Applicability assessment of molecular tools and identification of lines suitable for genetic transformation in the red alga Pyropia yezoensis
Article information
Abstract
Pyropia yezoensis, a red macroalga distributed in the intertidal zone, serves as an important model organism for studying the adaptive mechanisms of intertidal seaweeds in response to abiotic stressors. Additionally, it is a significant economic seaweed due to its high nutrient content and is widely cultivated in China, Japan, and Korea. Despite its importance, existing molecular tools for genetic transformation remain limited and require further development to enhance our understanding of P. yezoensis biology. To address this limitation, based on previous studies, we firstly evaluated the expression of eight fluorescent proteins (FPs) in P. yezoensis. Our findings confirmed that PyZsYellow, PyZsGFP, and PysGFP can be detected successfully their specific fluorescent signals within the cells of P. yezoensis thalli. Notably, PyZsGFP demonstrated effectiveness in sporophytic filaments. 2×SV40 and PyNP may function as nuclear localization signal sequences, effectively directing the FP to localize within the nucleus of cells in P. yezoensis. T2A linker can mediate protein cleavage, to express the multiple genes in one construct, enabling them to function independently in P. yezoensis. Furthermore, we identified that the RZ line exhibited the highest transformation potential, while the middle region of thalli showed the superior transformation efficiency. A significant correlation between cell size and transformation efficiency. These findings will contribute to developing a systematic toolkit for conducting genetic transformations and elucidating gene functions in P. yezoensis.
INTORDUCTION
Pyropia yezoensis, a member of the Rhodophyta phylum, is an economically significant seaweed due to its high economic value. This organism exhibits a complex life cycle that alternates between sporophyte and gametophyte generations (Takahashi and Mikami 2017). It naturally inhabits intertidal zone and demonstrates remarkable tolerance to a wide range of extreme environmental stresses, making it an ideal model red macroalga for physiological and genetic research related intertidal seaweed (Saga and Kitade 2002, Mao et al. 2019). Complete genome sequencing has revealed that approximately half of 12,855 protein-coding genes remain functionally uncharacterized (Wang et al. 2020). The establishment of a genetic transformation system would be beneficial for elucidating the functions of these unknown genes. Furthermore, such advancements could enhance our understanding of P. yezoensis biology and facilitate the development of new cultivars with desirable traits in breeding programs. Therefore, the identification and optimization of efficient molecular tools are critical for establishing a highly effective and stable genetic transformation system.
Reporter genes, also known as screenable or scorable markers, are genes that encode proteins which can be directly detected or catalyze specific reactions yielding easily detectable products (Anami et al. 2013). Currently, the most commonly utilized reporter genes in plant research for validating transgenic lines or visually tracking gene expression dynamics include those encoding β-glucuronidase (GUS), luciferase (LUC), and green fluorescent protein (GFP) (Ow et al. 1986, Stewart 2001). Since Jefferson et al. (1987) cloned the Gus gene from Escherichia coli, it has served as a sensitive and versatile reporter marker in higher plant molecular biology (Gustavsson et al. 2001). Due to its straightforward histochemical staining assays, GUS is utilized for in situ histochemical localizations of the β-glucuronidase activity within cells and tissues. However, the use of Gus as a reporter gene presents certain limitations. Specifically, positive transformants may not be directly obtained because cells expressing the Gus gene can undergo cell death following staining. Furthermore, stain diffusion complicates the differentiation between positive and false-positive cells (Guivarc’h et al. 1996, Santi et al. 2003). First discovered in jellyfish, fluorescent proteins (FPs) have been successfully optimized for application as effective biomarkers within living cells due to their non-toxic nature and nondestructive visualization capabilities alongside their small molecular size (Chalfie et al. 1994, Anami et al. 2013). The identification of FPs represents one of the most significant breakthroughs in biology (DeBlasio et al. 2010).
The nuclear localization signal (NLS) is a continuous segment of amino acid sequences that directs target proteins into the nucleus, serving as an essential tool for elucidating protein functions (Lange et al. 2007, Mehdi et al. 2011). Classical NLSs are characterized by either a single cluster of basic residues or two clusters connected by a linker region comprising approximately 10–12 residues (Chang et al. 2013). The initial identification of the monopartite NLS was made in the large T antigen of the SV40 virus, which contains a polypeptide with five basic amino acids (PKKKRKV) (Kalderon et al. 1984). Due to their remarkable efficiency, NLS sequences have been utilized for the artificial localization of GFP within the nucleus as positioning markers, as well as for fusion with functional nuclear proteins (Kitamura et al. 2015, Lu et al. 2021). Linkers are naturally occurring short amino acid sequences that serve to separate domains within a protein. Currently, three primary types of linkers have been identified, flexible linkers, rigid linkers, and cleavable linkers (Gustavsson et al. 2001). Cleavable linkers, such as 2A peptides, consist of 18–22 amino acid residues and are commonly found in viral genomes (Ahier and Jarriault 2014, Daniels et al. 2014). By linking multiple genes together to form a single transcript via the 2A peptide, this peptide can self-cleave through ribosome “skipping” at the 2A site. This process ultimately results in the separation of the end of the 2A sequence from downstream products (Luke et al. 2010, Lajevardi et al. 2022). The phenomenon of 2A-mediated co-expression is universal across all eukaryotic cells and offers several advantages, including a compact structure and an optimal balance of gene expression between upstream and downstream genes (Liu et al. 2017).
To date, numerous reports have documented advancements in genetic transformation in P. yezoensis, including both transient and stable transformation in the nucleus, plastid transformation, and genome editing. Various molecular tools have been employed, such as native promoters derived from Pyropia, antibiotic resistance genes, and reporter genes such as the GUS gene or FP gene (Fukuda et al. 2008, Mikami et al. 2009, Uji et al. 2010, Kong et al. 2017, Zheng et al. 2021, Cao et al. 2022, Wang et al. 2024). P. yezoensis cells contain substantial amounts of phycoerythrin, chlorophyll, and other compounds, resulting in pronounced autofluorescence distinct from that observed in higher plants and FPs used in those plants not suitable in P. yezoensis (Zhang et al. 2012). At present, several different FPs have been explored whether to be available as reporter within thalli cell, including AmCFP from Anemonia majano, ZsGFP and ZsYFP from reef coral Zoanthus, and plant-adapted sGFP (Mikami et al. 2009, Uji et al. 2010, Hirata et al. 2011). However, the expression efficiency of these FPs in different literature remains low. For instance, ZsGFP and sGFP were reported as effective reporters in thallus cell, whereas codons of these FPs were not optimized; consequently, leading to low expression efficiency with fewer than 200 FP-expressing cells detected per experiment (Mikami et al. 2009, Uji et al. 2010). Additionally, ZsYFP cannot be detected with significant expression in thallus cells (Uji et al. 2010). Therefore, it is still necessary to develop and extend more effective FPs suitable in P. yezoensis cells. Furthermore, there is limited information available regarding the screening of NLS and linker peptides in P. yezoensis. Only SV40 and P2A peptide were validated for their functionality (Wang et al. 2024). Whereas, it was reported that notable differences existed in preferences for various NLSs and various 2A peptides (E2A, P2A, and T2A) have different their cleavage efficiencies (Hicks and Raikhel 1995).
Among the factors determining genetic transformation efficiency, genotypes within the same species exert significant influences, as reported in various species such as wheat, maize, tomato, potato, and soybean (Olhoft et al. 2003, Chaudhry and Rashid 2012, Hensel et al. 2017, Bakhsh 2020). For example, Hi-II genotype of maize showed maximum transformation efficiency with a range of 12–18%, while Egyptian genotype line Gz643 exhibited 42.2% regeneration frequency, and IL3 line was found to be most amenable to transformation with a transformation frequency of 31.7% among tropical varieties (Vega et al. 2008, Yadava et al. 2017). Because P. yezoensis is an important economical seaweed, it is important to screen the lines with high transformation efficiency to develop new varieties with qualified traits using genome editing technology. At present, there are no reports regarding identifying the lines with high transformation efficiency.
Gene transfer methods are essential for enhancing transformation efficiency. Various techniques, including glass bead, electroporation, polyethylene glycol (PEG), PEG combined with electroporation (Mei et al. 1998), and Agrobacterium-mediated methods (Cheney et al. 2001), have been used for P. yezoensis; however, these transformation methods yield low transformation efficiency. With the exception of the Agrobacterium-mediated method, all other techniques require the preparation protoplasts, which increase challenges due to this organism’s thick cell walls. Currently, the transformation efficiency achieved through electroporation or protoplast transformation methods does not exceed 5% (Hado et al. 2003, Mizukami et al. 2004), and stable inheritance of transformed traits has yet to be achieved. Particle bombardment is the most commonly used transformation technology in P. yezoensis (Kong et al. 2017, Cao et al. 2022, Wang et al. 2024), which is not constrained by transformation material limitations. In our previous studies, we obtained stable transformants and achieved transformation efficiency exceeding 5% (Cao et al. 2022). Moreover, the transformation method itself is often not the limiting factor; codon usage and expression levels may exert greater influence on transgene functionality.
Therefore, in this research, using particle bombardment technique, our objective is to further develop and extend more effective and stable molecular tools, and screen the lines with high transformation efficiency in P. yezoensis. For FPs, except for AmCFP, ZsGFP, ZsYFP, and sGFP, we also selected another four FPs with different excitation/emission spectrums, including blue FP EBFP, red FP tdTomato, red FP DsRed2, and the far-infrared FP smURFP. For NLS and linker, we selected four NLSs (SV40, 2×SV40, PyNP, and PySV40), and another T2A linker peptide, further compared and evaluated their applicability within this context. To assess the impact of different lines on genetic transformation, we screened 39 different lines to compare their transformation capabilities. These investigations will provide valuable insights into genetic transformation and protein function validation, thereby establishing a foundation for molecular genetic breeding.
MATERIALS AND METHODS
Materials and culturing
A lab-cultured pure line of P. yezoensis, designated as RZ, along with thirty-eight additional lines preserved in the laboratory algae bank and sourced from various geographical locations (Supplementary Table S1), were utilized for the experiments.
Free-living filaments (sporophytes) were cultured in sterilized natural seawater supplemented with Provasoli’s enrichment solution (PES) medium at a temperature of 20 ± 1°C. The light intensity was maintained at 35–40 μmol photons m−2 s−1 under a photoperiod of 12 h light and 12 h dark (L: D). Continuous aeration was provided using filter-sterilized air, and the culture medium was renewed every three days for approximately one month. The free-living filaments were fragmented into branches measuring 20–30 μm in length and incubated in a 200 mL Petri dish containing shells, while maintaining the same light conditions used for culturing free-living filaments. After nearly two months, the shells filled with filaments were transferred to a culturing bottle placed in an incubator set at 25 ± 1°C (light intensity: 28–35 μmol photons m−2 s−1, photoperiod: 12 L: 12 D) to induce swelling of filamentous conchocelis, which may last for about one month. Subsequently, the temperature was reduced to 20 ± 1°C until conchospores were released. A sterilized nylon rope was introduced into the culturing bottle to facilitate attachment of the conchospores, allowing them to develop into small thalli.
After the thalli reached a length of 1 cm on the nylon rope, the temperature was further lowered to 10°C. The thalli were then continuously cultured for a period of 35–40 d for further experiments. Throughout the culturing process, light conditions were maintained at an intensity of 35–40 μmol photons m−2 s−1 with a photoperiod of 12 h light: 12 h dark. The culturing seawater was sterilized by adding PES medium and was continuously aerated with filter-sterilized air, being renewed every three days.
Gene synthesis and plasmid construction
Eight different FPs with varying excitation and emission wavelengths were selected to evaluate their applicability in P. yezoensis. These include EBFP (excitation at 383 nm, emission at 445 nm), FP AmCyan (excitation at 458 nm, emission at 489 nm), ZsGFP (excitation at 493 nm, emission at 505 nm), sGFP (excitation at 488 nm, emission at 515 nm), ZsYellow (excitation at 529 nm, emission at 539 nm), tdTomato (excitation at 554 nm, emission at 581 nm), DsRed2 (excitation at 550 nm, emission at 579 nm), as well as smURFP (excitation at 642 nm, emission at 670 nm). Considering that the average GC content of P. yezoensis genes is nearly 70%, the codons of these FP genes were optimized to enhance their expression in P. yezoensis cells (Wang et al. 2020). Full-length fragments of the aforementioned FP genes were synthesized by the commercial company (Sangon Biotech, Shanghai, China) and subsequently inserted into the backbone plasmid pBI121-pPyACT4-PyGUS-NOSt. Consequently, the PyGUS gene was replaced with each FP gene, which was placed under the control of the pPyACT4 promoter. The plasmids were constructed using homologous recombination technology with recombinant primers (Supplementary Table S2) and designated as pBI121-PyEBFP, pBI121-PyAmCyan, pBI121-PyZsGFP, pBI121-PyZsYellow, pBI121-PysGFP, pBI121-PysmURFP, pBI121-PyDsRed2, and pBI121-PytdTomato, respectively.
Four NLS sequences, namely SV40, 2×SV40, PySV40, and PyNP (see Supplementary Table S3), were selected to evaluate their nuclear-targeting abilities in P. yezoensis cells. The codons of SV40 and NP were optimized and designated as PySV40 and PyNP, respectively. The plasmid of PBI121-pPyACT4-PyZsYellow-NOSt served as the basic backbone for assessing NLS applicability. A fused NLS-PyZsYellow fragment was amplified using recombinant primers and then substituted for PyZsYellow to generate the destination plasmid, designated as PBI121-pPyACT4-NLS-PyZsYellow-NOSt (refer to Supplementary Table S4). The T2A peptide, derived from Toshea asigna virus 2A sequence, was used to employed to validate its self-cleaving property in P. yezoensis. The Py03626 gene encodes a histone acetylated reader protein that has been confirmed to localize within the nucleus (Pang et al. 2025). In this study, it was utilized as a nucleus marker gene to be fused with the T2A peptide and PyZsYellow to construct the vector pBI121-Py03626-T2A-PyZsYellow-NOSt (see Supplementary Tables S3 & S5).
Particle bombardment in filaments and thalli of Pyropia yezoensis
The concochelis in a healthy condition were fragmented into filamentous segments, each approximately 4–6 cells in length. Subsequently, around 1 × 105 filament cells were utilized for particle bombardment. After four days of recovery culture in a 20°C incubator, cell viability was assessed by staining with 0.5% Evans blue dye. Prior to the particle bombardment, the mortality rate of filament cells resulting from fragmentation was counted. Leafy gametophytes measuring 3 cm × 3 cm were employed for the particle bombardment. The bombardment parameters for thalli adhered to the previously established research protocols (Cao et al. 2022). For each bombardment, either 10 or 15 μg of plasmid DNA (for thalli and filament, respectively) along with 600 μg of gold particles (with a diameter of 0.6 μm) were utilized per construct. The bombardment of thalli was conducted at a helium pressure of 1,350 psi, a vacuum pressure of 30 mmHg, and a bombardment distance of 6 cm; for filaments, the corresponding parameters were 1,100 psi helium pressure, 29 mmHg vacuum pressure, and a bombardment distance of 9 cm.
The PDS-1000/He particle bombardment device (Bio-Rad, Foster City, CA, USA) was employed to perform transformation in both filaments and thalli. Following the bombardment procedure, algal tissues were transferred to a 96-well plate containing PES medium and incubated at temperatures of either 10 or 20°C (filaments at 20°C, thalli at 10°C) for 96 hours (including an initial period of 48 h in the darkness), after which they were observed under microscopy.
Microscopic observation
The gametophytic thalli and sporophytic filaments transformed with plasmids containing various FP were examined using a fluorescence microscope (Nikon Eclipse 80i; Tokyo, Japan). The following excitation and emission settings were used, PyEBFP and PyAmCyan through DAPI pass filter, ZsGFP, PysGFP, and PyZsYellow through FITC filter; PytdTomato, PyDsRed2, and PysmURFP through TRITC filter. Both fluorescence intensity and quantification of cell dimensions were respectively measured among 30 randomly selected cells under an optical microscope (BX53; Olympus, Tokyo, Japan). For the quantification of FP fluorescence, the autofluorescence was first quantified and then this fluorescence was subtracted from the total quantification, resulting in only the quantification of FP. ImageJ software was used to calculate the intensity of pixels. For each FP, the same observing parameters were conducted across constructs.
The transformation efficiency was measured by the number of cells expressing FP after genetic transformation/the total number of cells particle bombardment. First, we quantified the cell density (cells per mm2) using the semi-automatic Cell Counter plugin in ImageJ software. Subsequently, the total number of cells bombarded from each 3 cm × 3 cm thallus region was calculated. To minimize variability arising from differences in tissue thickness and intrinsic cell density across samples, thalli from all genotypes were cultured under identical environmental conditions; moreover, only the midsection of each thallus was selected for particle bombardment. All experiments were independently repeated three times to ensure data reliability.
Assays of chlorophyll fluorescence parameters
Chlorophyll fluorescence parameters were measured in the thalli of selected lines using a FluorCam chlorophyll meter (FluorCAM MF180; Photon Systems Instruments, Drásov, Czech Republic) following the methodology described in (Yin et al. 2025). Key parameters, including maximum photochemical efficiency of photosystem II (Fv/Fm), actual photochemical efficiency (Qy), non-photochemical quenching (NPQ), photochemical quenching (qP), and others, were extracted from the data generated by the instrument’s software during quenching curve analysis. Prior to measurements, samples were dark-adapted for 15 min. For each line, three biological replicates were analyzed, with each replicate subjected to three independent measurements.
Statistical analysis
All data in this study were presented as the mean ± standard deviation based on three biological replicates. A one-way analysis of variance (ANOVA) was performed to compare means across multiple independent groups. Prior to ANOVA, the assumptions of normality and homogeneity of variances were assessed using the Shapiro-Wilk test and Levene’s test, respectively. Post-hoc analyses were performed using the least significant difference method to evaluate significant differences among samples or treatments, with a significance level set at p < 0.05, as implemented in SPSS Statistics 17.0 (IBM Corp., Armonk, NY, USA). The correlation scatter plot was generated using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Regression analyses were performed using ordinary least squares (OLS) regression.
RESULTS
Comparisons of different FP expressed in Pyropia yezoensis cells
Previous studies have demonstrated that the endogenous promoter pPyACT4 can drive the expression of both GUS and the hygromycin resistance gene (Hyg) in P. yezoensis (Cao et al. 2022). Therefore, this study utilized the promoter pPyACT4 to control the expression of various FP genes. The schematic diagrams of the constructed vectors, which include pBI121-PyEBFP, pBI121-PyAmCyan, pBI121-PyZsGFP, pBI121-PyZsYellow, pBI121-PysGFP, pBI121-PysmURFP, pBI121-PyDsRed2, and pBI121-PytdTomato, are presented in Fig. 1A. Fluorescent signals emitted by each FP were observed 96 hours post-transformation via particle bombardment. Among these proteins, PyZsGFP, PyZsYellow, and PysGFP displayed pronounced green fluorescence within the cytoplasm of thalli cell (Fig. 1D–F), significantly surpassing the autofluorescence levels observed in these cells. No significant differences were noted regarding either fluorescence intensity or cell count among these three proteins (Fig. 2), indicating that they can serve as effective biomarkers for protein localization or non-invasively mark expression within living thallus cells. In contrast, the fluorescence signal from PyEBFP, PyAmCyan, PysmURFP, PyDsRed2, and PytdTomato did not exhibit significant difference in different cells (Fig. 1B, C & G–I). PyZsGFP has also been considered as a good reporter for transient gene expression in thalli by other researches (Mikami et al. 2009, Uji et al. 2010), which was inconsistent with the result of this research. We further investigated whether PyZsGFP could be also detected its specific signal in filament cells of P. yezoensis; results showed that PyZsGFP functions effectively as a reporter within sporophytic cells (Fig. 1J). Fig. 1K–M shows the autofluorescence of P. yezoensis under fluorescence filters DAPI, FITC, and TRITC.
The expression results of different fluorescent protein (FP) plasmids in Pyropia yezoensis. (A) A schematic diagram of FPs plasmids utilized in particle bombardment. (B–J) Observations of the expression of each FP under fluorescent microscopy. The expression plasmids, including pBI121-PyEBFP (B), pBI121-PyAmCyan (C), pBI121-PyZsGFP (D), pBI121-PyZsYellow (E), pBI121-PysGFP (F), pBI121-PysmURFP (G), pBI121-PyDsRed2 (H), and pBI121-PytdTomato (I), and pBI121-PyZsGFP (J). K–M shows the autofluorescence of P. yezoensis under fluorescence filters DAPI, FITC, and TRITC. Fluorescent images (B–M, upper) are displayed alongside bright field images (B–M, lower). FLU and BF represent fluorescence and bright-field images (same as below). The circle in red represents the cell showing fluorescent signal from FP (same as below). Scale bars represent: B–I, 20 μm; J–M, 50 μm.
The comparison of the number of cells expressing fluorescent proteins (FPs) and the average fluorescence intensity of various FPs in Pyropia yezoensis.
Screening for lines with high transformation efficiency in Pyropia yezoensis thalli
To identify lines of P. yezoensis exhibiting high transformation efficiency in thalli, we selected 39 lines from different geographic regions to compare their transformation efficiencies using PyZsYellow as reporter. The results demonstrated that yellow fluorescent signal of PyZsYellow FP were successfully detected in 23 lines (Fig. 3A). In contrast, no FP signals were observed in the remaining 16 lines (PYC-175, PYC-139, PYC-184, PYW-112, PYC-172, PYC-72, PYW-265, PYW-357, PYC-161, PYC-207, PYC-84, PYW-367, PYC-173, PYC-174, PYC-73, PYC-54, and PYC-157). Further analysis revealed that RZ line exhibited the highest transformation efficiency at 5.2 × 10−5, followed by PYC-144, PYC-64, PYC-359, PYC-78, and PYC-75 (Fig. 3C). We also compared transformation efficiency across different regions of the thalli and found that the middle region of thalli displayed higher transformation efficiency compared to the tip and basal regions (Fig. 3B).
The analysis of transformation efficiency in thalli from various lines of Pyropia yezoensis. Transformation efficiency is reported as mean ± standard deviation (×10−5). (A) Morphology of thalli with high transformation efficiency and observation of EP expression. Upper left, RZ; upper middle, PYC-64; upper right, PYC-144; lower left, PYC-359; lower middle, PYC-78; and lower right, PYC-8. (B) Comparison of expression efficiency of PyZsYellow protein from different regions of P. yezoensis thallus. **p < 0.01. (C) Comparison of transformation efficiency and average fluorescence intensity of PyZsYellow protein in different strains of P. yezoensis. The experiment was repeated three times (n = 3). *p < 0.05. (D) Correlation analysis between cell size and transformation efficiency in P. yezoensis thallus (Pearson correlation coefficient r = 0.8833, R2 = 0.7803, p < 0.0001, 95% CI, 0.7456–0.9487, n = 24, ordinary least squares [OLS] model: y = 197.3 + 591.6x). Scale bars represent: A, 50 μm.
To investigate factors influencing transformation efficiency among different lines, we measured cell size in the middle region of thalli from those with higher transformation efficiency. The results demonstrated a significant correlation between transformation efficiency and cell size (r = 0.8833, p < 0.0001) (Fig. 3D), while no correlation was found with chlorophyll fluorescence parameters, including Fv/Fm, Qy, NPQ, and qP (Supplementary Fig. S1). These findings suggest that cell size may play a critical role in determining transformation efficiency in thalli.
Application and validation of NLS and linker selected in Pyropia yezoensis
To identify suitable NLS that mediate the transport of proteins from the cytoplasm into the nucleus in P. yezoensis, four NLSs (SV40, 2×SV40, PySV40, and PyNP) were fused to PyZsYellow, and the resulting constructs were introduced into thalli cells. Subcellular localization was analyzed by visualizing PyZsYellow fluorescence. The green fluorescent signal was detected exclusively in the nuclei of cells transformed with plasmids containing PyZsYellow-2×SV40 and PyZsYellow-PyNP, indicating that 2×SV40 and PyNP can effectively mediate nuclear import of PyZsYellow in P. yezoensis (Fig. 4B & D). In contrast, both the nucleus and cytoplasm exhibited fluorescence in cells transformed with plasmid PyZsYellow-SV40 (Fig. 4A). No fluorescent signal was observed in cells transformed plasmid PySV40-PyZsYellow (Fig. 4C). To investigate whether the T2A peptide functions in P. yezoensis, it was inserted between Py03626 and PyZsYellow under the control of the promoter pPyACT4 to construct vector pBI121-pPyACT4-Py03626-T2A-PyZsYellow-NOSt. The Py03626 gene encodes a histone acetylated reader protein that has been confirmed to localize within the nucleus (Pang et al. 2025). Cells transformed with pBI121-Py03626-T2A-PyZsYellow exhibited green fluorescence throughout both the cytoplasm and nucleus (Fig. 4F); however, cells transformed with plasmid pBI121-Py03626-PyZsYellow showed green fluorescence only in the nucleus (Fig. 4E). These results indicate that T2A linker successfully cleaves the fused protein of Py03626-T2A-PyZsYellow into two separate proteins, allowing for distribution of PyZsYellow throughout the entire cell.
Observation of PyZsYellow gene expression fused with various nuclear localization signals (NLSs) and T2A in thalli cells of Pyropia yezoensis. (A–E) The plasmids contain different NLSs. (F) The plasmid contains T2A linker. Scale bars represent: A–F, 20 μm.
DISCUSSION
The GUS gene is commonly used as a reporter gene for molecular transformation in macroalgae, particularly in P. yezoensis (Gan et al. 2003, Mizukami et al. 2004, Fukuda et al. 2008, Hirata et al. 2011, Takahashi et al. 2011, Huddy et al. 2012, Ramessur et al. 2018, Cao et al. 2022, Wang et al. 2024). However, the identification of FP and its fluorescent derivatives has emerged as a powerful tool for studying various subcellular processes or serving as reporters. Several FPs were reported to be available as reporter within thalli cell, including AmCFP, ZsGFP, and sGFP (Mikami et al. 2009, Uji et al. 2010, Hirata et al. 2011). However, the codons of these FP genes were not optimized, leading to low expression efficiency. In this study, we selected eight FPs, including the above three FPs, that were individually optimized for codon usage significantly increasing the GC content in certain genes. PyZsYellow in this research is generated from ZsYFP. As a result, PyZsGFP, PysGFP, and PyZsYellow exhibited significantly higher expression efficiency and fluorescence intensity compared to previous research findings (Mikami et al. 2009, Uji et al. 2010). Notably, Uji et al. (2010) reported that ZsGFP was effective in P. yezoensis thallus cells; our results demonstrate PyZsGFP can function not only in thallus cells but also in filamentous cells. In addition, we selected other FPs to identify more reporters suitable in P. yezoensis, such as PyEBFP, PyAmCyan, PysmURFP, PyDsRed2, PytdTomato, which excitations/emission spectrum were different from those of PyZsGFP, PysGFP, and PyZsYellow, yet they didn’t show significant differences compared to the autofluorescence. These findings indicate that it is necessary to optimize codons for exogenous genes to increase their expression efficiency in P. yezoensis. Although we have not yet obtained cells with stable genetic transformation of FPs gene, our previous research has successfully achieved stable GUS gene transformation through particle bombardment. Therefore, obtaining cells with stable genetic transformation of FPs gene is feasible, and this will be the focus of our subsequent efforts.
Many studies have demonstrated that different explants within the same species, and different developmental stages of explants significantly influence transformation efficiency (Verma et al. 2014, Hashmi et al. 2022, Xia et al. 2023). Currently, thalli are commonly used as receptor materials in the genetic transformation of P. yezoensis, because positively transformed cells can easily develop into stable thalli (Cao et al. 2022, Wang et al. 2024). Although protoplasts derived from thalli have also been utilized in genetic transformation efforts, no stable transformants were obtained thus far (Hado et al. 2003, Mizukami et al. 2004). Furthermore, protoplast preparation is complex, time-consuming, and is associated with a low rate of development into thalli, which limits their application in genetic transformation for P. yezoensis. However, it is noteworthy that the diploid filament—an essential component of life cycle of P. yezoensis—could serve as alternative suitable receptor materials for genetic transformation. This approach may address the lethal effects caused by gene knockouts when using haploid thalli in genome editing of P. yezoensis. Nevertheless, an effective genetic transformation system for filaments has yet to be established. In this study, we successfully achieved PyZsGFP expression in filament cells by optimizing particle bombardment transformation parameters, resulting in a transformation efficiency reaching up to 7.0 × 10−5. Although this efficiency is lower than that observed in thallus-based genetic transformation, which exceeds 5% (Cao et al. 2022), this research nonetheless provides valuable insights and support for future improvement and advancements in filament-based genetic transformation.
It has been characterized that genotypes within the same species exert significant influences on genetic transformation efficiency, as reported in various species such as wheat, maize, tomato, potato, and soybean (Olhoft et al. 2003, Chaudhry and Rashid 2012, Hensel et al. 2017, Bakhsh 2020). In this study, 39 lines with different phenotypes from diverse geographic regions showed different transformation efficiencies. The RZ line developed by our laboratory exhibited the highest transient transformation efficiency of 5.2 × 10−5; further research should be conducted to elucidate the underlying mechanisms at both genetic and epigenetic levels. Additionally, the developmental status of explants also affects the optimal transformation efficiency. Unlike higher plants with differentiated roots, stems, and leaves, the thallus of P. yezoensis lacks organ differentiation in its thallus. However, cells located at different vertical regions of the thallus, namely the top, middle, and basal regions, exhibit different division rates and differentiation fates. We compared the transformation efficiencies of these three regions and found the highest efficiency as achieved in the middle region of thallus. Xia et al. reported that the explant over-expressing DNA replication-related gene, Baby boom (ZmBbm) and maize Wuschel2 (ZmWus2) exhibited greater transformation efficiency (Lowe et al. 2016, Xia et al. 2023). We discovered a significant correlation between cell size and transformation efficiency; this indicates that cell size may be a crucial factor influencing P. yezoensis’s transformation efficiency. Future studies are needed to further validate this correlation and clarify how cell size impacts transformation efficiency. Additionally, when compared to other algae, the transformation efficiency in P. yezoensis, whether in thalli or filaments, is relatively low, ranging from 5.2 to 7.0 × 10−5. For instance, recent studies on Chlamydomonas reinhardtii using electroporation technique have reported that transformation efficiencies of 5.04 ± 0.02% for cell-walled strains and 2.89 ± 0.29% for cell wall-deficient strains (Wan et al. 2025). In U. prolifera, by optimizaing PEG-mediated transformation method, a transformation efficiency up to 0.141% has been achieved (Qin et al. 2025). This may be associated with the characteristic of species. Therefore, future studies should continue to focus on enhancing transformation efficiency in P. yezoensis, surrounding transformation methods, material, and genotype.
In eukaryotes, the selective import of proteins into the nucleus is subject to stringent regulatory controls (Chang et al. 2013). While the general characteristics of the transport machinery are conserved across plants and other organisms, notable differences in preferences for NLSs have been observed (Hicks and Raikhel 1995). Both SV40 and 2×SV40 successfully localize YFP within the nuclei of Ulva mutabilis and Chlamydomonas (Blomme et al. 2021). In this study, we compared the nuclear localization levels of four different NLSs. When NLS was fused with C-terminal of PyZsYellow, 2×SV40 effectively facilitated the nuclear import of PyZsYellow, comparable to SV40 locating PyZsYellow throughout the cell. A similar nuclear localization pattern was also observed for PyNP. It has been confirmed that both the type and number of NLSs in diverse organisms influence their nuclear localization efficiency as well as the activity of the fused protein (Chang et al. 2013, Tsukamoto et al. 2025). Interestingly, when PySV40 with optimized genetic codons was fused with the N-terminal of PyZsYellow, no expression of the fused protein was detected. The reason for this inhibition remains unclear; however, our findings suggest that both 2×SV40 and PyNP hold potential for application in nuclear localization research when fused with target proteins in P. yezoensis, such as those involved in genome editing that necessitate nuclear localization of the Cas9 protein (Bernard et al. 2019).
2A self-cleaving peptides (2A peptides) can induce ribosomal skipping, thereby enabling the co-translational expression of multiple proteins from a single open reading frame through a cleavage event. This mechanism effectively addresses the challenge of uneven expression levels among different proteins. Although various 2A peptides (E2A, P2A, and T2A) have been widely used for co-expression purposes, their cleavage efficiencies vary significantly. For instance, in Ulva, E2A, F2A, and P2A can result in efficient separation of YFP from selective marker BleR, facilitating the simultaneous expression of multiple genes (Blomme et al. 2021). In Nannochloropsis salina, both T2A and E2A demonstrate optimal performance with a maximum cleavage rate approaching 45%, while also enhancing transformation and screening efficiency (Koh et al. 2018). Recently, P2A peptide was also validated for mediating protein cleavage of Cas9 nuclease and hygromycin resistance gene in P. yezoensis (Wang et al. 2024). In this study, the insertion of T2A between the Py03626 and PyZsYellow genes successfully relocated PyZsYellow from the nucleus to the cytoplasm. These findings indicate that the T2A peptide as well as P2A can facilitate the separation of upstream and downstream proteins via self-cleavage in P. yezoensis. Future research should be performed to compare the cleavage ability to identify the optimal linker sequence available in P. yezoensis. In the meantime. These molecular elements will also provide candidate choice in stable transformation and gene editing in the future.
In summary, we conducted further screening and validation of molecular tools for studying gene function and performing genetic transformation. Three FPs, namely PyZsYellow, PyZsGFP, and PysGFP, were further validated as effective reporters expressed in the cells of P. yezoensis. Both PyNP and 2×SV40 demonstrate potential for facilitating direct protein localization to the nucleus, while the T2A linker functions serve as a potential cleavable linker. Moreover, the RZ strain is established as an ideal model line for studying P. yezoensis biology, with the middle region of the thalli exhibiting the highest transformation efficiency. In our previous work, we established a stable genetic transformation system in thalli that provides efficient endogenous promoter and antibiotic selection markers while optimizing particle bombardment transformation parameters (Cao et al. 2022). Collectively, these findings contribute to the development of a comprehensive toolkit for genetic transformations and functional studies of genes in P. yezoensis.
Notes
ACKNOWLEDGEMENTS
This research was funded by the National Key Research and Development Program of China (2023YFD2400101), the National Natural Science Foundation of China (Grant No. 42276113, Grant No. 41976146), the Shandong Province Key Research and Development Program (Grant No. 2025LZGC037, Grant No. 2025LZGC010), and the Fundamental Research Funds for the Central Universities (Grant No. 202262001).
CONFLICTS OF INTEREST
The authors declare that they have no potential conflicts of interest.
SUPPLEMENTARY MATERIALS
Supplementary Table S1. The origin information of the 39 tested lines (https://www.e-algae.org).
algae-2026-41-3-11-Supplementary-Table-S1.pdfSupplementary Table S2. Homologous recombination primers of fluorescent protein plasmids construction (https://www.e-algae.org).
algae-2026-41-3-11-Supplementary-Table-S2.pdfSupplementary Table S3. The NLS and T2A nucleic acid sequences (https://www.e-algae.org).
algae-2026-41-3-11-Supplementary-Table-S3.pdfSupplementary Table S4. Homologous recombination primers sequence for plasmids construct with different NLS (https://www.e-algae.org).
algae-2026-41-3-11-Supplementary-Table-S4.pdfSupplementary Table S5. Homologous recombination primers sequence for plasmid construct with T2A (https://www.e-algae.org).
algae-2026-41-3-11-Supplementary-Table-S5.pdfSupplementary Fig. S1. The correlation analysis between chlorophyll fluorescence parameters and transformation efficiency of Pyropia yezoensis (https://www.e-algae.org).
algae-2026-41-3-11-Supplementary-Fig-S1.pdf