Studies On The Molecular Mechanism Of The Vitamin B₁₂-auxotrophy In Dinoflagellates And The Intraspecific Genetic Diversity In The Harmful Dinoflagellate Margalefidinium Fulvescens

Dinoflagellates are the major causative organisms of marine Harmful Algal Blooms(HABs;more than 75%HAB events are caused by dinoflagellates).The nutritional studies of dinoflagellates have mainly focused on the inorganic macro-nutrients such as nitrogen and phosphorus,while little attention has been paid to micro-nutrients such as vitamins and trace elements.However,over 90%of dinoflagellates have been proven to be vitamin B₁₂ auxotrophs,it is therefore important to understand the genetic basis of vitamin B₁₂ auxotrophy in dinoflagellates in the framework of the ecology of HABs,especially that of dinoflagellates HABs.Recent studies have shown that vitamin B₁₂ auxotrophy of some algae mainly depend on the presence of two methionine synthase genes:B₁₂-dependent METH,of which vitamin B₁₂ acts as the coenzyme,and B₁₂-independent METE,which can synthesize methionine without the involvement of B₁₂.Therefore,we obtained the full-length sequences of METH and METE for 14 species of dinoflagellates by rapid amplification of cDNA ends(RACE).In order to determine the functions of METH and METE in dinoflagellates,Quantitative real time polymerase chain reaction(qRT-PCR)were used to observe the molecular responses of METH and METE to various levels and re-supplements of vitamin B₁₂ in10 species of dinoflagellates.Then we constructed cDNA libraries of dinoflagellates by collecting from all publicly available databases of transcriptomes and genomes.Based on the abovementioned full-length sequences of METH and METE obtained from 14species of dinoflagellates,we retrieved the METH and METE sequences of 61dinoflagellate species via translated Basic Local Alignment Search Tool(tblastn)and analyzed the phylogenetic relationships among all sequences.Together,our experimental results and phylogenetic analyses revealed the genetic basis of vitamin B₁₂ auxotrophy of dinoflagellates,or provide a mechanistic explanation from the molecular level for this particular trophic mode of dinoflagellates.The major findings of our study are itemized as follows:(1)Full-length cloning of METH and METE in 14 species of dinoflagellatesA total of 38 full-length cDNA sequences of METH and METE genes from 14species of dinoflagellates maintained in the laboratory were cloned.All 14 species have METHs with the same conserved domain as found in other model organisms.However,only 12 of the 14 dinoflagellate species have METEs(note METE is the only annotation for the gene in all current databases),all of which have C-terminal domain only but lack the N-terminal domain,suggesting a loss of their function as a methionine synthase gene.We did not find any evidence for the existence of METE gene in the other two species of dinoflagellates.(2)Re-confirming the vitamin B₁₂ auxotrophy of dinoflagellatesVia physiological experiments,we found that the growth of 8 dinoflagellate species depended on the availability and levels of vitamin B₁₂ and directly responded to limitation and supplement of vitamin B₁₂.Re-supplement of vitamin B₁₂ in the cultures of the 8 dinoflagellate species showing growth-limiting effects of lower levels of vitamin B₁₂ could recover the growth of all cultures,while the supplement of methionine could not do so.Based on these results,we reconfirmed these 8 species of dinoflagellates are auxotrophs of vitamin B₁₂.(3)Transcriptional responses of METH and METE to the changing level of vitamin B12Based on the results of qRT-PCR measurement for 10 species of dinoflagellates,the transcriptional responses of METH and METE to the changing level and re-supplement of vitamin B₁₂ can be briefly summarized as follows:the expression of METH exhibited a negative feedback to the level of vitamin B₁₂,i.e.,the expression of METH increased when the concentration of vitamin B₁₂ decreased to limit the growth of dinoflagellates,while the expression of METH decreased after the concentration of vitamin B₁₂ was increased via re-supplementing to the initial level of f/2 medium;The expression of METE,however,exhibited not to respond to the change in vitamin B₁₂concentration and particularly,did not appear a high expression to take over the function of METH in response to limited levels of vitamin B₁₂.The expression of METE was also not repressed when vitamin B₁₂was restored to a high level.Judged on the basis of the expression patterns of METH and METE in those algal(e.g.diatoms)species that have METH and METE functioning in fact as methionine synthases genes and the fact that the growth of 8 dinoflagellates were inhibited by low vitamin B₁₂levels,the METE gene in our dinoflagellates appeared not to function as a B₁₂-independent methionine synthase.(4)Probing the possible functions of METE in dinoflagellatesSince the METE gene appeared not to be a real methionine synthase in all dinoflagellates we investigated but exhibited considerable expressions at all levels of vitamin B₁₂,we,in hope of probing the possible function of METE,observed the expression patterns of both METH and METE in the B₁₂-auxotroph Karlodinium veneficum along the growth cycle and different time points of the light-dark cycle via qRT-PCR.The expression levels of METH and METE decreased gradually with the growth of K.veneficum during the entire exponential growth stage,but were higher during the dark cycle(12 h)than that during the light period(12 h).The expression patterns of METE along the growth cycle and light-dark cycle suggest that METE may be involved in some physiological processes rather than methionine synthesis in dinoflagellates.(5)Phylogenetic analyses of METH and METE genes of dinoflagellates and the species from other taxaWe constructed cDNA libraries of dinoflagellates by collecting from all publicly available databases of transcriptomes and genomes and then,based on the full-length sequences of METH and METE obtained from the 14 dinoflagellate species in this study,we retrieved the METH and METE sequences of 61 species of dinoflagellates All 61dinoflagellates contained METH genes that have the same conserved and functional domains,suggesting a genuine function of B₁₂-dependent methionine synthase.The phylogenetic analysis of METH sequences exhibited clustering pattern consistent to the phylogenetic relationships among all species in general.However,METE gene sequences could be obtained from only 43 of the 61 dinoflagellate species,and there was no evidence for the presence of METE gene in the other 18 species.More importantly,all the obtained METE sequences have the C-terminal domain only and lack the N-terminal domain that is required for a genuine function of methionine synthase as demonstrated in non-dinoflagellate taxa(e.g.diatoms).The phylogenetic analysis of all METE sequences together with organisms from other groups that have functional METE exhibited that all METE sequences formed two major clades in accordance with presence or absence of the N-terminal domain.Therefore,METE in dinoflagellates are not functioning as a methionine synthase due to the absence of N-terminal domain.In conclusion,we elucidated the genetic basis of vitamin B₁₂ auxotrophy of dinoflagellates via cloning the full-length sequences and phylogenetic analyses of the methionine synthases genes METH and METE and the transcriptional responses of the two genes to the changing level of vitamin B₁₂ in 10 dinoflagellate cultures.We hypothesize that all dinoflagellates are probably vitamin B₁₂ auxotrophs because of the absence of N-terminal domain in METE needed for the gene to encode a genuine B₁₂-independent methionine synthase,or possibly an absence of the incomplete METE gene in some dinoflagellates,and thus have to rely on the B₁₂-dependent synthase gene METH and consequently the availability of vitamin B₁₂.Revealing the molecular mechanisms of vitamin B₁₂ auxotrophy of dinoflagellates is believed to have greatly deepened our understanding of the role played by vitamin B₁₂ in regulating the growth and consequently the dynamics of HABs,especially that of dinoflagellates.We also anticipate the outputs of this research will provide insights for the forecasting,prevention,and control of HABs.In addition to the work described above,we also assessed the intrapopulational and intraindividual genetic diversity of the naked dinoflagellate Margalefidinium fulvescens in the thesis.Large subunit ribosomal DNA(LSU rDNA)sequences have been increasingly used to infer the phylogeny and species identity of organisms,a few previous studies,however,have observed high intraspecific and even intraindividual variability in LSU rDNA in some dinoflagellate species due to,assumably,large copy numbers of rDNA in dinoflagellates.Since the copy number of LSU rDNA varies tremendously among dinoflagellate species,the intraspecific and intraindividual diversity for a species of particular interest thus needs to be investigated individually.As a toxic and HABs-forming dinoflagellate,Margalefidinium fulvescens has been observed to approach blooming density in Jiaozhou Bay,China since 2015 after numerous blooms having been reported from other countries.In trying to identify the source of this newly observed HABs-forming species in China by sequencing the LSU rDNA for both field samples and clonal cultures,we noticed and thus further investigated high intrapopulational and intraindividual genetic diversities of the dinoflagellate.The D1–D6 region of the LSU rDNA(1,435 bases)was amplified from7 field samples(pooled cells)and 11 clonal cultures,cloned,sequenced,and analyzed phylogenetically for 2,341 sequences obtained.All the numbers of sequences obtained from each clonal culture were far less than the estimated rDNA copy number in M.fulvescens.In the clone library,only one unique sequence was contained in all samples as the most dominant sequence.We found high intrapopulational and intraindividual genetic diversity in M.fulvescens as reflected in the number of polymorphic sites and unique sequences in the clone library for different field samples and clonal cultures in comparison to other species.The mean number of nucleotide differences of each sequence from different field samples and clonal cultures were 6.43 and 4.42 bases,respectively,with the highest being 132 bases,nearly 10%.The sequences with highest variability may be easily annotated as different species if they were obtained from environmental genomic studies because sequence-based species identification in meta-barcoding studies often use"97%identity"threshold.Based on that the mean and overall intrapopulational genetic diversity calculated for 7 field samples was equivalent to the mean and overall intraindividual variability for 11 clonal cultures in indices of genetic diversity,together with the result of AMOVA analysis,we infer that the variability within individual cells(i.e.variability among LSU rDNA polymorphic copies)caused both the intraindividual and intrapopulational genetic diversities observed in the M.fulvescens population,and a higher interpopulational diversity may exist among different geographic populations.The results provide an insightful basis for such a comprehensive interpopulational comparison and important implications for identifying species and establishing new taxa based on the similarity comparison to reference sequences deposited in databases.