Suppression Subtractive Hybridization Reveals Transcript Profiling of Chlorella under Heterotrophy to Photoautotrophy Transition

<div><h3>Background</h3><p>Microalgae have been extensively investigated and exploited because of their competitive nutritive bioproducts and biofuel production ability. <em>Chlorella</em> are green algae that can grow well heterotrophically and photoautotrophically. Previous studies proved that shifting from heterotrophy to photoautotrophy in light-induced environments causes photooxidative damage as well as distinct physiologic features that lead to dynamic changes in <em>Chlorella</em> intracellular components, which have great potential in algal health food and biofuel production. However, the molecular mechanisms underlying the trophic transition remain unclear.</p> <h3>Methodology/Principal Findings</h3><p>In this study, suppression subtractive hybridization strategy was employed to screen and characterize genes that are differentially expressed in response to the light-induced shift from heterotrophy to photoautotrophy. Expressed sequence tags (ESTs) were obtained from 770 and 803 randomly selected clones among the forward and reverse libraries, respectively. Sequence analysis identified 544 unique genes in the two libraries. The functional annotation of the assembled unigenes demonstrated that 164 (63.1%) from the forward library and 62 (21.8%) from the reverse showed significant similarities with the sequences in the NCBI non-redundant database. The time-course expression patterns of 38 selected differentially expressed genes further confirmed their responsiveness to a diverse trophic status. The majority of the genes enriched in the subtracted libraries were associated with energy metabolism, amino acid metabolism, protein synthesis, carbohydrate metabolism, and stress defense.</p> <h3>Conclusions/Significance</h3><p>The data presented here offer the first insights into the molecular foundation underlying the diverse microalgal trophic niche. In addition, the results can be used as a reference for unraveling candidate genes associated with the transition of <em>Chlorella</em> from heterotrophy to photoautotrophy, which holds great potential for further improving its lipid and nutrient production.</p> </div

<i>Chlorella</i> growth and the cellular component temporal pattern characteristics under diverse nutrition transition.

<p>A: Biomass concentration; B: Protein content; C: Chlorophyll content. Error bars represent the mean ± standard deviation of three independent biological replicates.</p

Functional classification of the differentially expressed genes in reverse library.

<p>Numbers in parentheses represent the number of ESTs categorized into each group.</p

Gene Ontology (GO) annotation of genes obtained from the SSH libraries.

<p>GO predictions identified several categories based on the three terms cellular component, molecular function, and biological process, and were plotted by WEGO. LI represents forward library under the light-induced treatment; HC represents the reverse library under the heterotrophic culture process.</p

Assembly and annotation statistics of the two SSH libraries.

<p>Assembly and annotation statistics of the two SSH libraries.</p

Time course changes on <i>Chlorella</i> physiological features under heterotrophy to photoautotrophy transition.

<p>A: Changes of photosynthesis fluorescence parameters on different light intensity; B: Production of ROS and lipid peroxidation; C: Activities of ROS scavenging enzymes. H: heterotrophy; P: photoautotrophy. Error bars represent the mean ± standard deviation of three independent biological replicates.</p

Functional classification of the differentially expressed genes in forward library.

<p>Numbers in parentheses represent the number of ESTs categorized into each group.</p

Time-course expression patterns of the selected differentially expressed genes from the forward SSH library.

<p>All expression values were normalized to the value of actin gene. Relative amount was calibrated based on the transcript amount of the corresponding gene in control (aliquots of the cDNA samples were mixed from different time points). Left of the dotted line represent the mixed photoautotrophic cells as control and heterotrophic cells as treatment. Right of the dotted line is just the opposite. Values are mean ± standard deviation obtained from three independent biological experiments. Hetero- and H: heterotrophy; Photo- and P: photoautotrophy.</p

Table_1_Enhanced phycocyanin production of Arthrospira maxima by addition of mineral elements and polypeptides using response surface methodology.docx

IntroductionAs a pigment protein, phycocyanin has been widely used in the fields of food, nutraceutical and biotechnology due to its excellent biological activities of antioxidant. So far, Arthrospira has been generally considered as a great species for phycocyanin production.MethodsIn this study, independent and interactive effect of three factors, NaCl, Bainengsi (BS) and Bainengtai (BT) on microalgae growth and phycocyanin production were explored by response surface methodology (RSM). Using Box-Behnken design (BBD) method, the well-fitting quadratic models were established based on experimental results.ResultsMoreover, the maximum phycocyanin concentration reached 704.66 mg L-1 at the optimal condition (185 mM NaCl, 200 mg L-1 BS, and 20 mg L-1 BT), while the maximum phycocyanin content of 19.03% was obtained at another optimal condition (136 mM NaCl, 200 mg L-1 BS, and 50 mg L-1 BT). Compared to control, the concentration and content of phycocyanin were increased by 22.98% and 16.73%, respectively.DiscussionOverall, this study demonstrated that addition of exogenous substances (BS, BT) into culture medium optimized by RSM was an effective approach to increase phycocyanin production, which paved a potential way to realizing high efficient production of algal biomass and bioactive substances.</p