Analysis of Hyperosmotic Tolerance Mechanisms in Gracilariopsis lemaneiformis Based on Weighted Co-Expression Network Analysis.

We conducted transcriptome sequencing on salt-tolerant mutants X5 and X3, and a control (Ctr) strain of Gracilariopsis lemaneiformis after treatment with artificial seawater at varying salinities (30‱, 45‱, and 60‱) for 3 weeks. Differentially expressed genes were identified and a weighted co-expression network analysis was conducted. The blue, red, and tan modules were most closely associated with salinity, while the black, cyan, light cyan, and yellow modules showed a close correlation with strain attributes. KEGG enrichment of genes from the aforementioned modules revealed that the key enrichment pathways for salinity attributes included the proteasome and carbon fixation in photosynthesis, whereas the key pathways for strain attributes consisted of lipid metabolism, oxidative phosphorylation, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) interactions in vesicular transport, and porphyrin and chlorophyll metabolism. Gene expression for the proteasome and carbon fixation in photosynthesis was higher in all strains at 60‱. In addition, gene expression in the proteasome pathway was higher in the X5-60 than Ctr-60 and X3-60. Based on the above data and relevant literature, we speculated that mutant X5 likely copes with high salt stress by upregulating genes related to lysosome and carbon fixation in photosynthesis. The proteasome may be reset to adjust the organism's proteome composition to adapt to high-salt environments, while carbon fixation may aid in maintaining material and energy metabolism for normal life activities by enhancing carbon dioxide uptake via photosynthesis. The differences between the X5-30 and Ctr-30 expression of genes involved in the synthesis of secondary metabolites, oxidative phosphorylation, and SNARE interactions in vesicular transport suggested that the X5-30 may differ from Ctr-30 in lipid metabolism, energy metabolism, and vesicular transport. Finally, among the key pathways with good correlation with salinity and strain traits, the key genes with significant correlation with salinity and strain traits were identified by correlation analysis.

Macropinocytosis in Gracilariopsis lemaneiformis (Rhodophyta).

Macropinocytosis is an endocytic process that plays an important role in animal development and disease occurrence but until now has been rarely reported in organisms with cell walls. We investigated the properties of endocytosis in a red alga, Gracilariopsis lemaneiformis. The cells non-selectively internalized extracellular fluid into large-scale endocytic vesicles (1.94 ± 0.51 µm), and this process could be inhibited by 5-(N-ethyl-N-isopropyl) amiloride, an macropinocytosis inhibitor. Moreover, endocytosis was driven by F-actin, which promotes formation of ruffles and cups from the cell surface and facilitates formation of endocytotic vesicles. After vesicle formation, endocytic vesicles could be acidified and acquire digestive function. These results indicated macropinocytosis in G. lemaneiformis. Abundant phosphatidylinositol kinase and small GTPase encoding genes were found in the genome of this alga, while PI3K, Ras, and Rab5, the important participators of traditional macropinocytosis, seem to be lacked. Such findings provide a new insight into endocytosis in organisms with cell walls and facilitate further research into the core regulatory mechanisms and evolution of macropinocytosis.

Breeding of New Strains of Gracilariopsis lemaneiformis with High Agar Content by ARTP Mutagenesis and High Osmotic Pressure Screening.

ARTP (atmospheric and room temperature plasma mutagenesis) mutagenesis was tried on G. lemaneiformis, and mutagenesis conditions were confirmed. An osmotic pressure screening program was established. Mutants were identified and characterized of relevant physiological traits. The aim of the study is to try to use ARTP mutagenesis and osmotic pressure screening for the breeding of high-agar G. lemaneiformis. Treatment time of 46 s was found to be an optimal mutagenesis time. The mutagenized spores were initially screened with 58‰ salinity artificial seawater, and then, the surviving spores were screened twice with 60‰ salinity artificial seawater in their vertical growth phase and branch growth phase, respectively. Four fast-growing and hypertonic resistance gametophytes were selected. The actual photosynthetic efficiency [Y(PSII)], photochemical quenching (qL), and non-photochemical quenching (NPQ) of four mutants were measured. The values of Y(PSII) and qL of HAGL-X3 and HAGL-X5 were higher than those of the control in the early stage of salt stress. NPQs of HAGL-X3 and HAGL-X5 were higher than control in most of the times. The growth rates of the four mutants were higher than that of the control. HAGL-X4 was the highest. The agar content was measured; HAGL-X5 displayed the highest agar content among the tested strains. HAGL-X5 was more in line with expectations, because of its high agar content and good hypertonic resistance. In this study, the mutant of G. lemaneiformis with high agar content was obtained by the procedure, which provided a certain reference for the selection of G. lemaneiformis strains with high agar content.

Ploidy Identification by Flow Cytometry and Application of the Method to Characterize Seasonal Ploidy Variation of Wild Populations of the Red Alga Gracilariopsis lemaneiformis.

Gracilariopsis lemaneiformis (Gp. lemaneiformis) is an economically important alga. At present, there is no way to quickly and easily determine its ploidy and life cycle dynamics in wild populations, which affects the process of genetic breeding. In this study, we developed and verified a ploidy identification method using flow cytometry and then used it to explore the seasonal fluctuation of the ploidy ratio and the environmental factors that influence it in wild populations of this species. Of the three methods we tested for nucleus extraction, quick chopping was the best because of its high extraction efficiency, low debris background, obvious subcellular scatter plot, and clear typical histogram. Samples from the tip of the alga were more suitable for preparing the nuclear suspension than samples from the base. Generalized linear model analysis based on diagnosis of multicollinearity revealed a negative correlation between temperature and ploidy ratio. Among the environmental factors tested, temperature had the greatest influence on the ploidy ratio, whereas precipitation and sunshine duration had no effect on the ploidy ratio fluctuation. Our study will be useful for material collection and studies of utilization and life cycle dynamics. Moreover, understanding of ploidy dynamics may provide a theoretical basis for improving variety and breeding of Gp. lemaneiformis in the future.

Estimating the ploidy of Gracilariopsis lemaneiformis at both the cellular and genomic level1.

The determination of the ploidy level of an organism is a prerequisite for studies of evolution, cellular function, and genomic construction. Identification of the ploidy of the economically important red alga Gracilariopsis lemaneiformis has been hindered by its small genome and large number of chromosomes. Therefore, in the current study, PloidyNGS, a tool that calculates the number of reads supporting different alleles at each position along the genome sequence, and fluorescence in situ hybridization coupled with tyramide signal amplification (TSA-FISH) were used to clarify the ploidy of G. lemaneiformis. In addition, flow cytometry (FCM) was used to estimate the ploidy of different somatic cells. The PloidyNGS results showed that most of the alleles in the gametophyte were monomorphic, whereas the TSA-FISH results showed that one hybridization signal was observed in gametophytic nuclei and two in tetrasporophytic nuclei when the nuclei were hybridized by single copy gene probes. These results confirmed that G. lemaneiformis is a haploid in the gametophytic generation and diploid in the sporophytic generation. Moreover, the FCM result suggested that G. lemaneiformis was not an endopolyploid. Based on previous studies, we hypothesize that the nuclear number is important for the cellular differentiation and function of this species. We also suggest that G. lemaneiformis evolved from a paleopolyploid, the genome of which has been diploidized, and that traces of genomic doubling are no longer apparent. Thus, this study provides important evidence for further studies on the evolution and genomes of red algae.

Heterozygous Single Nucleotide Polymorphic Loci in Haploid Gametophytes of Gracilariopsis lemaneiformis (Rhodophyta).

Gracilariopsis lemaneiformis is an important commercial macroalga. Whole-genome resequencing was conducted to identify single nucleotide polymorphisms (SNPs) in parental gametophytes and 60 F1 gametophytes of Gp. lemaneiformis in this study, and 9,989 SNPs located in nonrepetitive sequences were obtained. Among these SNPs, 92.02% of loci were identified as having a heterozygous genotype in at least one gametophyte, and 48.07% of loci had identical heterozygous genotypes in the 62 gametophytes. For each gametophyte, the proportions of homozygous and heterozygous loci ranged between 13.74 and 21.61% (mean of 17.04%) and between 66.36 and 83.59% (mean of 77.12%), respectively. The remainder were missing loci, representing an average 5.84%. Sources of heterozygous SNPs were free of exogenous DNA contamination, cross contamination among individuals, plastid and mitochondrial sequences, chimeras of different thallus parts or different cells, and repetitive sequences. Genotypes of heterozygous SNPs were verified by Sanger sequencing of PCR products and monoclones. Duplications of chromosomal rearrangements in the genome of Gp. lemaneiformis might explain the presence of heterozygous SNPs in haploid gametophytes.