Study On The Growth,lipid Accumulation And Transcriptome Dynamics Of Nannochloris Sp.JB17 Under Carbonate Stress

The extremely saline-alkaline soil in the northeast of China is characterized by extremely high pH in addition to high salinity.Soil salinization and alkalization are major threats to crop development.However,many studies have focused on the molecular mechanisms of plant response to salt stress,and much less attention has been paid to carbonate stress.Carbonate stress is becoming an increasingly important stress factor,which is much more harmful to plants than any single neutral salt stress.Therefore,studying on the mechanisms of plants responded and adapted to carbonate stress provides a theoretical basis for effectively improving the saline-alkaline tolerance of crops.As lower plants,microalgae are important primary producers,have the advantages of wide distribution,easy availability,short growth cycle and strong adaptability.Nannochloris sp.JB17(JB17)was isolated from the saline-alkaline soil in the northeast of China and was found to be resistant to high concentrations of NaHCO3.Therefore,JB17 is an excellent material for studying the resistance mechanism of plants under carbonate stress.Here,we mainly studied on the following aspects:1.We found that JB17 was a single-celled eukaryotic organism,which was closely related to the Chlorella-clade,and cell proliferation was through multiple divisions.By observing the color of the culture and cell morphology,we found that the color of the JB17 cell cultures became green with time after NaHCO3 treatments,and the cell growth and division were vigorous,while the color of the Chlorella vulgaris cultures began to turn white,and more chlorotic and disintegrated cells were found.The cell density and biomass were further measured,and we found that the final cell density and maximum dry weight of JB17 cultured with 50,100 and 300 mM NaHCO3 increased,and were higher than that of C.vulgaris.The results show that exogenous NaHCO3 promotes the growth of JB17.2.The trypan blue staining found that the cell viability of JB17 was significantly improved after 50,100 and 300 mM NaHCO3 treatments,while the cell viability under the same treatment concentrations were significantly higher than that of C.vulgaris.The results indicate that JB17 has a liking of bicarbonate.Although the malondialdehyde content of JB 17 cells were increased after 50 mM NaHCO3 treatments,the cell damage of JB17 after NaHCO3 treatments was less severe than C.vulgaris.In addition,the content of proline and soluble sugar in JB17 increased significantly after NaHCO3 treatments,and the activities of superoxide dismutase,catalase and peroxidase also increased significantly,indicating that JB17 responded to carbonate stress through osmotic regulation and antioxidant enzyme systems.3.We studied the effect of exogenous NaHCO3 on photosynthesis and found that the chlorophyll a content of JB17 increased with time and concentration after NaHCO3 treatments,and the oxygen evolution rate increased significantly after NaHCO3 treatments for 24 hours.According to the curve fitting of the rapid light curve,we found that the addition of NaHCO3 could increase the relative electron transport rate and the maximum relative electron transport rate of JB17.These results indicate that exogenous NaHCO3 promotes the photosynthesis of JB17,and JB17 may use high concentrations of NaHCO3 as a carbon source.4.The total starch and lipids contents of JB17 significantly increased after the addition of 100 and 300 mM NaHCO3.NaHCO3 in the culture medium greatly stimulated the biomass concentration and productivity,percentage of lipids out of total dry weight,and lipid productivity of JB17.The total saturated fatty acids production in JB17 was significantly lower and monounsaturated fatty acids and polyunsaturated fatty acids contents were significantly higher in the 100 and 300 mM NaHCO3;and the proportion of C18:2 and C18:3n3 increased significantly.These results indicate that high concentrations of NaHCO3 enhance the photosynthesis in JB17 by using it as a carbon source,leading to the accumulation of starch and lipids.JB17 has considerable potential as a raw material for the production of edible oil or health care products.5.We constructed the transcriptome of JB17 through de novo-assembled using Trinity software,which produced 18,746 genes.Benchmarking Universal Single-Copy Orthologs was used to assess the completeness of the conserved content in the transcriptome assemblies,indicating that the JB17 transcriptome data were highly credible.For the 50 mM NaHCO3 treatments,pairwise comparisons of 4 h,24 h,and 72 h versus the control revealed that 3,612,3,682,and 3,404 differentially expressed genes(DEGs),respectively.In the 300 mM NaHCO3 treatments,there were 2,317,4,026,and 4,735 DEGs between the control and the samples collected at 4 h,24 h,and 72 h,respectively.Interestingly,the 134 highly expressed(RSEM>1000)DEGs were mainly enriched in photosynthesis related processes,indicating that JB17 might respond to carbonate stress by enhancing photosynthesis.6.There were 33 modules correlated with distinct treatments constructed by Weighted Gene Co-expressed Network Analysis.The brown module(1,281 genes)was highly associated with 72 h NaHCO3 treatments under both 50 mM and 300 mM concentration.The cyan module,representing 165 genes,was highly associated with the 300 mM NaHCO3 treatments ignoring treatment time.Notably,the enriched GO terms in the two most important modules included reactive oxygen species scavenging-related GO terms including oxidoreductase activity and peroxisome.These results indicate that JB17 responds to carbonate stress through reactive oxygen species scavenging.7.One hub gene was UGP3,which encodes UTP-glucose-1-phosphate uridylyltransferase 3,involved in carbohydrate metabolism and sulfolipid synthesis.It implies that an increasing carbohydrate and lipid metabolism may also be one of the strategies of JB17 to deal with carbonate stress.Another hub gene was MUS81,which encodes crossover junction endonuclease,involved in cell division and DNA damage repairing.It shows that JB17 may respond to the carbonate stress through rapid cell division.These results indicate that JB17 can cope with the carbonate stress by the photosynthesis improving,reactive oxygen species scavenging,and carbohydrate and lipid metabolism enhancing mechanisms.Our results provide helpful information for elucidating the regulatory mechanism of carbonate tolerance in Nannochloris sp.JB17.