Genome-wide characterization and identification of Trihelix transcription factors and expression profiling in response to abiotic stresses in Chinese Willow (Salix matsudana Koidz).

Trihelix transcription factors (TTF) are a class of light-responsive proteins with a typical triple-helix structure (helix-loop-helix-loop-helix). Members of this gene family play an important role in plant growth and development, especially in various abiotic stress responses. Salix matsudana Koidz is an allotetraploid ornamental forest tree that is widely planted for its excellent resistance to stress, but no studies on its Trihelix gene family have been reported. In this study, the Trihelix gene family was analyzed at the genome-wide level in S. matsudana. A total of 78 S. matsudana Trihelix transcription factors (SmTTFs) were identified, distributed on 29 chromosomes, and classified into four subfamilies (GT-1, GT-2, SH4, SIP1) based on their structural features. The gene structures and conserved functional domains of these Trihelix genes are similar in the same subfamily and differ between subfamilies. The presence of multiple stress-responsive cis-elements on the promoter of the S. matsudana Trihelix gene suggests that the S. matsudana Trihelix gene may respond to abiotic stresses. Expression pattern analysis revealed that Trihelix genes have different functions during flooding stress, salt stress, drought stress and low temperature stress in S. matsudana. Given that SmTTF30, as a differentially expressed gene, has a faster response to flooding stress, we selected SmTTF30 for functional studies. Overexpression of SmTTF30 in Arabidopsis thaliana (Arabidopsis) enhances its tolerance to flooding stress. Under flooding stress, the leaf cell activity and peroxidase activity (POD) of the overexpression strain were significantly higher than the leaf cell activity and POD of the wild type, and the malondialdehyde (MDA) content was significantly lower than the MDA content of the wild type. Thus, these results suggest that SmTTF30 enhances plant flooding tolerance and plays a positive regulatory role in plant flooding tolerance.

Stability and Chemical Conversion of the Purified Reference Material of Gymnodimine-A under Different Temperature and pH Conditions.

Gymnodimines (GYMs) are a group of fast-acting phycotoxins and their toxicological effects on human beings are still unclear due to the lack of sufficiently well-characterized large quantities of purified toxins for toxicology studies. In this study, a certified reference material (CRM) of GYM-A was prepared from the dinoflagellate Karenia selliformis, followed by multi-step chromatography separation and purification. Subsequently, the stability of GYM-A in methanolic media was evaluated at different temperature (-20, 4, and 20 °C) and pH (3, 5, and 7) conditions for 8 months, and the conversion products of GYM-A were explored by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The results show that the stability of GYM-A decreased with increasing temperature and pH values. The GYM-A was stable during storage at -20 °C regardless of pH, but it decreased rapidly (81.8% ± 9.3%) at 20 °C in pH 7 solution after 8 months. Moreover, the concentrations of GYM-A did not significantly change at all temperatures in solutions with pH 3 (p > 0.05). It is recommended that GYM-A should be stored at low temperature (≤-20 °C) and pH (≤3) conditions for long-term storage in aqueous methanolic media. In addition, two conversion products of GYM-A, tentatively named as GYM-K (m/z 540) and GYM-L (m/z 524), were identified in the samples stored at high levels of pH and temperature. Based on the LC-HRMS data, the hypothetical chemical structures of both converting derivatives were proposed. A useful strategy for long-term storage of GYM-A CRM in aqueous methanolic media was suggested and two hypothesized conversion products of GYM-A were discovered in this study.

Comparative study on the esterification of gymnodimine in different shellfish exposed to the dissolved toxin in seawater.

Some lipophilic phycotoxins dissolved in seawater can be accumulated by bivalves via the filtering process. To explore the relationship between the bioaccumulation of gymnodimine-A (GYM-A) and free fatty acids (FFAs) of shellfish, three species of bivalves (venus clam Meretrix meretrix, mussel Mytilus galloprovincialis, and ark shell Anadara kagoshimensis) were exposed to dissolved GYM-A for 7 days in the same seawater system. Results indicated that GYM-A can be accumulated by these bivalves from the dissolved phase and esterified with FFAs reaching over 90% in most tissues of bivalves. Gymnodimine-A and its esters mainly distributed in the gills of shellfish, and the highest concentration of toxins occurred in mussel, followed by ark shell and venus clam. Similar percent of different fatty acid esters occurred in the experimental shellfish, in which the C16:0, C17:0, C18:0, C18:1, C20:1, C20:2, C22:2, and C22:6-GYM-A esters were the main metabolites of GYM-A. The binding capacity of fatty acids and GYM-A varied in different FFAs, which can explain why the C20:1-GYM-A ester dominated the ester profile while C16:0 was the most abundant fatty acid in all samples. Comparing with the FFA profile of shellfish in the control groups, overexpression of some FFAs occurred in the tissues of shellfish exposed to GYM-A in the experimental groups, which suggested that biosynthesis of FFAs was affected by the accumulation and metabolism of GYM-A in bivalves. Multiple fatty acids including some valuably nutritional FFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were consumed in the esterification metabolism of GYM-A, which hinted that the lipid metabolism and nutritional quality of shellfish affected by the contamination of GYMs should be explored and assessed in future works.

Prevalence and distribution of domoic acid and cyclic imines in bivalve mollusks from Beibu Gulf, China.

Beibu Gulf is an important shellfish aquaculture area in the northwest of the South China Sea, China. In this study, the toxin profile and spatial-temporal distribution of domoic acid (DA) and 10 lipophilic phycotoxins were systematically analyzed in the bivalve mollusks collected in Beibu Gulf from October 2018 to October 2020. Neurotoxin DA was first detected in the mollusks from the investigative regions with a prevalence of 17.7%, peaking at 401 µg kg-1. Cyclic imines (CIs) including gymnodimine-A (GYM-A, 46.6%) and 13-desmethyl-spirolide-C (SPX1, 15.8%) predominated the lipophilic phycotoxins in shellfish, peaking at 10.1 µg kg-1 and 19.6 µg kg-1, respectively. Gymnodimine-A partially accompanied by SPX1 was detected in all batches of shellfish samples, suggesting that Alexandrium ostenfeldii and Karenia selliformis were possible sources of CIs-group toxins in Beibu Gulf. During the investigative period, relatively higher levels of DA occurred in shellfishes from March to August, while slightly higher contents of CIs in mollusks appeared in October and December. Spatial distribution of the targeted phycotoxins demonstrated that shellfishes tended to accumulate relatively higher contents of toxins in Lianzhou, Qinzhou and Tieshan bays.

Development of an Efficient Extraction Method for Harvesting Gymnodimine-A from Large-Scale Cultures of Karenia selliformis.

Gymnodimine-A (GYM-A) is a fast-acting microalgal toxin and its production of certified materials requires an efficient harvesting technology from the large-scale cultures of toxigenic microalgae. In this study the recoveries of GYM-A were compared between several liquid-liquid extraction (LLE) treatments including solvents, ratios and stirring times to optimize the LLE technique for harvesting GYM-A from Karenia selliformis cultures, of which the dichloromethane was selected as the extractant and added to microalgal cultures at the ratio 55 mL L-1 (5.5%, v/v). The recovery of GYM-A obtained by the LLE technique was also compared with filtration and centrifugation methods. The stability of GYM-A in culture media were also tested under different pH conditions. Results showed that both the conventional filter filtration and centrifugation methods led to fragmentation of microalgal cells and loss of GYM-A in the harvesting processes. A total of 5.1 µg of GYM-A were obtained from 2 L of K. selliformis cultures with a satisfactory recovery of 88%. Interestingly, GYM-A obviously degraded in the culture media with the initial pH 8.2 and the adjusted pH of 7.0 after 7 days, but there was no obvious degradation in the acidic medium at pH 5.0. Therefore, the LLE method developed here permits the collection of large-volume cultures of K. selliformis and the high-efficiency extraction of GYM-A. This work provides a simple and valuable technique for harvesting toxins from large-scale cultures of GYM-producing microalgae.

Effects of Polystyrene Microplastics on Growth and Toxin Production of Alexandrium pacificum.

Microplastics (MP) widely distributed in aquatic environments have adverse effects on aquatic organisms. Currently, the impact of MP on toxigenic red tide microalgae is poorly understood. In this study, the strain of Alexandrium pacificum ATHK, typically producing paralytic shellfish toxins (PST), was selected as the target. Effects of 1 and 0.1 µm polystyrene MP with three concentration gradients (5 mg L-1, 25 mg L-1 and 100 mg L-1) on the growth, chlorophyll a (Chl a), photosynthetic activity (Fv/Fm) and PST production of ATHK were explored. Results showed that the high concentration (100 mg L-1) of 1 µm and 0.1 µm MP significantly inhibited the growth of ATHK, and the inhibition depended on the size and concentration of MP. Contents of Chl a showed an increase with various degrees after MP exposure in all cases. The photosynthesis indicator Fv/Fm of ATHK was significantly inhibited in the first 11 days, then gradually returned to the level of control group at day 13, and finally was gradually inhibited in the 1 µm MP treatments, and promotion or inhibition to some degree also occurred at different periods after exposure to 0.1 µm MP. Overall, both particle sizes of MP at 5 and 25 mg L-1 had no significant effect on cell toxin quota, and the high concentration 100 mg L-1 significantly promoted the PST biosynthesis on the day 7, 11 and 15. No significant difference occurred in the cell toxin quota and the total toxin content in all treatments at the end of the experiment (day 21). All MP treatments did not change the toxin profiles of ATHK, nor did the relative molar percentage of main PST components. The growth of ATHK, Chl a content, Fv/Fm and toxin production were not affected by MP shading. This is the first report on the effects of MP on the PST-producing microalgae, which will improve the understanding of the adverse impact of MP on the growth and toxin production of A. pacificum.