyov, O.; Altman, A. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 2004, 9, 244–252. [Google Scholar] [CrossRef]
  7. Ul Haq, S.; Khan, A.; Ali, M.; Khattak, A.M.; Gai, W.X.; Zhang, H.X.; Wei, A.M.; Gong, Z.H. Heat shock proteins: Dynamic biomolecules to counter plant biotic and abiotic stresses. Int. J. Mol. Sci. 2019, 20, 5321. [Google Scholar] [CrossRef]
  8. Radons, J. The human HSP70 family of chaperones: Where do we stand? Cell Stress Chaperones 2016, 21, 379–404. [Google Scholar] [CrossRef]
  9. Sable, A.; Agarwal, S.K. Plant heat shock protein families: Essential machinery for development and defense. J. Biol. Sci. Med. 2018, 4, 51–64. [Google Scholar]
  10. Kohler, V.; Andréasson, C. Hsp70-mediated quality control: Should I stay or should I go? Biol. Chem. 2020, 401, 1233–1248. [Google Scholar] [CrossRef] [PubMed]
  11. Kampinga, H.H.; Craig, E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat. Rev. Mol. Cell Biol. 2010, 11, 579–592. [Google Scholar] [CrossRef] [PubMed]
  12. Renner, T.; Waters, E.R. Comparative genomic analysis of the Hsp70s from five diverse photosynthetic eukaryotes. Cell Stress Chaperones 2007, 12, 172–185. [Google Scholar] [CrossRef]
  13. Yu, A.; Li, P.; Tang, T.; Wang, J.; Chen, Y.; Liu, L. Roles of Hsp70s in stress responses of microorganisms, plants, and animals. Biomed. Res. Int. 2015, 2015, 510319. [Google Scholar] [CrossRef] [PubMed]
  14. Boorstein, W.R.; Ziegelhoffer, T.; Craig, E.A. Molecular evolution of the HSP70 multigene family. J. Mol. Evol. 1994, 38, 1–17. [Google Scholar] [CrossRef]
  15. Karlin, S.; Brocchieri, L. Heat shock protein 70 family: Multiple sequence comparisons, function, and evolution. J. Mol. Evol. 1998, 47, 565–577. [Google Scholar] [CrossRef]
  16. Scharf, K.D.; Berberich, T.; Ebersberger, I.; Nover, L. The plant heat stress transcription factor (Hsf) family: Structure, function and evolution. Biochim. Biophys. Acta. 2012, 1819, 104–119. [Google Scholar] [CrossRef]
  17. Guo, M.; Liu, J.H.; Ma, X.; Luo, D.X.; Gong, Z.H.; Lu, M.H. The plant heat stress transcription factors (HSFs): Structure, regulation, and function in response to abiotic stresses. Front. Plant Sci. 2016, 7, 114. [Google Scholar] [CrossRef] [PubMed]
  18. Andrási, N.; Pettkó-Szandtner, A.; Szabados, L. Diversity of plant heat shock factors: Regulation, interactions, and functions. J. Exp. Bot. 2021, 72, 1558–1575. [Google Scholar] [CrossRef] [PubMed]
  19. Tsuda, K.; Tsuji, T.; Hirose, S.; Yamazaki, K. Three Arabidopsis MBF1 homologs with distinct expression profiles play roles as transcriptional co-activators. Plant Cell Physiol. 2004, 45, 225–231. [Google Scholar] [CrossRef]
  20. Jaimes-Miranda, F.; Chávez Montes, R.A. The plant MBF1 protein family: A bridge between stress and transcription. J. Exp. Bot. 2020, 71, 1782–1791. [Google Scholar] [CrossRef]
  21. Yoshida, T.; Sakuma, Y.; Todaka, D.; Maruyama, K.; Qin, F.; Mizoi, J.; Kidokoro, S.; Fujita, Y.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Functional analysis of an Arabidopsis heat-shock transcription factor HsfA3 in the transcriptional cascade downstream of the DREB2A stress-regulatory system. Biochem. Biophys. Res. Commun. 2008, 368, 515–521. [Google Scholar] [CrossRef] [PubMed]
  22. Ohama, N.; Sato, H.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Transcriptional regulatory network of plant heat stress response. Trends Plant Sci. 2017, 22, 53–65. [Google Scholar] [CrossRef] [PubMed]
  23. Saidi, Y.; Finka, A.; Goloubinoff, P. Heat perception and signaling in plants: A tortuous path to thermotolerance. New Phytol. 2011, 190, 556–565. [Google Scholar] [CrossRef] [PubMed]
  24. Bourgine, B.; Guihur, A. Heat shock signaling in land plants: From plasma membrane sensing to the transcription of small heat shock proteins. Front. Plant Sci. 2021, 12, 710801. [Google Scholar] [CrossRef]
  25. Abdelrahman, M.L.; Ishii, T.; El-Sayed, M.; Tran, L.S.P. Heat sensing and lipid reprograming as a signaling switch for heat stress responses in wheat. Plant Cell Physiol. 2020, 61, 1399–1407. [Google Scholar] [CrossRef]
  26. Carratù, L.; Franceschelli, S.; Pardini, C.L.; Kobayashi, G.S.; Horvath, I.; Vigh, L.; Maresca, B. Membrane lipid perturbation modifies the set point of the temperature of heat shock response in yeast. Proc. Natl. Acad. Sci. USA 1996, 93, 3870–3875. [Google Scholar] [CrossRef]
  27. Horváth, I.; Glatz, A.; Varvasovszki, V.; Török, Z.; Páli, T.; Balogh, G.; Kovács, E.; Nádasdi, L.; Benkö, S.; Joó, F.; et al. Membrane physical state controls the signaling mechanism of the heat shock response in Synechocystis PCC 6803: Identification of hsp17 as a “fluidity gene”. Proc. Natl. Acad. Sci. USA 1998, 95, 3513–3518. [Google Scholar] [CrossRef]
  28. Vigh, L.; Maresca, B.; Harwood, J.L. Does the membrane’s physical state control the expression of heat shock and other genes? Trends Biochem. Sci. 1998, 23, 369–374. [Google Scholar] [CrossRef]
  29. Balogh, G.; Horváth, I.; Nagy, E.; Hoyk, Z.; Benkõ, S.; Bensaude, O.; Vígh, L. The hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock protein response. FEBS J. 2005, 272, 6077–6086. [Google Scholar] [CrossRef]
  30. Nagy, E.; Balogi, Z.; Gombos, I.; Akerfelt, M.; Björkbom, A.; Balogh, G.; Török, Z.; Maslyanko, A.; Fiszer-Kierzkowska, A.; Lisowska, K.; et al. Hyperfluidization-coupled membrane microdomain reorganization is linked to activation of the heat shock response in a murine melanoma cell line. Proc. Natl. Acad. Sci. USA 2007, 104, 7945–7950. [Google Scholar] [CrossRef]
  31. Königshofer, H.; Tromballa, H.W.; Löppert, H.G. Early events in signalling high-temperature stress in tobacco BY2 cells involve alterations in membrane fluidity and enhanced hydrogen peroxide production. Plant Cell Environ. 2008, 31, 1771–1780. [Google Scholar] [CrossRef] [PubMed]
  32. Tóth, M.E.; Vígh, L.; Sántha, M. Alcohol stress, membranes, and chaperones. Cell Stress Chaperones 2014, 19, 299–309. [Google Scholar] [CrossRef] [PubMed]
  33. Hofmann, N.R. The plasma membrane as first responder to heat stress. Plant Cell 2009, 21, 2544. [Google Scholar] [CrossRef] [PubMed]
  34. Mittler, R.; Finka, A.; Goloubinoff, P. How do plants feel the heat? Trends Biochem. Sci. 2012, 37, 118–125. [Google Scholar] [CrossRef] [PubMed]
  35. Török, Z.; Crul, T.; Maresca, B.; Schütz, G.J.; Viana, F.; Dindia, L.; Piotto, S.; Brameshuber, M.; Balogh, G.; Péter, M.; et al. Plasma membranes as heat stress sensors: From lipid-controlled molecular switches to therapeutic applications. Biochim. Biophys. Acta. 2014, 1838, 1594–1618. [Google Scholar] [CrossRef] [PubMed]
  36. Saidi, Y.; Finka, A.; Muriset, M.; Bromberg, Z.; Weiss, Y.G.; Maathuis, F.J.; Goloubinoff, P. The heat shock response in moss plants is regulated by specific calcium-permeable channels in the plasma membrane. Plant Cell 2009, 21, 2829–2843. [Google Scholar] [CrossRef]
  37. Gao, F.; Han, X.; Wu, J.; Zheng, S.; Shang, Z.; Sun, D.; Zhou, R.; Li, B. A heat-activated calcium-permeable channel—Arabidopsis cyclic nucleotide-gated ion channel 6—Is involved in heat shock responses. Plant J. 2012, 70, 1056–1069. [Google Scholar] [CrossRef]
  38. Finka, A.; Goloubinoff, P. The CNGCb and CNGCd genes from Physcomitrella patens moss encode for thermosensory calcium channels responding to fluidity changes in the plasma membrane. Cell Stress Chaperones 2014, 19, 83–90. [Google Scholar] [CrossRef]
  39. Rütgers, M.; Muranaka, L.S.; Schulz-Raffelt, M.; Thoms, S.; Schurig, J.; Willmund, F.; Schroda, M. Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii. Plant Cell Environ. 2017, 40, 2987–3001. [Google Scholar] [CrossRef]
  40. Zhang, H.; Li, W.; Li, J.; Fu, W.; Yao, J.; Duan, D. Characterization and expression analysis of hsp70 gene from Ulva prolifera J. Agardh (Chlorophycophyta, Chlorophyceae). Mar. Genom. 2012, 5, 53–58. [Google Scholar] [CrossRef]
  41. Lago-Lestón, A.; Mota, C.; Kautsky, L.; Pearson, G.A. Functional divergence in heat shock response following rapid speciation of Fucus spp. in the Baltic Sea. Mar. Biol. 2009, 157, 683–688. [Google Scholar] [CrossRef]
  42. Smolina, I.; Kollias, S.; Jueterbock, A.; Coyer, J.A.; Hoarau, G. Variation in thermal stress response in two populations of the brown seaweed, Fucus distichus, from the Arctic and subarctic intertidal. R. Soc. Open. Sci. 2016, 3, 150429. [Google Scholar] [CrossRef]
  43. Reith, M.; Munholland, J. An hsp70 homolog is encoded on the plastid genome of the red alga, Porphyra umbilicalis. FEBS Lett. 1991, 294, 116–120. [Google Scholar] [CrossRef]
  44. Brawley, S.H.; Blouin, N.A.; Ficko-Blean, E.; Wheeler, G.L.; Lohr, M.; Goodson, H.V.; Jenkins, J.W.; Blaby-Haas, C.E.; Helliwell, K.E.; Chan, C.X.; et al. Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta). Proc. Natl. Acad. Sci. USA 2017, 114, E6361–E6370. [Google Scholar] [CrossRef] [PubMed]
  45. Zhou, X.; Wang, P.; Yan, B.; Li, X.; Yi, L. Characterization and expression patterns of two 70-kDa heat shock protein genes in the intertidal red alga Porphyra yezoensis. Bot. Mar. 2011, 54, 447–455. [Google Scholar] [CrossRef]
  46. Yu, X.; Mo, Z.; Tang, X.; Gao, T.; Mao, Y. Genome-wide analysis of HSP70 gene superfamily in Pyropia yezoensis (Bangiales, Rhodophyta): Identification, characterization and expression profiles in response to dehydration stress. BMC Plant Biol. 2021, 21, 435. [Google Scholar] [CrossRef]
  47. Ji, D.; Li, B.; Xu, Y.; Chen, C.; Xie, C. Cloning and quantitative analysis of five heat shock protein 70 genes from Pyropia haitanensis. J. Appl. Phycol. 2015, 27, 499–509. [Google Scholar] [CrossRef]
  48. Wang, L.; Mao, Y.; Kong, F.; Li, G.; Ma, F.; Zhang, B.; Sun, P.; Bi, G.; Zhang, F.; Xue, H.; et al. Complete sequence and analysis of plastid genomes of two economically important red algae: Pyropia haitanensis and Pyropia yezoensis. PLoS ONE 2013, 8, e65902. [Google Scholar] [CrossRef]
  49. Cao, M.; Bi, G.; Mao, Y.; Li, G.; Kong, F. The first plastid genome of a filamentous taxon ‘Bangia’ sp. OUCPT-01 in the Bangiales. Sci. Rep. 2018, 8, 10688. [Google Scholar] [CrossRef] [PubMed]
  50. Khoa, H.V.; Mikami, K. Membrane-fluidization-dependent and -independent pathways are involved in heat-stress-inducible gene expression in the marine red alga Neopyropia yezoensis. Cells 2022, 11, 1486. [Google Scholar] [CrossRef]
  51. Mikami, K.; Li, C.; Irie, R. A unique life cycle transition in the red seaweed Pyropia yezoensis depends on apospory. Commun. Biol. 2019, 2, 299. [Google Scholar] [CrossRef] [PubMed]
  52. Lee-Yoon, D.; Easton, D.; Murawski, M.; Burd, R.; Subjeck, J.R. Identification of a major subfamily of large hsp70-like proteins through the cloning of the mammalian 110-kDa heat shock protein. J. Biol. Chem. 1995, 270, 15725–15733. [Google Scholar] [CrossRef]
  53. Easton, D.P.; Kaneko, Y.; Subjeck, J.R. The hsp110 and Grp1 70 stress proteins: Newly recognized relatives of the Hsp70s. Cell Stress Chaperones 2000, 5, 276–290. [Google Scholar] [CrossRef] [PubMed]
  54. Tang, T.; Yu, A.; Li, P.; Yang, H.; Liu, G.; Liu, L. Sequence analysis of the Hsp70 family in moss and evaluation of their functions in abiotic stress responses. Sci. Rep. 2016, 6, 33650. [Google Scholar] [CrossRef]
  55. Junprung, W.; Norouzitallab, P.; De Vos, S.; Tassanakajon, A.; Nguyen Viet, D.; Van Stappen, G.; Bossier, P. Sequence and expression analysis of HSP70 family genes in Artemia franciscana. Sci. Rep. 2019, 9, 8391. [Google Scholar] [CrossRef] [PubMed]
  56. Wang, H.; Goffreda, M.; Leustek, T. Characteristics of an Hsp70 homolog localized in higher plant chloroplasts that is similar to DnaK, the Hsp70 of prokaryotes. Plant Physiol. 1993, 102, 843–850. [Google Scholar] [CrossRef]
  57. Lin, B.L.; Wang, J.S.; Liu, H.C.; Chen, R.W.; Meyer, Y.; Barakat, A.; Delseny, M. Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana. Cell Stress Chaperones 2001, 6, 201–208. [Google Scholar] [CrossRef]
  58. Tanaka, K. Chloroplast transcriptional machinery of red algae: Conservation of four types of transcriptional regulators in non-green chloroplasts. In Prophyra yezoensis: Frontiers in Physiological and Molrcular Biological Research; Mikami, K., Ed.; Nova Science Publishers: New York, NY, USA, 2012; pp. 39–59. [Google Scholar]
  59. Mikami, K.; Kanesaki, Y.; Suzuki, I.; Murata, N. The histidine kinase Hik33 perceives osmotic stress and cold stress in Synechocystis sp. PCC 6803. Mol. Microbiol. 2002, 46, 905–915. [Google Scholar] [CrossRef]
  60. Mikami, K.; Murata, N. Membrane fluidity and the perception of environmental signals in cyanobacteria and plants. Prog. Lipid Res. 2003, 42, 527–543. [Google Scholar] [CrossRef]
  61. Kropat, J.; Oster, U.; Rüdiger, W.; Beck, C.F. Chlorophyll precursors are signals of chloroplast origin involved in light induction of nuclear heat-shock genes. Proc. Natl. Acad. Sci. USA 1997, 94, 14168–14172. [Google Scholar] [CrossRef]
  62. Schroda, M.; Vallon, O.; Wollman, F.A.; Beck, C.F. A chloroplast-targeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition. Plant Cell 1999, 11, 1165–1178. [Google Scholar] [CrossRef] [PubMed]
  63. Ding, F.; Li, F.; Zhang, B. A plastid-targeted heat shock cognate 70-kDa protein confers osmotic stress tolerance by enhancing ROS scavenging capability. Front. Plant Sci. 2022, 13, 1012145. [Google Scholar] [CrossRef]
  64. Tian, F.; Hu, X.L.; Yao, T.; Yang, X.; Chen, J.G.; Lu, M.Z.; Zhang, J. Recent advances in the roles of HSFs and HSPs in heat stress response in woody plants. Front. Plant Sci. 2021, 12, 704905. [Google Scholar] [CrossRef] [PubMed]
  65. Haider, S.; Raza, A.; Iqbal, J.; Shaukat, M.; Mahmood, T. Analyzing the regulatory role of heat shock transcription factors in plant heat stress tolerance: A brief appraisal. Mol. Biol. Rep. 2022, 49, 5771–5785. [Google Scholar] [CrossRef] [PubMed]
  66. Thiriet-Rupert, S.; Carrier, G.; Chénais, B.; Trottier, C.; Bougaran, G.; Cadoret, J.P.; Schoefs, B.; Saint-Jean, B. Transcription factors in microalgae: Genome-wide prediction and comparative analysis. BMC Genom. 2016, 17, 282. [Google Scholar] [CrossRef] [PubMed]
  67. Petroll, R.; Schreiber, M.; Finke, H.; Cock, J.M.; Gould, S.B.; Rensing, S.A. Signatures of transcription factor evolution and the secondary gain of red algae complexity. Genes 2021, 12, 1055. [Google Scholar] [CrossRef]
  68. Finka, A.; Cuendet, A.F.; Maathuis, F.J.; Saidi, Y.; Goloubinoff, P. Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance. Plant Cell 2012, 24, 3333–4338. [Google Scholar] [CrossRef]
  69. Wu, H.C.; Jinn, T.L. Oscillation regulation of Ca2+ /calmodulin and heat-stress related genes in response to heat stress in rice (Oryza sativa L.). Plant Signal Behav. 2012, 7, 1056–1057. [Google Scholar] [CrossRef]
  70. Weigand, C.; Kim, S.H.; Brown, E.; Medina, E.; Mares, M., 3rd; Miller, G.; Harper, J.F.; Choi, W.G. A ratiometric calcium reporter CGf reveals calcium dynamics both in the single cell and whole plant levels under heat stress. Front. Plant Sci. 2021, 12, 777975. [Google Scholar] [CrossRef]
  71. Li, C.; Ariga, I.; Mikami, K. Difference in nitrogen starvation-inducible expression patterns among phylogenetically diverse ammonium transporter genes in the red seaweed Pyropia yezoensis. Am. J. Plant Sci. 2019, 10, 1325–1349. [Google Scholar] [CrossRef]
Plants 12 02070 g001 550
Figure 1. Phylogenetic analysis of HSP70s from the Bangiales and unicellular red algae. Each DDBJ/EMBL/GenBank accession number is indicated before the species names in the tree. Red algal HSP110 proteins were used for the outgroup. The cytoplasmic, ER, CP, and MT HSP70 clades and the HSP110 outgroup are indicated by colored boxes. The HSP70 homologs from N. yezoensis are boxed. The percentage support for each branch from 1000 bootstrap replicates is indicated at the nodes of the tree. The scale bar indicates 0.2 substitutions per site.
Figure 1. Phylogenetic analysis of HSP70s from the Bangiales and unicellular red algae. Each DDBJ/EMBL/GenBank accession number is indicated before the species names in the tree. Red algal HSP110 proteins were used for the outgroup. The cytoplasmic, ER, CP, and MT HSP70 clades and the HSP110 outgroup are indicated by colored boxes. The HSP70 homologs from N. yezoensis are boxed. The percentage support for each branch from 1000 bootstrap replicates is indicated at the nodes of the tree. The scale bar indicates 0.2 substitutions per site.
Plants 12 02070 g001
Plants 12 02070 g002 550
Figure 2. Effects of temperature changes on the expression of NyBiP1 and NyBiP2 in three life stages of Neopyropia yezoensis. Values on the y-axis represent the fold-change of the expression of each gene relative to that at 0 h. Shading indicates dark period. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Figure 2. Effects of temperature changes on the expression of NyBiP1 and NyBiP2 in three life stages of Neopyropia yezoensis. Values on the y-axis represent the fold-change of the expression of each gene relative to that at 0 h. Shading indicates dark period. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Plants 12 02070 g002
Plants 12 02070 g003 550
Figure 3. Effects of temperature changes on the expression of NycpDnaK and NymtDnaK in three life stages of Neopyropia yezoensis. Values on the y-axis represent the fold-change of the expression of each gene relative to that at 0 h. Shading indicates dark period. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Figure 3. Effects of temperature changes on the expression of NycpDnaK and NymtDnaK in three life stages of Neopyropia yezoensis. Values on the y-axis represent the fold-change of the expression of each gene relative to that at 0 h. Shading indicates dark period. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Plants 12 02070 g003
Plants 12 02070 g004 550
Figure 4. Relationship between the changes in the physical state of the membrane and the expression of NyBiP1 and NyBiP2 in three life stages of Neopyropia yezoensis. Membrane fluidization or rigidification was induced by pharmacological treatment with 2.5 mM benzyl alcohol (BA) or 4% dimethyl sulfoxide (DMSO), respectively, at 15 °C for 5, 15, and 30 min, after which the expression of the genes of interest was compared to their expression at 25 °C. Values on the y-axis represent the fold-change in the expression of each gene relative to that at 0 h. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Figure 4. Relationship between the changes in the physical state of the membrane and the expression of NyBiP1 and NyBiP2 in three life stages of Neopyropia yezoensis. Membrane fluidization or rigidification was induced by pharmacological treatment with 2.5 mM benzyl alcohol (BA) or 4% dimethyl sulfoxide (DMSO), respectively, at 15 °C for 5, 15, and 30 min, after which the expression of the genes of interest was compared to their expression at 25 °C. Values on the y-axis represent the fold-change in the expression of each gene relative to that at 0 h. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Plants 12 02070 g004
Plants 12 02070 g005 550
Figure 5. Relationship between the changes in the physical state of the membrane and the expression of NycoDnaK and NymtDnaK in three life stages of Neopyropia yezoensis. Membrane fluidization or rigidification was induced by pharmacological treatment with 2.5 mM benzyl alcohol (BA) or 4% dimethyl sulfoxide (DMSO), respectively, at 15 °C for 5, 15, and 30 min, after which the expression of these genes was compared to their expression at 25 °C. Values on the y-axis represent the fold-change in the expression of each gene relative to that at 0 h. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Figure 5. Relationship between the changes in the physical state of the membrane and the expression of NycoDnaK and NymtDnaK in three life stages of Neopyropia yezoensis. Membrane fluidization or rigidification was induced by pharmacological treatment with 2.5 mM benzyl alcohol (BA) or 4% dimethyl sulfoxide (DMSO), respectively, at 15 °C for 5, 15, and 30 min, after which the expression of these genes was compared to their expression at 25 °C. Values on the y-axis represent the fold-change in the expression of each gene relative to that at 0 h. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Plants 12 02070 g005
Plants 12 02070 g006 550
Figure 6. Requirement for membrane fluidization in the dark-induced second gene expression peaks in the three life stages of Neopyropia yezoensis. The expression of genes for ER and organellar HSP70 was examined at 8 and 12 h into heat stress (25 °C) exposure under continuous light with or without a treatment of 2.5 mM BA. Values on the y-axis represent the relative fold-change in the expression of each gene. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Figure 6. Requirement for membrane fluidization in the dark-induced second gene expression peaks in the three life stages of Neopyropia yezoensis. The expression of genes for ER and organellar HSP70 was examined at 8 and 12 h into heat stress (25 °C) exposure under continuous light with or without a treatment of 2.5 mM BA. Values on the y-axis represent the relative fold-change in the expression of each gene. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Plants 12 02070 g006
Plants 12 02070 g007 550
Figure 7. Effects of membrane rigidification on the dark-induced second gene expression peaks in the three life stages of Neopyropia yezoensis. The expression of genes encoding ER and organellar HSP70 was examined at 8 and 12 h into heat stress (25 °C) exposure under continuous light with or without a treatment of 4% DMSO. Values on the y-axis represent the relative fold-change in the expression of each gene. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Figure 7. Effects of membrane rigidification on the dark-induced second gene expression peaks in the three life stages of Neopyropia yezoensis. The expression of genes encoding ER and organellar HSP70 was examined at 8 and 12 h into heat stress (25 °C) exposure under continuous light with or without a treatment of 4% DMSO. Values on the y-axis represent the relative fold-change in the expression of each gene. Significant differences in the expression level in the three life stages, indicated by different letters, were defined from triplicate independent replicate data using a one-way ANOVA with a Tukey’s test (p < 0.05). G, gametophyte; S, sporophyte; CS, conchosporophyte.
Plants 12 02070 g007
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Mikami, K.; Khoa, H.V. Membrane Fluidization Governs the Coordinated Heat-Inducible Expression of Nucleus- and Plastid Genome-Encoded Heat Shock Protein 70 Genes in the Marine Red Alga Neopyropia yezoensis. Plants 2023, 12, 2070. https://doi.org/10.3390/plants12112070

AMA Style

Mikami K, Khoa HV. Membrane Fluidization Governs the Coordinated Heat-Inducible Expression of Nucleus- and Plastid Genome-Encoded Heat Shock Protein 70 Genes in the Marine Red Alga Neopyropia yezoensis. Plants. 2023; 12(11):2070. https://doi.org/10.3390/plants12112070

Chicago/Turabian Style

Mikami, Koji, and Ho Viet Khoa. 2023. "Membrane Fluidization Governs the Coordinated Heat-Inducible Expression of Nucleus- and Plastid Genome-Encoded Heat Shock Protein 70 Genes in the Marine Red Alga Neopyropia yezoensis" Plants 12, no. 11: 2070. https://doi.org/10.3390/plants12112070

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop