Hsp70 is one of the most conserved proteins (Lindquist and Craig 1988) and all eukaryotes have multiple hsp70 gene products that differ from each other in gene structure, subcellular location, and expression level (Brocchieri et al. 2008; Murpy 2013). In the present study, the sequence similarities of the three Hsp70s cloned from A. vermiculophyllum were all at least 90% higher than their corresponding proteins in Gracilariopsis lemaneiformis. However, the similarities among them were low, i.e., 63% (Hsp70-1 vs. Hsp70-2), 48% (Hsp70-1 vs. Hsp70-3), and 49% (Hsp70-2 vs. Hsp70-3). This indicates both their conserved nature and functional divergence.
Previous studies have shown that different Hsp70 paralogs have both overlapping and diverse functions (Boorstein et al. 1994; Daugaard et al. 2007). The divergent C-terminal SBDs (substrate-binding domains) are necessary for certain co-chaperone interactions and probably define their distinctive function (Brocchieri et al. 2008; Demand et al. 1998; Sung et al. 2001). For example, cytosolic eukaryotic Hsp70s possess GGMP repeats and the EEVD motif at the carboxyl terminus, whereas other Hsp70 family members lack such structural elements (Boorstein et al. 1994; Freeman et al. 1995). It has been shown that in photosynthetic eukaryotes: (1) the Hsp70s are located in four different cell compartments: the cytoplasm, mitochondria, endoplasmic reticulum (ER), and chloroplast (Bukau and Horwich 1998; Sung et al. 2001), and; (2) the most common motif for the cytosolic is EEVD, for the endoplasmic reticulum (ER) is HDEL, for the mitochondrion is PEAEYEEAKK and for the plastid is PEGDVIDADFTDSK (Guy and Li 1998). In the present study, the phylogenetic tree shows that the three Hsp70s cloned from A. vermiculophyllum were clustered into three different groups (Fig. 1). Hsp70-1 and Hsp70-2 are associated with cytoplasmic and endoplasmic reticulum locations, respectively, since each of them had the typical localization tag (motif EEVD or HDEL) at their C-terminus. For Hsp70-3 (AX191579.1), although the sequence of the C-terminus motif was not perfectly matched with the plastid motif sequence mentioned above (Table 1), the sequence structures for all the quoted Gracilariaceae species in this cluster were the same. Some of the sequences were explicitly acquired from chloroplast genome research, such as YP_063608.1 from G. tenuistipitata and YP_009509315.1 from A. vermiculophyllum. Therefore, it can be inferred that the Hsp70-3 in this cluster is associated with a chloroplast location. It has been shown that there are two putative cpHsp70s (chloroplast Hsp70s) in fully sequenced genomes of land plants, such as Arabidopsis and rice, but the green alga Chlamydomonas only harbors a single cpHsp70 (Su and Li 2008). It is currently unknown whether this is also true for red algae.
Gracilariaceae species The sequence of C-terminus Gracilariopsis lemaneiformis heat shock protein 70-3 ALJ33148.1 KQQETDNTDTDSVID-------TNSKEA Gracilaria tenuistipitata heat shock protein 70 (chloroplast) YP_063608.1 TQQDNSKTEDGSVID-------TNSKEA Agarophyton vermiculophyllum Hsp70-type chaperone (chloroplast) YP_009509315.1 TQEENKKTEDDSVID-------TKSKEA Agarophyton vermiculophyllum heat shock protein 70-3 AX191579.1 QLKKNKKTEDDSVID-------TKSKGS Chloroplast C-terminal motif PEGDVID ADF TDSK
Table 1. Sequence alignment of the C-terminus of Hsp70-3
These three hsp70 genes (hsp70-1, hsp70-2 and hsp70-3) were previously cloned and tested for their transcriptional level under heat shock in Gracilariopsis lemaneiformis (Gu et al. 2012; Liu et al. 2018) and the results indicated that in the wild type Hsp70-1 was the most active Hsp70 after the heat shock, because its transcriptional level increased dramatically. On the other hand, the transcriptional levels for hsp70-2 and especially for hsp70-3 were low, so it is supposed that under heat shock, Hsp70-2 and Hsp70-3 may play only a supporting role. Hsp70-3 is probably employed more actively in reaction to stresses other than heat. In the current study for A. vermiculophyllum as mentioned above, the transcriptional levels of all three hsp70 genes were highly upregulated in gametophytes as compared to tetrasporophytes, and the highest increase was the hsp70-3 in gametophytes, especially in female gametophytes. Chen et al. (2011) have previously shown that hsp70-3 is one of the differentially expressed gene clones in the female gametophytic SSH (suppression subtractive hybridization) library constructed between matured female and male gametophytes of G. lemaneiformis. Furthermore, it was reported that in the green algae Chlamydomonas and Dunaliella, chloroplast stromal Hsp70B has a functional relationship with photosynthesis (Schroda et al. 1999; Yokthongwattana et al. 2001), and in Arabidopsis, cpHsp70 has been shown to be essential in plant development with knockout mutants having variegated malformed cotyledons and roots (Su and Li 2008).
All these results suggest that Hsp70-3 is located in the chloroplast and, in A. vermiculophyllum, plays an important role as a chaperon molecule in the development of haploid gametophytes, especially in the female gametophyte.
It has been previously reported that the cytoplasmic protein Hsp70 is more highly expressed in invasive populations (mainly tetrasporophytes) of A. vermiculophyllum than in native mixed-ploidy populations not only after, but also before, heat shock (Hammann et al. 2016; Krueger-Hadfield et al. 2016). There are, however, at least two possible explanations for the difference between these findings and those of the present study. Firstly, the methods used differed in the different studies. In Hammann et al. (2016), the Hsp70 content was checked using Western Blot, while here the quantities of transcribed hsp70 genes were detected by RT-qPCR. The correlation of transcription level of a gene to its protein level is not always consistent. Secondly, the environmental conditions of the algae were different. The invasive population of A. vermiculophyllum were actually under stress due to the unfamiliar conditions of their new environment and that may induce the expression of cytoplasmic Hsp70-1. But our experiments were focused on the activities of different hsp genes in different ploidy plants of native specimens without stress, so the stress-resistant trait was not the primary one induced.
In contrast to Hsp-70, it has previously been suggested that hsp90 is active during the development of female gametophytes in Griffithsia japonica (Lee et al. 1998). In the present study, however, we did not find significant differences for the transcriptional levels of hsp90 among tetrasporaphytes and male/female gametophytes.
In conclusion, in A. vermiculophyllum and probably other related red algae, cytoplasmic Hsp70-1 and ER Hsp70-2 might be more active in heat resistance, and Hsp70-3, as the chloroplast Hsp70 (cpHsp70), might participate as a chaperon molecule important for haploid development. Further studies are needed to understand the precise function(s) of cpHsp70 in A. vermiculophyllum.
Wild thalli of A. vermiculophyllum were collected from Fushan Bay, Qingdao, China (36.0° N, 120.2° E) during their fertile period. Three types of mature thalli—tetrasporophytes (diploid), female and male gametophytes (haploid)—were separated and thoroughly rinsed with sterilized seawater and cultured in seawater supplemented with 100 μmol/L NaNO3 and 10 μmol/L NaH2PO4 (final concentration) at 20 ℃ with 50-60 μmol photons/m-2s-1 for a light/dark period of 12 h:12 h for 1-2 days.
Total RNAs were extracted from 200 mg fresh thalli for each sample, using the GeneJET Plant RNA Purification Kit (Thermo Fisher Scientific, USA). The extracted RNAs were quantified using a spectrophotometer and verified by running samples on 1.0% agarose gels. To remove the remaining genomic DNA, total RNAs were treated with DNaseI (30 U) for 15 min at 37 ℃. Then 1 μg total RNAs were subject to cDNA synthesis using SuperScriptTM Ⅱ reverse transcription (RT)-PCR kit (TaKaRa, Japan) in 25 μl volume according to instruction manual, using the Anchored Oligo(dT)18 Primer. The first strands of cDNA obtained were used for partial cDNA fragment cloning and real-time PCR. For 3' and 5' RACE, the first strands of cDNA were obtained using 3'CDS primer (Table 3) according to SMARTer RACE 5'/3' Kit (Clontech, USA).
Function Name of the primers Primer sequences (5′–3′) 3′ RACE Antisense 3′ CDS primer AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTVN Sense hsp70-1 3′ GSP 1 CTTCTACTCATCCGTCACTCGTGCCAA hsp70-1 3′ GSP 2 GAACATCAACCCGGACGAGGCTGTG hsp70-1 3′ GSP 3 GCCTCGTCCGGGTTGATGTTCTTGC hsp70-2 3′ GSP 1 TGATGAAGCTCTTCAAGCGCAAGTACGGC hsp70-2 3′ GSP 2 CGAGGTTGTGCTAGTCGGCGGTTC hsp70-2 3′ GSP 3 GACATGTCGGCGTCCTTGAGAACCTTCT hsp70-3 3′ GSP 1 TATTACTTCTACCGATACAGGCCCCAA hsp70-3 3′ GSP 2 GTGTTAATCCTGATGAAGTAGTTGCAATT hsp70-3 3′ GSP 3 CAGGGAGAAAGAGAGTTTACTAAAGACA hsp90 3′ GSP 1 CAGCCGTGAGATGCTCCAGCAGAATAAGA hsp90 3′ GSP 2 CGTATGAAGGAGGGTCAGAAGAACATCTACTAC hsp90 3′ GSP 3 GCGCGAAGAGGAGAAGAAGGCTTGTG 5′ RACE Sense 5′ CDS primer a TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 5′ CDS primer b CTAATACGACTCACTATAGGGC Antisense hsp70-1 5′ GSP 1 CAATGGTGAGGAGGGAGACATCGAAAGTTC hsp70-1 5′ GSP 2 TCTCCCTCCTCACCATTGAAGACGGTAT hsp70-1 5′ GSP 3 CTTCTACTCATCCGTCACTCGTGCCAA hsp70-2 5′ GSP 1 GACAGCGTTCTTGACTTCCTTGCCGAG hsp70-2 5′ GSP 2 GGAGTACCTCGGCAAGGAAGTCAAGAAC hsp70-2 5′ GSP 3 TGATGAAGCTCTTCAAGCGCAAGTACGGC hsp70-3 5′ GSP 1 CCATATGATAAAGATGCTGCTGTTGGTTC hsp70-3 5′ GSP 2 TGAACCAACAGCAGCATCTTTATCATATGG hsp70-3 5′ GSP 3 TATTACTTCTACAGATACAGGCCCTAAACA hsp90 5′ GSP 1 CCTCCTCAGTGTCCTCCAACTCCATAC hsp90 5′ GSP 2 CTGGTCAGCTCATCAGTGCTCTTAGTGG hsp90 5′ GSP 3 GGCAAATCCTCAGAGTCGACAACACCC
Table 3. Primers used in the 3' and 5' RACE for whole CDS sequences of Hsp genes
Three hsp70 genes, i.e., hsp70-1, hsp70-2, and hsp70-3, were previously cloned from Gracilariopsis lemaneiformis, which is a red algal species in a closely related genus to Gracilaria (Gu et al. 2012; Liu et al. 2018). Therefore, we used these three genes and their related sequences as the templates to design the degenerate primers for cloning the partial cDNA fragment of each hsp70 gene from A. vermiculophyllum. The coding sequences (CDSs) used as templates were: G. lemaneiformis (KR534897.1), Chondrus crispus (XM_005712356.1), Pyropia haitanensis (KF715267.1), and Pyropia yezoensis (KF574043.1) for cloning hsp70-1; Gracilariopsis lemaneiformis (KR534898.1), Chondrus crispus (XM_005718442.1), Pyropia haitanensis (KF715271.1), and Galdieria sulphuraria (XM_005707399.1) for cloning hsp70-2, Gracilariopsis lemaneiformis (KR534899.1), Gracilaria salicornia (NC_023785.1), Gracilaria tenuistipitata (NC_006137.1), and Gelidium elegans (NC_029858.1) for cloning hsp70-3.
The degenerate primers for cloning the partial cDNA fragment of hsp90 from A. vermiculophyllum were designed following the same procedure for hsp70 cloning. The three complete CDSs of hsp90 gene used were Chondrus crispus (XM_005715072.1), Pyropia yezoensis (GU301885.1), and Pyropia haitanensis (KF732652.1).
All the designed degenerate primers mentioned above are shown in Table 2. Nest PCR was utilized to enhance the specificity of the products.
Name of the primers Primer sequences (5′–3′) hsp70-1 sense 1 CTACTCDTGYGTSGGYRTGG hsp70-1 sense 2 GTSCCSGCCTACTTYAAYGACTC hsp70-1 sense 3 GCTGGTGAYACTCAYYTKGGDGG hsp70-1 antisense 3 GTCATVACSRCACCRGCVGTCTC hsp70-1 antisense 2 TGGTTGTCRGMGTAVGTVGARAAGA hsp70-1 antisense 1 CCTCYTCCTCAGCCTTGWAYTTCTC hsp70-2 sense 1 CGCCAGGCBACWAAGGAYGC hsp70-2 sense 2 GGWGGWGARGACTTTGACCAGCG hsp70-2 sense 3 CTCATTGGCGACGCCGCAAAGAAC hsp70-2 antisense 3 GATCTTGSTCATSACACCBCCAACMGT hsp70-2 antisense 2 CCCTTGTCATTGGTGRTAGTGATGRTG hsp70-2 antisense 1 ATKCCAYTGAGRTCRAAYTTSCCG hsp70-3 sense 1 GACAAGCWGTAATKAATCCRGAAAATAC hsp70-3 sense 2 GCTCCTGARGAAATTTCTGCWCA hsp70-3 antisense 2 ATRCCATCYAATCTAAAAGTNCCRAAAC hsp70-3 antisense 1 GTAATWGATTGKTCTTTACCWGTTCCTTTATC hsp90 sense 1 GACACCAAGGTCGAAGACATCNCTGAGGATGA hsp90 sense 2 CAGCTCGAATTCAAGGCSATCAT hsp90 sense 3 GGGTGTTGTCGACTCTGAGGAT hsp90 antisense 3 TCCAGTRATGTAGTARATGTTCTTCTG hsp90 antisense 2 GCCTGCGCCTTCATGATRCGYTCCATGT hsp90 antisense 1 ACTTCMTCCATGTTRGAGGCAGCRG
Table 2. Degenerate primers designed for cloning the partial cDNA fragments of Hsp genes from A. vermiculophyllum
The PCR for each reaction was performed with 20 μl of a mixture containing 2 μl of 10 × buffer, 2.0 mmol/L MgCl2, 0.25 mmol/L of each dNTP, 0.25 U of TaqDNA polymerase (TaKaRa, Japan) and 1 μmol/L of each primer. PCR products as the templates for next round of Nest PCR reaction needed to be diluted 50-fold. The final PCR products were purified and inserted into the pMD18-T vector (TaKaRa, Japan) which was used to transform Escherichia coli DH5α competent cells. Products were then sequenced by Shanghai Invitrogen.
Nest PCR was applied in the 3' and 5' RACE to obtain the whole CDS sequences of Hsp genes. The first strands of cDNA obtained with SMARTer RACE 5'/3' Kit (Clontech, USA) was used as the template for RACE. The 3' CDS primer and 5' CDS primers contained in the kit and the designed GSPs (gene specific primers) are listed in Table 3. The PCR reaction process was used following the manufacturer's instructions.
The phylogenetic tree based on the results of multiple alignments of amino acid sequences among the deduced Hsp70 with other related proteins were constructed using MEGA 5.22.
Real-time PCR analysis of the mRNA relative transcription levels were performed using RealMaster Mix (SYBR Green) (TIANGEN, China) and an ABI 7500 Fast real-time PCR platform. The real-time PCR amplification profile was 95 ℃ for 30 s and then 40 cycles of 95 ℃ for 5 s, 54 ℃ for 20 s, and 72 ℃ for 30 s using the designed primers listed in Table 4. After amplification, the melting curve was generated with a ramp speed of 0.5 ℃ every cycle from 60 to 95 ℃ by heating 30 s each cycle. For each gene, a standard amplification curve was constructed with five serial dilution points (in steps of tenfold) of cDNA. All reactions were carried out in three technical replicates and each group also had three replicates using different individual plants. The data were collected at the end of each extension step. The relative transcriptions of genes for each treatment group were analyzed by the 2-ΔΔCt method (Livak and Schmittgen 2001). The housekeeping gene CoI (cytochrome oxidase) was used as the internal reference genes.
Gene Primer sequence (5′–3′) (sense/antisense) hsp70-1 CTTCTACTCATCCGTCACTCGTGCCAA TCTCCCTCCTCACCATTGAAGACGGTAT hsp70-2 CGAGGTTGTGCTAGTCGGCGGTTC GACAGCGTTCTTGACTTCCTTGCCGAG hsp70-3 TATTACTTCTACAGATACAGGCCCTAAACA TGAACCAACAGCAGCATCTTTATCATATGG hsp90 TGCTGGAGCATCTCACGG CAGCTCGAATTCAAGGCCAT Rbc-L AGTAACTCCTGTTGCTTC GTAGAGGACCACATGTTT CoI CTCGATCCAAAGCCATT CAGCCAACAGTAACAGAAAA
Table 4. Primer sequences of candidate internal control genes and hsp genes