Biochemical Mechanisms Of Dissolved Organic Phosphorus Utilization By Skeletonema Costatum

Phosphorus is an essential macronutrient for phytoplankton proliferation in the ocean.Observation data show that the growth of phytoplankton in some sea areas is subjected to the seasonal limitation of dissolved inorganic phosphorus?DIP?.Other studies have found that some species can compete to obtain dissolved organic phosphorus?DOP?under DIP-deficient conditions to outgrow other species in interspecific competition and even to form harmful algal blooms.DOP exists in numerous chemical forms in the ocean,and the physiological and biochemical pathways of DOP utilization and the types of DOP that are preferentially used by phytoplankton are still not fully explained and need to be further clarified from the molecular level.In this study,we selected Skeletonema costatum as the study species and used adenosine triphosphate?ATP?;sodium glycerophosphate?SG-P?;glucose 6-phosphate?G-6-P?,which contains theester bond;and glyphosate?G-P?,which contains thebond as the different DOP sources.The physiological responses of S.costatum to different DOP utilization were detected through a culture experiment in a laboratory.We further studied the utilization characteristics of the different DOP sources in S.costatum at four temperatures,compared the gene transcription and expression between ATP and Na H₂PO₄ conditions in the whole genome level,and discussed the molecular mechanism of ATP utilization in S.costatum.The main results are as follows:1)The physiological responses of S.costatum to ATP,SG-P,G-6-P and G-P were studied.S.costatum maintained its growth under ATP,SG-P and G-6-P conditions,but the growth of algal cells was considerably low under G-P conditions.The results showed that the utilization of the different types of DOP was different in S.costatum,and DOP with C–O–P bond was utilized more easily than the C–P types.A large amount of DIP was produced in the culture medium of the ATP treatment group;thus ATP may be utilized after enzymatic hydrolysis outside the cell.By contrast,no remarkable increase in DIP was detected in the SG-P and G-6-P treatment groups.This result suggested that SG-P and G-6-P may be directly utilized by S.costatum.However,no remarkble increase in alkaline phosphatase?AP?activity was detected in the ATP,SG-P and G-6-P treatment groups;hence the utilization of ATP,SG-P and G-6-P was independent of AP.2)The effects of temperature change on the utilization of ATP,SG-P,G-6-P and G-P in S.costatum were studied.The results showed that temperature had a significant effect on the growth of algal cells under different DOP conditions?P<0.01?,and had a significant interaction with DOP types?P<0.01?.The temperature range of this experiment was.Warming promoted the growth of algae cells under the ATP,SG-P and G-6-P conditions,but had no obvious effect on the growth of algal cells under the G-P condition.Temperature had a significant effect on the DIP concentration of the ATP treated group?P<0.01?.Warming could accelerate the production of DIP in the ATP-treated group,but not in the other DOP-treated groups.In addition,the AP activities of the ATP,SG-P,and G-6-P groups were low at different high(up to 83 fmol p NP cell^-1 h^-1)and decreased with the increase of temperature.3)The molecular mechanism of ATP utilization in S.costatum was studied by transcriptome technology.High-throughput RNA sequencing was used to compare the genome-wide transcriptional expression of S.costatum in the ATP and Na H₂PO₄conditions.A total of 603 genes?136 up-expressed genes and 467 down-regulated genes?were differentially expressed in the ATP treatment group compared with the Na H₂PO₄ treatment group.Six genes were randomly selected for real-time quantitative PCR?RT-q PCR?to further verify the accuracy of the results of the RNA transcriptome sequencing.In accordance with the previous results of the AP activity measurement,the expression of AP-coding genes in the ATP-treated group was unremarkably upregulated,but the expression of NAD+diphosphatase and nucleotide pyrophosphatase/phosphodiesterase genes were substantially increased.This finding suggested that these hydrolases are involved in ATP utilization.In addition,Gene Ontology?GO?and Kyoto Encyclopedia of Genes and Genomes?KEGG?enrichment analyses showed that S.costatum can adapt to the external ATP environment and maintain its normal growth by down-regulating purine metabolism,glyceride metabolism and photosynthesis.This study elucidated the physiological responses of different DOP types utilization and temperature changes and further revealed the molecular utilization mechanism of ATP in S.costatum.The results showed that ATP,SG-P and G-6-P were more easily utilized by S.costatum,and the growth of algal cells under these three DOP conditions can be greatly promoted by warming.Howere,the utilization process and the effects of warming were independent of AP activity.The transcriptome result showed that ATP may be utilized by other hydrolases?NAD+diphosphate and nucleotide pyrophosphatase/phosphodiesterase?.This utilization mechanism of DOP is uniquely reported in diatoms.This study will help uncover the utilization pathways of DOP in S.costatum and other diatoms.