Chlorophyll a Synthesis by an Animal Using Transferred Algal Nuclear Genes
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See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/226053724 Chlorophyll a synthesis by an animal using transferred algal nuclear genes. Symbiosis Article in Symbiosis · December 2010 Impact Factor: 1.44 · DOI: 10.1007/s13199-009-0044-8 CITATIONS READS 26 167 3 authors, including: Nicholas E Curtis Julie Schwartz Ave Maria University 11 PUBLICATIONS 215 CITATIONS 24 PUBLICATIONS 369 CITATIONS SEE PROFILE SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Nicholas E Curtis letting you access and read them immediately. Retrieved on: 24 May 2016 SYMBIOSIS (2009) 49, 121–131 DOI 10.1007/s13199-009-0044-8 ©Springer Science+Business Media B.V. 2009 ISSN 0334-5114 Chlorophyll a synthesis by an animal using transferred algal nuclear genes Sidney K. Pierce*, Nicholas E. Curtis, and Julie A. Schwartz Department of Integrative Biology, SCA 110, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620, USA, Email. [email protected] (Received September 22, 2009; Accepted October 13, 2009) Abstract Chlorophyll synthesis is an ongoing requirement for photosynthesis and a ubiquitous, diagnostic characteristic of plants and algae amongst eukaryotes. However, we have discovered that chlorophyll a (Chla) is synthesized in the symbiotic chloroplasts of the sea slug, Elysia chlorotica, for at least 6 months after the slugs have been deprived of the algal source of the plastids, Vaucheria litorea. In addition, using transcriptome analysis and PCR with genomic DNA, we found 4 expressed genes for nuclear-encoded enzymes of the Chla synthesis pathway that have been horizontally transferred from the alga to the genomic DNA of the sea slug. These findings demonstrate the first discovery of Chla production in an animal using transferred nuclear genes from its algal food. Keywords: Horizontal gene transfer, chlorophyll synthesis, chloroplast symbiosis, kleptoplasty, Elysia chlorotica, Vaucheria litorea 1. Introduction chloroplast. In spite of this plant/animal dichotomy, we have discovered the long term ability to synthesize Chla in In plants and algae, the chlorophyll a (Chla) synthesis an animal. pathway, starting with 5-aminolevulinic acid (ALA), is A few years ago, we reported the first discovery of the contained within the chloroplast, and regulated by intricate transfer of functional, nuclear genes between multicellular interactions among the production of plastid- and nuclear- species (Pierce et al., 2007). The sacoglossan sea slug, encoded, cytoplasmically-processed enzymes used in the Elysia chlorotica, eats the chromophytic alga, Vaucheria reactions of the synthesis, the presence or absence of light, litorea. Certain cells that line the slug’s digestive as well as cofactor and substrate level kinetics. While diverticula are able to phagocytize undigested chloroplasts chlorophyll synthesis does not occur in animals, the which are maintained intracellularly, and continue to be synthesis of the identical porphyrin moieties of chlorophyll photosynthetically active for as long as the 10–11 month and cytochrome/hemoglobin proceeds from ALA to the life cycle of the slug (West et al., 1984). Although there is level of protoporphyrin IX along an identical synthesis no evidence of chloroplast division in the slug cytoplasm, pathway in both plants and animals, albeit in different synthesis of several chloroplast proteins occurs during the intracellular compartments using enzymes of identical endosymbiotic association, including proteins that are name, but distinctive sequence. At that point, the pathway encoded by algal nuclear genes (Pierce et al., 1996; Hanten directions diverge to either the chlorophylls or and Pierce, 2001). Our original discovery found expressed cytochrome/hemoglobin (see the review by Tanaka and genes for three of the algal light-harvesting complex Tanaka, 2007). Thus, while the porphyrin synthesis proteins, LHCV-1, LHCV-2 and FCP, in genomic DNA of enzymes in the initial portion of the pathway to Chla are the slug (Pierce et al., 2007). In addition, we located the present in the mitochondria or cytoplasm of animal cells, a same genes in the genomic DNA of unhatched veliger plant/algae-specific set of several, nuclear-encoded larvae, which lack symbiotic chloroplasts and do not feed enzymes is required to complete the synthesis inside the on V. litorea, confirming that the transferred algal genes were vertically transmitted in the slugs. Transferred gene sequences were also present in slug cDNA, indicating their *The author to whom correspondence should be sent. transcription (Pierce et al., 2007). 122 S.K. PIERCE ET AL. Furthermore, transfer of another algal nuclear gene on the sides of the aquarium. When found, they were gently between these species has recently been confirmed by removed and placed immediately into small culture dishes others (Rumpho et al., 2008). All of the above results were containing sterile artificial sea water containing rifampicin, produced using PCR-based experiments which were greatly where the embryos were maintained until they had impeded by the lack of slug and alga reference sequences in developed into veliger larvae (West et al., 1984), but had the public databases for both primer design and product not hatched. At the point DNA was extracted (see below), identification, as well as low sequence conservation of the veliger larvae, which do not contain symbiotic plastids, many of the genes of interest and contamination from the had not fed and were still inside their egg capsules. copious amounts of mucus produced by the highly mucogenic slugs. Genomic DNA purification In order to facilitate the hunt for additional transferred genes, we have recently turned our efforts to the production Genomic DNA was purified from pre-hatched and analysis of a transcriptome [expressed sequence tags E. chlorotica veliger larvae and V. litorea using the (EST)] library database from V. litorea (Schwartz et al., Nucleon® genomic DNA extraction kit, PhytoPure® (Tepnel submitted). Since EST’s are RNA-based, they are only a Life Sciences, Manchester, UK) following manufacturer’s representation of the genes being expressed at the moment instructions. the RNA is extracted. However, they provide the exact coding sequence of genes of interest, which greatly RNA isolation and mRNA purification facilitates not only identification, but also primer design and comparison with sequences in the slug. Using EST E. chlorotica: Total RNA was isolated from >2 month analysis, subsequently confirmed by PCR, we have already starved slugs as follows. Slugs were homogenized in located several additional transferred genes in the slug Trizol® Reagent (Invitrogen, Carlsbad, CA) and the genomic DNA (lhcv-3, lhcv-4, prk) (Schwartz et al., homogenate was centrifuged at 12,000 × g at 4oC to pellet submitted), as well as four genes in the Chla synthesis cellular debris. The supernatant was extracted with 1:6 (v/v) pathway. chloroform and centrifuged at 12,000 × g at 4oC. RNA was precipitated from the aqueous phase by adding 1:4 (v/v) isopropanol followed by 1:4 (v/v) 0.8M Na citrate/1.2 M 2. Materials and Methods NaCl solution and spun at 12,000 × g at 4oC. The RNA pellet was washed twice with 75% ethanol, air dried, Animals resuspended in diethylpyrocarbonate (DEPC)-treated water and quantified spectrophotometrically (260 nm). mRNA Specimens of Elysia chlorotica were collected in a salt was purified from total RNA using the Dynabeads® Oligo marsh on Martha’s Vineyard, MA. They were shipped to (dt)25 mRNA Purification Kit (Invitrogen) following Tampa, FL where they were kept in aquaria, without access manufacturer’s instructions and quantified spectrophoto- to algae, containing sterilized, aerated, artificial sea water metrically (260 nm). o (Instant Ocean, 1,000 mosm/kg H2O) at 10 C under a 14/10 V. litorea: Total RNA was isolated from the alga using hr light/dark cycle using fluorescent tubes (cool white). the Nucleon® genomic DNA extraction kit, PhytoPure® The slugs were starved for at least 2 months before use in following the manufacturer’s instructions, taking advantage the experiments. of the co-purification of RNA in that methodology. mRNA was purified as described above. Algae cDNA preparation The Vaucheria litorea used in the experiments came from a culture maintained in modified f/2 medium as E. chlorotica and V. litorea 1st and 2nd strand cDNAs described previously (Pierce et al., 1996). The original were synthesized from purified mRNA using the Mint inoculum for this culture came from the same salt marsh cDNA Synthesis Kit (Evrogen, Moscow, Russia) according that provided the slugs. At the time of the experiments the to manufacturer’s instructions. slugs and algae had not been in contact with each other for months and had undergone several biweekly changes of PCR and sequencing sterile media. Specific primers (Eurofins MWG/Operon, Huntsville, Veliger larvae AL) were designed from our EST sequences. Touchdown PCR reactions were done using 100 ng of genomic DNA or The aquaria containing the adult slugs were monitored cDNA, 12.5 pmol of each primer, 0.25 mM dNTP mix (ID daily for egg masses. Generally, the masses were deposited Labs, London, Ontario, Canada) and 1.25 units of CHLOROPHYLL SYNTHESIS BY AN ANIMAL 123 IDProof™ DNA polymerase (ID Labs). Initial denaturation still in the 14C-ALA containing media as above. Following was done at 95oC for 2 min, followed by 20 cycles of the incubation, the slugs or filaments were removed from denaturing at 95oC (30 s) and annealing (30 s) where the their media, washed in medium without isotope and Chla annealing temperature was reduced 1oC every other cycle, was extracted immediately. To test the effect of light on then a 72oC extension period. This was followed by an Chla synthesis, the experiment was done the same way additional 20 cycles of denaturing at 95oC for 30 s, then 30 except that the 18 hr incubation under lights was replaced s at the lowest annealing temperature obtained in the with 18 hrs in the dark.