Identification of Indicator Genes for Agar Accumulation in Gracilariopsis lemaneiformis (Rhodophyta).

Agar, as a seaweed polysaccharide mainly extracted from Gracilariopsis lemaneiformis, has been commercially applied in multiple fields. To investigate factors indicating the agar accumulation in G. lemaneiformis, the agar content, soluble polysaccharides content, and expression level of 11 genes involved in the agar biosynthesis were analysed under 4 treatments, namely salinity, temperature, and nitrogen and phosphorus concentrations. The salinity exerted the greatest impact on the agar content. Both high (40‱) and low (10‱, 20‱) salinity promoted agar accumulation in G. lemaneiformis by 4.06%, 2.59%, and 3.00%, respectively. The content of agar as a colloidal polysaccharide was more stable than the soluble polysaccharide content under the treatments. No significant correlation was noted between the two polysaccharides, and between the change in the agar content and the relative growth rate of the algae. The expression of all 11 genes was affected by the 4 treatments. Furthermore, in the cultivar 981 with high agar content (21.30 ± 0.95%) compared to that (16.23 ± 1.59%) of the wild diploid, the transcriptional level of 9 genes related to agar biosynthesis was upregulated. Comprehensive analysis of the correlation between agar accumulation and transcriptional level of genes related to agar biosynthesis in different cultivation conditions and different species of G. lemaneiformis, the change in the relative expression level of glucose-6-phosphate isomerase II (gpiII), mannose-6-phosphate isomerase (mpi), mannose-1-phosphate guanylyltransferase (mpg), and galactosyltransferase II (gatII) genes was highly correlated with the relative agar accumulation. This study lays a basis for selecting high-yield agar strains, as well as for targeted breeding, by using gene editing tools in the future.

Expression Profiling Reveals the Possible Involvement of the Ubiquitin-Proteasome Pathway in Abiotic Stress Regulation in Gracilariopsis lemaneiformis.

Gracilariopsis lemaneiformis is an economically important red macroalga, the cultivation of which is affected by abiotic stresses. This research intends to study the response mechanism of various components of the ubiquitin-protease pathway to abiotic stress in G. lemaneiformis. The algae were treated with five common external stresses (high temperature, low temperature, O3, PEG, and water shortage) to study the macroscopic and microscopic manifestations of the ubiquitin-proteasome pathway. Firstly, the changes in soluble protein and ubiquitin were detected during the five treatments, and the results showed that the content of soluble protein and ubiquitin significantly increased under most stresses. The content of the soluble protein increased the most on the second day after 20% PEG treatment, which was 1.38 times higher than that of the control group, and the content of ubiquitin increased the most 30 min after water shortage treatment, which was 3.6 times higher than that of the control group. Then, 12 key genes (E1, E2, UPL1, HRD1, UFD1, Cul3, Cul4, DDB2, PIAS1, FZR1, APC8, and COP1) of the ubiquitin-proteasome pathway were studied, including an estimation of the probably regulatory elements in putative promoter regions and an analysis of transcript levels. The results showed that CAAT box, LTR, GC motif, and MBS elements were present in the putative promoter regions, which might have endowed the genes with the ability to respond to stress. The transcript analysis showed that under high temperature, low temperature, PEG, O3, and water shortage, all of the genes exhibited instant and significant up-regulation, and different genes had different response levels to different stresses. Many of them also showed the synergistic effect of transcript up-regulation under various stress treatments. In particular, E1, E2, Cul3, Cul4, UPL1, HRD1, and COP1 performed most significantly under the five stresses. Collectively, our exploration of the ubiquitin-proteasome pathway and the transcript levels of key genes suggest a significant role to cope with adversity, and potential candidate genes can be selected for transformation to obtain stress-resistant strains.