Abstract

   The influence of harvest time on the photosynthetic protein quality of
   the red alga Porphyra dentata was determined using label-free
   proteomics. Of 2716 differentially abundant proteins that were
   identified in this study, 478 were upregulated and 374 were
   downregulated. The top enriched Kyoto Encyclopedia of Genes and Genomes
   (KEGG) and gene ontology (GO) pathways were metabolic processes and
   biosynthetic pathways such as photosynthesis, light harvesting, and
   carbon fixation in photosynthetic organisms. Nine important
   photosynthetic proteins were screened. Correlations among their
   expression levels were contrasted and verified by western blotting.
   PSII D1 and 44-kDa protein levels increased with later harvest time and
   increased light exposure. Specific photoprotective protein expression
   accelerated P. dentata growth and development. Biological processes
   such as photosynthesis and carbon cycling increased carbohydrate
   metabolism and decreased the total protein content. The results of the
   present study provide a scientific basis for the optimization of the
   culture and harvest of P. dentata.

   Keywords: different harvest, formation mechanism, photosynthetic,
   Porphyra dentata, proteomics

1. Introduction

   Porphyra dentata (P. dentata) is an edible red seaweed belonging to the
   Phylum Rhodophyta (Class Protoflorideae). In recent years, it has been
   widely cultivated, harvested, processed, and consumed in East-Asian
   countries. Southern China extensively cultivates and produces seaweeds
   [[42]1,[43]2,[44]3]. As consumer health awareness increases, the demand
   for Porphyra in food processing is increasing rapidly. Porphyra is rich
   in proteins, carbohydrates, vitamins, micronutrients, and essential
   amino acids [[45]4,[46]5]. It is also abundant in vitamin B12,
   porphyrans, and taurine [[47]6,[48]7,[49]8]. P. dentata has
   significantly higher mineral and amino acid content than other Porphyra
   species [[50]9]. Certain constituents of P. dentata are bioactive and
   may have antineoplastic and other pharmacological efficacy. This alga
   is also rich in polysaccharides and is used to prepare various peptide
   products and tea beverages [[51]10,[52]11,[53]12].

   The P. dentata harvest period is from November to March. It is
   cultivated by the split stubble method, and it is harvested four to
   five times annually. The harvests are designated the first to the
   fourth or fifth harvests [[54]13,[55]14]. The protein levels from the
   first and fifth harvest are approximately 35% and 31%, respectively.
   Meanwhile, the carbohydrate levels in the first and fifth harvest are
   29% and 31%, respectively (Yang M, Zhao Y, Wang J, et al., unpublished
   data). The nutritive value gradually decreased with harvest time.

   In addition to changes due to longer harvest times, different
   processing methods had a considerable impact on P. dentata nutrient
   composition and content. The thalli of the first harvest are crisp,
   have a high protein content, and are rich in bioactive ingredients.
   They may be eaten raw or processed into various products such as
   sun-dried, baked, or sliced laver and salads. The thalli of the fifth
   harvest are thick, black, and cheap. They are used as food additives or
   non-food biological materials. Hence, the harvest period of P. dentata
   determines the industrial application of this alga.

   Sunlight provides the energy required for the growth of P. dentata and
   affects its temperature and light stress responses [[56]15]. P. dentata
   growth, development, and physiology are all influenced by ambient
   temperature, ultraviolet (UV) irradiation, and light wavelength and
   intensity. A previous study showed that the total soluble protein (TSP)
   content decreased while the growth rate increased with increasing light
   intensity and sunlight duration [[57]16]. The relative photosynthetic
   response of the alga varies with regional climate and aquatic
   physicochemistry [[58]17]. Observed differences in P. dentata
   photosynthesis are associated with biotic and abiotic stressors,
   including temperature extremes, ocean acidification, carbon fixation
   rates, tides, salinity level [[59]18,[60]19,[61]20].

   Recently, proteomics has been widely used to evaluate red algal edible
   quality. For example, these methods revealed that Porphyra haitanensis
   counteracts drought stress mainly by downregulating its own
   photosynthetic and energy metabolism activity. They were also used to
   identify the antioxidant enzymes involved in photosynthesis and protein
   synthesis in wild-type Porphyra yezoensis as well as
   irradiation-induced mutants [[62]21,[63]22] Label-free proteomics
   operates stably and can significantly reveal detailed information about
   the changes in proteins that occur in response to different conditions.
   It can also identify potential biomarkers [[64]23]. To the best of our
   knowledge, the present study was the first to apply proteomics to P.
   dentata during different harvest periods and assess differential
   expression of the proteins related to the observed variations in
   quality between first-harvest and fifth-harvest P. dentata. The factors
   implicated in algal harvest quality formation were investigated from
   the perspective of photosynthesis. The objectives of this study
   included the provision of a theoretical basis for quantifying the
   quality grading standards of P. dentata and a scientific basis for its
   effective development and utilization at different harvest times.

2. Materials and Methods

2.1. Samples

   First- and fifth-harvest P. dentata were obtained from Shen’ao Bay
   (23.46° N, 117.09° E), Nan’ao Island, Shantou City, Guangdong Province,
   China, in December 2020. They were cleaned with sterile water to remove
   sediment, appressoria, and other impurities, placed in sterile sealed
   tubes, transported to the laboratory in liquid nitrogen, and stored at
   −80 °C.

2.2. Total Protein Extraction

   The samples were ground in liquid nitrogen and BPP (borax,
   polyvinylpolypyrrolidone [PVPP], and phenol) mixed at a 1:10 ratio. The
   suspensions were centrifuged at 12,000× g and 4 °C for 20 min, and the
   supernatants were collected. An equal volume of Tris-saturated phenol
   was added to each supernatant, and each mixture was vortexed at 4 °C
   for 10 min. The mixtures were centrifuged (3K30, Sigma, St. Louis, MO,
   USA) at 12,000× g and 4 °C for 20 min, and the phenol phases were
   collected. An equal volume of BPP was added to each supernatant and the
   mixtures were vortexed at 4 °C for 10 min. The solutions were
   centrifuged at 12,000× g and 4 °C for 20 min, and the phenol phases
   were collected. Five volumes pre-cooled 0.1 M onium acetate in methanol
   were added, and the proteins were precipitated at −20 °C overnight. The
   supernatants were discarded by centrifugation (12,000× g), and the
   precipitates were washed twice with 90% (v/v) acetone and then
   air-dried. The precipitates were resuspended in lysis buffer (1% (v/v)
   SDS plus 8 M urea) and sonicated on ice for 3 min. The lysates were
   centrifuged and the supernatants were collected to determine the
   protein content by the bicinchoninic acid (BCA) method in a BCA Protein
   Assay Kit (Beyotime Biotechnology, Shanghai, China). The proteins were
   quantified according to the kit protocol.

2.3. Protein Digestion

   Protein digestion was performed according to a standard procedure
   [[65]24]. Each sample tube contained 100 μg protein, and Tris
   (2-carboxyethyl) phosphine (TCEP) was added to a final concentration of
   10 M. The tubes were incubated at 37 °C for 60 min. Appropriate amounts
   of iodoacetamide (IAM) were added to a final concentration of 40 M and
   reacted in the dark for 40 min. Then six volumes of cold acetone were
   added to each sample tube. The tubes were inverted three times and
   incubated at −20 °C for 4 h or until precipitates formed. The acetone
   was removed by centrifugation at 10,000× g for 20 min, and the
   precipitated protein was resuspended in 150 µL of 100 M
   triethylammonium bicarbonate (TEAB) buffer. Then 1:50 trypsin solution
   was added to each sample tube, and the mixtures were incubated at 37 °C
   overnight.

2.4. Peptide Desalination and Quantification

   The peptides were vacuum-dried (LNG—T83, Huamei, Jiangsu, China) and
   resuspended in 2% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic
   acid (TF). The samples were desalted with Sep-Pak (Waters Corp.,
   Milford, MA, USA) and vacuum-dried. The peptide concentrations were
   determined with a Peptide Quantification Kit (No. 23275; Thermo Fisher
   Scientific, Waltham, MA, USA). Loading buffer was added to each tube to
   prepare the samples for mass spectrometry (MS) analysis. The
   concentration of each sample was 0.5 µg/µL.

2.5. Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) Analysis

   The peptides were dissolved in mass spectrometry loading buffer and
   analyzed by LC–MS/MS. The peptide samples were separated in an EASY-nLC
   1200 Liquid System (Thermo Fisher Scientific). A C18 chromatographic
   column was used (75 μm × 25 cm; Thermo Fisher Scientific). Mobile phase
   A was 2% (v/v) acetonitrile plus 0.1% (v/v) formic acid. Mobile phase B
   was 80% (v/v) acetonitrile plus 0.1% (v/v) formic acid. The separation
   gradient was (1) 0–2 min: mobile phase B linearly increased from 0 to
   6%; (2) 2–105 min: mobile phase B linearly increased from 6% to 23%;
   (3) 105–130 min: mobile phase B linearly increased from 23% to 29%; (4)
   130–147 min: mobile phase B linearly increased from 29% to 38%; (5)
   147–148 min: mobile phase B linearly increased from 38% to 48%; (6)
   148–149 min: mobile phase B linearly increased from 48% to 100%; (7)
   149–155 min: mobile phase B held at 100%. Mass spectrometry was
   conducted in Q-Exactive HF-X (Thermo Fisher Scientific). The MS
   scanning range (m/z) was 350–1300, the acquisition mode was DDA, the
   fragmentation mode was HCD, the primary MS resolution was 70,000, and
   the secondary MS resolution was 17,500.

2.6. Protein Identification

   The original MS data output files were imported into the Proteome
   Discoverer^TM v. 2.2 software System (Thermo Fisher Scientific) for
   database searching and analysis. The fixed modification was
   carbamidomethyl, the variable modification was oxidation, the digested
   protein was trypsin, the maximum allowable error range of the parent
   ion mass was ±10 ppm, and the false discovery rate (FDR) of the peptide
   identification was ≤0.01.

   The identified protein contained at least one specific peptide. The
   protein abundance information obtained from the database search was
   used to perform statistical tests and analyses on the differentially
   abundant proteins. Student’s t-test was used to identify significant
   differences between treatment means at p < 0.05. Proteins with content
   that differed by ≥1.2-fold were considered differentially abundant
   proteins (DAPs).

2.7. Bioinformatics Analysis

   The GO ([66]http://geneontology.org, accessed on 22 October 2021) and
   KEGG pathway ([67]https://www.genome.jp/kegg/, accessed on 22 October
   2021) databases were used to perform GO function annotations and
   metabolic pathway analysis, respectively, on the DAPs to obtain
   information regarding their associated biological functions and
   processes and their cellular locations.

2.8. Western Blotting

   Two key proteins were selected as targets to corroborate the results of
   the proteomics analyses and verify photosynthetic protein expression.
   The extracted P. dentata proteins were quantitated by SDS-PAGE, and the
   protein strips were transferred by a semi-dry method [[68]23]. At a
   constant 400 mA, 45 min was required to transfer the target protein to
   the activated polyvinylidene fluoride (PVDF) membrane. The membrane was
   blocked with fast blocking solution for 10 min, primary rabbit
   anti-photosynthesis protein serum (1:3000) was added, and the membrane
   was incubated at 4 °C overnight. The secondary antibody was horseradish
   peroxidase (HRP)-labeled goat anti-rabbit serum (1:3000). The mixture
   was incubated for 2 h, and the membrane was immersed in western blot
   HRP-DAB substrate color developing solution, incubated in the dark for
   5 min, and photographed with an automatic gel imager (3500R, Tanon,
   Shanghai, China).

3. Results

3.1. Identified by Label-Free Proteomic Analysis

   We identified a total of 5151 proteins in P. dentata. There were 2574
   proteins at the first harvest, of which 90 were uniquely expressed.
   There were 2577 proteins at the fifth harvest, of which 93 were
   uniquely expressed ([69]Figure 1a). Student’s t-test analysis
   identified 2716 DAPs, of which 478 were upregulated and 374 were
   downregulated. The criteria were as follows: FC > 1.2 indicated an
   upregulated protein while FC < 0.83 indicated a downregulated protein
   (p < 0.05) ([70]Figure 1b).

Figure 1.

   [71]Figure 1
   [72]Open in a new tab

   Label-free proteomic analysis: (a) First/Fifth harvest protein Venn of
   P. dentata. (b) Volcano plot of differentially abundant proteins (DAPs)
   from P. dentata (threshold = | log2 (fold change) |>1.2 or <0.83 and p
   < 0.05).

3.2. GO and KEGG Pathway Annotation Analysis of the DAPs

   The GO classification of the ADPs between the first and fifth harvest
   P. dentata groups is shown in [73]Figure 2a. The annotations of the
   DAPs in biological processes were significantly increased and focused
   on cellular processes (GO: 0009987, 323), metabolic processes (GO:
   0008152, 295), and regulation of biological processes (GO: 00050789,
   51). In cell composition, cell (GO: 0005623, 308) and cell part (GO:
   00044464, 297) were enriched. There were no significant differences in
   the cell membrane, membrane transport, or organelle expression. Under
   molecular functions, catalytic activity (GO: 00003824, 387), binding
   (GO: 0005488, 307), and other subsidiary proteins were enriched. The
   activation energy of the proteome continuously regulated P. dentata
   growth and development with the extension of the harvest period. The
   KEGG pathway annotation showed that metabolic proteins occupied the
   highest proportion, followed by genetic information processing and
   environmental information processing ([74]Figure 2b). Under metabolic
   proteins, amino acid metabolism (34), carbohydrate metabolism (64), and
   energy metabolism (47) were enriched. These were presumed to be related
   to photosynthesis and respiration during P. dentata growth and
   development. The carbohydrates were converted into nucleic acids,
   proteins, lipids, and structural polysaccharides and contributed to the
   differences in algal quality among harvest periods [[75]25]. Under
   genetic information, the folding, classification, and degradation (48)
   and translation (48) annotations were enriched. Under environmental
   information processing, guide protein signal transduction was presumed
   to be influenced by seawater and the ambient environment and gradually
   adapted to growth regulation.

Figure 2.

   [76]Figure 2

   [77]Figure 2
   [78]Open in a new tab

   Annotation analysis of the DAPs: (a) Protein GO analysis of First/Fifth
   harvest (MF: Molecular function; CC: Cellular component; BP: Biological
   process). (b) Protein KEGG analysis of First/Fifth harvest.

3.3. GO and KEGG Pathway Enrichment Analysis of the DAPs

   The molecular characteristics of the P. dentata proteins were
   established. A GO enrichment analysis was performed using goatools
   ([79]https://pypi.org/project/goatools, accessed on 22 October 2021),
   and the molecular functions were elucidated and taxonomically
   categorized by Fisher’s exact test. The GO functional enrichment
   annotations found by the enrichment analysis are presented in
   [80]Figure 3a. Photosynthesis, light harvesting (GO: 0007965), and
   organellar protein localization (GO: 0072594) had similar significance
   levels in the distribution. The activity levels of oxidoreductases
   using other nitrogenous compounds as donors and NAD or NADP as
   acceptors (Oxidoreductase activity, with NAD or NADP as acceptor, GO:
   0016717) and metabolite precursor and energy generation (GO: 0006091)
   were the most highly enriched and significantly differently expressed
   (p < 0.01). The changes in respiratory metabolism and self-regulation
   influenced by exogenous sources were enhanced during P. dentata growth.
   During its growth, Porphyra is sensitive to environmental conditions
   (current, salinity, tides, pH, light intensity, and nutrient levels),
   and these contribute to variations in growth and quality. At the
   single-cell level, multicellular fertilization, attachment,
   germination, thallus development, and damage to the light-trapping
   pigment system and the algal body in response to external stressors all
   affect metabolite production and light energy transmission and, by
   extension, the levels of ATP and NADP required to reduce CO[2] for
   carbohydrate biosynthesis. For these reasons, the two proteins classes
   significantly differed among P. dentata harvest times [[81]26,[82]27].
   Based on the GO analysis, a KEGG pathway enrichment annotation was also
   performed. p < 0.05 was the correction threshold ([83]Figure 3b).
   Proteins were significantly upregulated in the following pathways:
   protein processing in the endoplasmic reticulum (map04141), ascorbate
   and aldarate metabolism (map00053), N-glycan biosynthesis (map00510),
   and carbon fixation in photosynthetic organisms (map00710). Protein
   enrichment was also detected in betalain biosynthesis (map00965),
   linoleic acid metabolism (map00591), photosynthesis-antenna proteins
   (map00196), and others associated with nutrient biosynthesis.

Figure 3.

   [84]Figure 3
   [85]Open in a new tab

   Enrichment analysis of the DAPs DAPs: (a) GO function of enrichment
   analysis of First/Fifth harvest. (b) DAPs KEGG function of enrichment
   analysis of First/Fifth harvest. (* p < 0.05, ** p < 0.01).

3.4. Differential Expression of Photosynthesis-Related Proteins

   Photosynthesis is essential for algal growth and energy production.
   Half the products of CO[2] fixation in the global ecosystem are derived
   from algal photosynthesis [[86]28]. P. dentata is the main alga in the
   intertidal zone, and its quality may change during growth until harvest
   in response to changes in light intensity. Weather conditions, changes
   in the angle of light inclination, and other exogenous factors account
   for observed differences in the perceived stress responses manifested
   as protein upregulation, downregulation, and new protein emergence. A
   portion of the protein decomposes and ultimately disappears. Here, we
   selected nine DAPs associated with photosynthesis and calculated the
   correlations among them ([87]Figure 4, [88]Table 1).

Figure 4.

   [89]Figure 4
   [90]Open in a new tab

   Correlation changes of important photosynthetic differential protein.

Table 1.

   Differentially abundance proteins for photosynthesis in First/Fifth
   harvest identified by label-free proteomics.
   Accession Protein Name Ko Name First/Fifth Harvest FC p-Value
   TRINITY_DN4391_c0_g1_i1_orfp1 Extrinsic protein in photosystem II Xdh
   129.68 0
   TRINITY_DN20824_c0_g2_i1_orf1 Photosystem I P700 chlorophyll a
   apoprotein A1 PsaA 1.625 0.012
   TRINITY_DN43883_c0_g1_i1_orf1 Photosystem II protein D1 PsbA 1.363
   0.031
   TRINITY_DN8817_c0_g1_i1_orf1 Photosystem II 44 kDa protein psbC 1.485
   0.020
   TRINITY_DN14993_c0_g1_i1_orf1 Photosystem II protein W Psb28 1.235
   0.093
   TRINITY_DN12291_c0_g1_i1_orfp1 Photosystem II 12 kDa extrinsic protein,
   chloroplastic PsbU 129.68 0
   TRINITY_DN376_c0_g2_i1_orfp1 NADH-ubiquinone oxidoreductase 129.68 0
   TRINITY_DN31442_c0_g1_i1_orf1 Chlorophyll a/b-binding protein Lhca1
   1.891 0.321
   TRINITY_DN2334_c0_g3_i3_orf1 Light-harvesting protein Lhca1 0.762 0.142
   [91]Open in a new tab

   In the photosynthetic system of P. dentata, the DAPs were distributed
   mainly in the chloroplasts and thylakoid membranes. The levels of PSI
   P700 chlorophyll a apoprotein A1, PSII protein D1, PSII 44kDa protein,
   PSII protein W, and chlorophyll a/b-binding protein were upregulated
   while the light-harvesting protein was downregulated at the first/fifth
   harvest. Extrinsic protein in PSII, NADH-ubiquinone oxidoreductase, and
   PSII 12 kDa extrinsic chloroplastic protein was specifically expressed
   at the fifth harvest.

3.5. Western Blotting

   When using β-actin as the internal reference, the changes in the
   expression level of certain proteins at the first/fifth harvest were
   verified by western blotting ([92]Figure 5). Two parallel experiments
   were conducted, and the DAPs were D1 and 44-kDa in PSII. The target
   DAPs were upregulated at the first/fifth harvest. Hence, the western
   blotting results were in accordance with those of the proteomic assay.

Figure 5.

   [93]Figure 5
   [94]Open in a new tab

   Differential proteins D1 and 44k Da verification result of western
   blotting (M: Marker 1~2: Fifth, 3~4: First).

4. Discussion

   In algae, aerobic photosynthesis occurs mainly in Photosystems I and II
   (PSI and PSII), which dynamically absorb and balance light energy. The
   PSI and PSII comprise chloroplasts, light-harvesting antenna proteins,
   redox proteins, and other elements. It actively distributes the
   absorbed light energy for photosynthesis [[95]29].

   Photosystem II protein W (PSII W) belongs to the Psb28 protein family.
   It protects PSII catalyzing light-induced water oxidation, converting
   light energy into chemical energy, and generating O[2]. During
   assembly, Psb28 prevents PSII from being damaged by light. Gradual PSII
   W upregulation may occur as a result of a prolonged harvest period and
   gradual darkening of the algae. The latter potential confirmed
   increases in the efficiency of photosynthesis and the biosynthesis of
   light-harvesting pigment proteins. Excessive light energy absorption
   increases the synthesis of photoprotective PSII W, thereby ensuring
   normal algal growth and development. The lack of this protein may
   result in growth retardation, especially under naturally occurring
   intermittent high light/dark conditions. The regulation of this protein
   confirms the importance of the PSII assembly factors in light
   adaptability [[96]30,[97]31].

   Factors such as high-intensity light and high temperature reduce CO[2]
   availability, induce reactive oxygen species (ROS) formation, and
   significantly reduce the photosynthetic rate of Porphyra chloroplasts
   [[98]32]. Protective extrinsic proteins are attached to the periphery
   of PSII. The PSII 12-kDa extrinsic protein, which is present in the
   chloroplast and is a member of the Psbu, is an extrinsic protein in
   PSII of cyanobacteria and red algal and was specifically expressed at
   the fifth harvest. It stabilizes the photosynthetic environment in the
   presence of oxygen [[99]33]. It is possible that prolonged
   photosynthesis stimulates photoprotection, mitigating the adverse
   impact of external abiotic stress. In addition, the extrinsic protein
   in PSII reduces harvest loss that is caused by high ROS levels. This is
   a self-defense mechanism in algae [[100]34,[101]35]. NADH ubiquinone
   oxidoreductase is an energy-transducing, ATP-synthesizing enzyme that
   participates in the PSI electron transport chain (ETC) during light
   energy conversion. This enzyme is specifically expressed during the
   fifth harvest as PSII activity is inhibited from balancing the ETC and
   normal ATP synthesis. NADH ubiquinone oxidoreductase maintains normal
   ATP cycle activity while inhibiting excessive substrate reduction and
   ensuring normal biosynthesis [[102]36,[103]37].

   In P. dentata, PSII D1 is an important protein in light energy
   conversion. The synthesis and degradation of PSII D1 play decisive
   roles in algal quality and recovery. When the light intensity exceeds
   the energy synthesis requirement, it can readily cause mechanical
   damage to the PSII system and trigger photoinhibition [[104]38].
   Consequently, the pigment content of the laver thallus decreases as the
   synthesis of carotenoids, phycoerythrins, phycocyanins, and other
   pigments is reduced. The algal thalli may undergo discoloration, decay,
   and deterioration [[105]39]. Chloroplast thylakoid membrane fluidity is
   the key to damaged D1 protein degradation and synthesis [[106]40]. The
   western blotting and proteomic analyses showed that the D1 protein
   content was significantly higher at the fifth than the first harvest.
   As the harvest period extended, the laver thalli gradually became
   wider, thicker, and darker. The amount of light energy absorbed by the
   photosynthetic pigment protein was reduced, but the relative increase
   in energy conversion efficiency nonetheless maintained the rate of
   photosynthetic oxidation. The thick algal thalli mitigated
   light-induced damage and promoted D1 protein synthesis. There were
   higher levels of Na+-ATPase at the fifth than the first harvest, and
   high ATP conversion efficiency was maintained. The results of a prior
   study on the photosynthetic characteristics of Porphyra yezoensis at
   different harvesting periods were similar to those of the present work
   [[107]41].

   Photosynthesis is affected by the carbon cycle and UV irradiation. The
   latter inhibits algal carotenoid and chlorophyll biosynthesis,
   especially in dehydrated Porphyra. Thence, the loss of photosynthetic
   pigment significantly lowers photochemical efficiency and
   photosynthetic carbon fixation by PSII. However, gradual increases in
   atmospheric CO[2] and UV adaptability help improve P. dentata growth
   and development. These modifications induce photosynthetic activity and
   repress photoinhibition. Therefore, prolongation of the algal harvest
   period significantly increases the D1 protein content. Rubisco is the
   key protein in carbon fixation and was upregulated in the first/fifth
   algal harvests. Under natural conditions, UV irradiation is conducive
   to the formation and accumulation of amino acids required for protein
   synthesis. Additionally, mycosporine-like amino acid (MAA) proteins
   unique to Porphyra increase throughout the harvest period and mitigate
   the effects of UV radiation and oxidative stress, promoting PSII D1
   protein synthesis [[108]42]. Previous research on Porphyra haitanensis
   reported that UV irradiation significantly increased the proline (Pro),
   serine (Ser), histidine (His), and alanine (Ala) content which, in
   turn, could offset stress-related cell damage, remove ROS, protect
   membrane integrity, enhance metabolic pathways, and increase the total
   sugar content [[109]43,[110]44]. These findings were consistent with
   our results (data not published). Glutamic acid (Glu) was downregulated
   in Porphyra haitanensis but upregulated in P. dentata, possibly because
   each species has a unique background antioxidant capacity. An increase
   in the glutamate content can stimulate glutathione metabolism and
   increase the ascorbate content. Light stress induces the production of
   dehydroascorbic acid (DHA), which reduces oxidative damage. Increases
   in amino acid content enhance the high-quality protein content of algal
   supplements. Moreover, the fifth algal harvest has a strong seaweed
   flavor and can be used as high-quality raw material for seaweed
   processing.

   The exogenous ion content in P. dentata significantly affects its
   photosynthetic rate. The Ca^2+ and Fe^3+ content significantly
   increased in P. dentata with prolongation of the harvest period (data
   not published). Increases in the concentrations of these cations
   maintain cellular homeostasis and redox capacity, mitigate
   light-induced temperature elevation, improve antioxidant capacity, and
   favor D1 protein synthesis [[111]45]. Mn^2+ is also a key cofactor in
   the latter process. The C-terminus of mature D1 protein is critical for
   Mn^2+ cluster assembly. The affinity of the PSII external proteins is
   significantly reduced in the absence of Mn^2+ and its cluster elements.
   The loss of D1 protein hinders further biosynthesis of this substance.
   Free Ca^2+ is also necessary for D1 protein assembly. For this reason,
   the Mn^2+ content was significantly higher at the fifth than the first
   algal harvest [[112]46].

   The 44-kDa CP43 antenna protein in PSII is the core component of the
   antenna system and is vital to light energy absorption and
   transmission, and pigment biosynthesis. CP43 transfers light energy to
   the PSII reaction center (RC) and help protect the optical system
   structure. The PSII D1 and PSII 44-kDa protein levels are mutually
   correlated. D1 maturation is severely impeded when photoinhibition
   alters the PSII steady-state. Hence, the interaction between D1 and
   CP43 is hindered, as our western blotting results demonstrated
   [[113]47]. However, the CP43 and D1 proteins accumulate and repair, the
   pigment proteins are synthesized and accumulate, and the algal color
   deepens with the gradual extension of the growth and harvest periods.
   In addition, CP43 forms a complex domain with the surrounding CP47
   protein. This mechanism is conducive to light energy storage, increases
   the content of pigment proteins such as cytochrome b6, and accelerates
   color accumulation, as our measurements indicated [[114]48].

   Members of the Lhc pigment protein family are differentially expressed
   in P. dentata. They all capture light energy and rapidly transmit
   energy. Lhc is regulated by the photochemical reactions in PSI and PSII
   [[115]49]. In P. dentata, Lhc localized mainly to PSI and various Lhch1
   species. The latter are light-harvesting antenna pigment protein
   complexes that participate in chlorophyll biosynthesis and color
   assembly in P. dentata. Lhca1 protein forms an LHCI-730 heterodimer
   when Lhca4 and PSI core proteins combine with PsaG and PsaF after
   delivery to PSII. They function in tandem with PSII D1, PSII D2, CP43,
   and CP47 proteins to catalyze NADP synthesis [[116]50].
   Light-harvesting protein content gradually decreased, and chlorophyll
   a/b-binding protein gradually increased at the first/fifth harvests. In
   the initial growth stage, a large amount of light must be absorbed to
   capture sufficient light energy to support the energy response and ATP
   synthesis and conduction. Later in the season, reactions decline in the
   efficiency of photosynthesis induces algal senescence and pigment
   accumulation [[117]51]. Potential description the quality of P. dentata
   was lower at the fifth than the first harvest.

5. Conclusions

   In the present study, a label-free proteomics approach was used to
   investigate the changes in photosynthetic protein content of P. dentata
   harvested at different times. A total of 2716 DAPs were identified. Of
   these, nine major photosynthetic DAPs were screened and confirmed.
   Western blotting verified that the PSII D1 and 44-kDa protein levels
   gradually increased throughout the harvest period. The results of this
   study showed that the expression levels of specific proteins protecting
   the photosystem increased with harvest time, accelerating algal growth
   and pigment accumulation, gradually causing algal hypertrophy, reducing
   organoleptic quality, increasing carbohydrate accumulation, lowering
   the total protein content, and diminishing overall edible quality. The
   findings of this work emphasize the importance of photosynthetic
   protein changes during the harvest season, which sheds light on the
   molecular mechanism of nutritional changes in P. dentata during this
   period. At the same time, a protein database was constructed to support
   the future quality classification of this commercially valuable red
   algae. This lays a theoretical foundation for research on the breeding,
   cultivation, and processing of P. dentata, and provides an important
   reference for better understanding the physiological response
   mechanisms of marine algae to natural environmental stresses.

Acknowledgments