Protein sequences and alignments
Four putative trehalose-6-phosphate synthase genes from P. haitanensis were cloned and named as PhTPS1, PhTPS2, PhTPS3, and PhTPS4. Their GenBank accession no. are KF147832.1, KM519457.1, KM519458.1, and KF245464.1,
respectively. The open reading frames (ORFs) of PhTPS1–4 are 3462 bp, 4029 bp, 3324 bp, and 3024 bp in length and encode polypeptides of 1154
aa, 1343 aa,1108 aa, and 1008 aa, respectively (Fig. 1). The molecular weights of
the PhTPS1–4 deduced amino acid sequences are 124, 145, 117, and 112 kDa, with a theoretical
isoelectric point (pI) of 6.73, 6.05, 5.99, and 5.77, respectively.
By searching in the NCBI Conserved Domain Search tool, two conserved structural domains
named TPS domain (Glyco_transf_20) and TPP domain (Trehalose_PPase) were discovered
in PhTPS1, PhTPS2, and PhTPS3 (Fig. 1). The TPS domain in the three PhTPS comprises
the main length of the protein and is present near the N-terminal and is annotated
as trehalose-6-phosphate synthase. The TPP domain annotated as trehalose-6-phosphate
phosphatase (TPP domain) is located at the C-terminal, whereas in PhTPS4, with the
exception of the two domains, there is a special domain named CBM20 at the N-terminal
known to be involved in starch binding.
Currently, only the crystal structures of Escherichia coli TPS (PDI No. 1GZ5)  and Candida albicans TPS (PDI No. 5HUT)  proteins have been elucidated. Here, we compared the data
of these two proteins and used multiple sequence alignment to evaluate the TPS domains
from different species and GGPS domain sequences from cyanobacteria. It was found
that the TPS domains of PhTPS1–3 were homologous with the C. albicans TPS (PDI No. 5HUT) protein, with 53%, 50%, and 34% identity, respectively. However,
PhTPS4 showed a low identity of only 10%. Based on the alignment, we found nine sites
conserved with the UDP-glucose substrate binding sites, and four sites conserved with the glucose-6-phosphate binding sites (Additional file 1: Figure S1). Sites G157, D274, H298, R406, D505, M507, N508, L509,
and E513 of PhTPS1, and sites G181, D410, H434, R542, D641, M643, N644, L645, and
E649 of PhTPS2 were associated with the substrate UDP-glucose binding sites and are
highly conserved, without any mutated sites. However, in the two proteins PhTPS3 and
PhTPS4, there are three different sites. For example, in PhTPS3, D201, H225, and N441
are changed to N, Y, and S, respectively. In PhTPS4, the mutated sites are G198K,
R464D, and M564L, but the other six sites (D328, H352, D562, N565, L566, E570) are
conserved. For the substrate glucose-6-phosphate binding sites, only four sites in
PhTPS1 (R136, Y213, W222, and R440) are highly conserved. In PhTPS2-4, site mutations
were present, including Y247H and R580Q in PhTPS2, R63H and Y141F in PhTPS3, while
R501 in PhTPS4 is deleted. In addition to the binding sites of the two substrates,
multiple sequence alignment showed that the sequences of the four PhTPS members were
highly similar to the highly conserved fragments (homology > 90%) of other species
TPSs and cyanobacteria GGPSs. An insert fragment (309–397 aa) was found in PhTPS2.
This insert was also found in P. yezoensis TPS-3 (contig_27879) (350–427 aa, with 48% identity with the PhTPS2 insertion fragment).
No other species were detected.
Phylogenetic analysis of trehalose-6-phosphate synthase in P. haitanensis.
In this study, a phylogenetic tree of the fused protein from bacteria, fungi, algal, animals, and higher plants was constructed
based on the TPS/TPP, TPS, and GGPS domain to investigate the evolutionary relationships
among them (Fig. 2). Single domain TPS proteins were mainly located in the group of
prokaryotic sequences. TPS/TPP fused proteins existed extensively.
The tree is separated into two main clades. The TPSs of animals and some prokaryotes and the GGPSs of the cyanobacteria form one clade. In this clade, the special GGPSs
are grouped at the end of a single branch. The TPSs of some prokaryotes, fungi, algae,
and higher plants form another cluster. Prokaryotic fused TPS/TPP proteins are located
between the single domain prokaryotic sequences and all of the eukaryotic sequences.
The TPS genes of plants are divided into two very distant clades that belong to plant
Class I enzymes and plant Class II enzymes. It is clear that the proteins from red algae are closely related to each other, but
group in clusters; for example, P. yezoensis, Chondrus crispus, G. sulphuraria, and Cyanidioschyzon merolae. PhTPS1 and 2 are close to the proteins of P. yezoensis (Contig 4636 and Contig 27879) with homologies of 81.28% and 72.3%. Four clusters,
namely, PhTPS1–4 are dispersed along different branches instead of clustering together.
The cluster including PhTPS1–2 is along the branch with plant Class I, and PhTPS3–4
is along the branch with plant Class II. PhTPS4 is relatively distant from the other
three PhTPSs and is closely associated with Class II proteins. It forms a small cluster
with some red and brown algae. Some TPS proteins containing the N-terminus CBM20 domain
were noted. They are relatively close in the phylogenetic tree, involving proteins
from red algae, diatoms, and brown alga (S. japonica); for example, P. umbilicalis (OSX79290.1, 85.84%), G. sulphuraria (EME31717.1, 48.05%), and C. merolae CM3596 (BAM80147.1, 41.25%) from Rhodophyta, S. japonica(AGT20052.1, 23.45%) from Phaeophyta, and Phaeodactylum tricornutumCCAP 1055/1 (XP_002180425.1, 28.36%) from Bacillariophyta, but are not found in the TPS genes of other species.
The phylogenetic tree of the only TPS and TPP domain were also constructed, respectively
(Additional file 2: Figure S2A, B). It could be found that the phylogenetic tree for
only TPS domain is nearly the same as that of TPS/TPP. While, the phylogenetic tree
for only TPP domain is different from that of TPS/TPP. Instead of forming two large
clades, all clades branched from the root and the clades position changed. For example,
the PhTPP domains are divided into three clades. The clades of PhTPP 1 and PhTPP 3
are separated by plant Class I. Among them, the cluster of red alga including PhTPP
1and plant Class I to form a clade. Besides, the species of each small clade is basically
Expression and enzymatic function of PhTPS1–4 proteins.
To verify the function of four proteins from P. haitanensis PhTPS1–4, we expressed their TPS domain by E. coli and separated the purified proteins by SDS-PAGE. We observed bands in the position
of the corresponding molecular weight (PhTPS1, 77.9 kDa; PhTPS2, 82.3 kDa; PhTPS3,
65.4 kDa; PhTPS4, 75.9 kDa). To confirm the expression, four recombinant His-tagged
proteins were confirmed by Western blotting using an anti-His-tag-antibody (Fig. 3).
To detect the biochemical activity of PhTPS1–4, UDP-Gal and G3P were allowed to react
with them, and the resulting products were respectively analyzed (Fig. 4). First,
the retention times of the two purified standards, floridoside (retention time = 20.83
min) and isofloridoside (retention time = 26.14 min), were obtained and identified
using the [M-H]− ions at m/z 253.0925 by HPLC-MS.
In MS/MS spectra, the characteristic fragment ion at
m/z 89.02 and 119.03 from [M-H]− ions were also utilized for qualitative analysis of floridoside and isofloridoside
. It was found that the reaction products floridoside and isofloridoside were
generated which were catalyzed by PhTPS1 and PhTPS4 using HPLC-MS. While catalyzing
by PhTPS3, only the isofloridoside was produced. However, the floridoside and isofloridoside
were not detected after catalyzing by PhTPS2.
Quantitative analysis of the catalytic products of PhTPS1, PhTPS3, and PhTPS4 showed
that the conversion ratios of the four enzymes were all low. The enzyme activities
of PhTPS1 and PhTPS4 producing floridoside were 0.26 and 0.22 μmol·h−1·mg−1, respectively. The enzyme activities of PhTPS1 and PhTPS4 producing isofloridoside
were 0.50 and 0.61 μmol·h−1·mg−1, respectively. The enzyme activity of PhTPS3 was 0.23 μmol·h−1·mg−1, and only the isofloridoside was biosynthesized (Table 3).
Expression of PhTPS1–4 under different abiotic stimuli
The expression of four PhTPS genes was analyzed under desiccation, high temperature, and different salinity treatments
(Fig. 5). Following 35°C
high temperature stress treatment for 30 min, the expression of PhTPS1–4 was significantly increased. The increase in PhTPS3 and PhTPS4 was the most obvious, reaching 20.5- and 26.6-fold (P < 0.01) that of the control, followed by PhTPS1, which was increased by 9.2-fold (P < 0.01) that of the control. After recovery under normal temperature for 1 h following
the thermal shock, the upregulation of PhTPS2, PhTPS3, and PhTPS4 was reduced, but the upregulation of PhTPS1 was significantly enhanced, reaching 11.5-fold that of the control. Compared with
recovery for 1 h, recovery for 3 h did not elicit any major changes (Fig. 5A).
During the first 1 h of desiccation, the expression of four PhTPSs increased significantly and remained at high levels throughout the process. Among
them, PhTPS1 and PhTPS4 showed the strongest responses. When treated for 1 h, the increased expression multiple
reached more than 30 times (P < 0.01), and the expression level gradually decreased with the extension of desiccation
time. However, the up-regulation of PhTPS2 and PhTPS3 was slightly weaker than that of PhTPS1 and PhTPS4, and the up-regulation peaked at 2 h, but the up-regulation remained at around 5–12
fold of the control during the entire desiccation process (Fig. 5B).
The expression of the PhTPS1–4 genes was examined when the P. haitanensis thalli were grown under different NaCl concentrations ranging from
500 mM to 1400 mM (Fig. 5C). Pyropia haitanensis is mainly grown in the East China sea, and the seaweed used in this study is from
Xiangshan, China, where the salinity is
500 mM. Therefore, here we compare gene expression under different salinity stress concentrations
with that under
500 mM NaCl as a control. According to the results, the expressions of four PhTPS genes varied under different salinity stresses, but their overall expression was not very high. Among them, PhTPS4 was most sensitive to changes in salinity, and under
700 mM NaCl, PhTPS4 showed slight salt-stimulated expression and was upregulated to 1.86-fold that of
500 mM NaCl group (P < 0.01). The levels of PhTPS1, PhTPS3, and PhTPS4 were increased under
1400 mM NaCl stress, being 2.22-, 2.04-, and 2.16-fold higher than that of the
500 mM NaCl group (P < 0.01). PhTPS3 and PhTPS4 all reacted relatively strongly at high salinity. PhTPS2 was not upregulated with the increase in salinity in comparison to the
500 mM NaCl group.
The accumulation of (iso)floridoside in P. haitanensis under various NaCl concentrations ranged from
500 mM to 1400 mM for 1 h. LC–MS revealed that floridoside and isofloridoside all accumulated (Fig.
5D). The concentration of isofloridoside rose proportionally with the external NaCl
500 mM to 700 mM (P < 0.01), but decreased under highly hypersaline conditions. Floridoside plays a rather
minor role as an osmolyte, because its change trend was the same as isofloridoside
under salt stress and even decreased at a high salt concentration.