Tandem Repeats, High Copy Number and Remarkable Diel Expression Rhythm of Form II RuBisCO in Prorocentrum donghaiense (Dinophyceae)

Xinguo Shi,Huan Zhang,Senjie Lin,Shin Yamazaki

doi:10.1371/journal.pone.0071232

Xinguo Shi, Huan Zhang + Show 2 more

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https://doi.org/10.1371/journal.pone.0071232

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Abstract

Gene structure and expression regulation of form II RuBisCO (rbcII) in dinoflagellates are still poorly understood. Here we isolated this gene (Pdrbc) and investigated its diel expression pattern in a harmful algal bloom forming dinoflagellate Prorocentrum donghaiense. We obtained cDNA sequences with triple tandem repeats of the coding unit (CU); the 5′ region has the sequence of a typical dinoflagellate plastid gene, encoding an N-terminus with two transmembrane regions separated by a plastid transit peptide. The CUs (1,455 bp except 1464 bp in last CU) are connected through a 63 bp spacer. Phylogenetic analysis showed that rbcII CUs within species formed monophyletic clusters, indicative of intraspecific gene duplication or purifying evolution. Using quantitative PCR (qPCR) we estimated 117±40 CUs of Pdrbc in the P. donghaiense genome. Although it is commonly believed that most dinoflagellate genes lack transcriptional regulation, our RT-qPCR analysis on synchronized cultures revealed remarkable diel rhythm of Pdrbc expression, showing significant correlations of transcript abundance with the timing of the dark-to-light transition and cell cycle G2M-phase. When the cultures were shifted to continuous light, Pdrbc expression remained significantly correlated with the G2M-phase. Under continuous darkness the cell cycle was arrested at the G1 phase, and the rhythm of Pdrbc transcription disappeared. Our results suggest that dinoflagellate rbcII 1) undergoes duplication or sequence purification within species, 2) is organized in tandem arrays in most species probably to facilitate efficient translation and import of the encoded enzyme, and 3) is regulated transcriptionally in a cell cycle-dependent fashion at least in some dinoflagellates.

Highlights

Ribulose bisphosphate carboxylase/oxygenase (RuBisCO) is the first major enzyme responsible for photosynthetic fixation of inorganic carbon and is critical in sustaining the ecosystem and in carbon biogeochemical cycling
BLASTX of the sequence against GenBank database showed that its 39 region (606 bp) was 91% identical to that of P. minimum RBCII gene (rbcII) (PmrbcII; AAO13049) at the amino acid level, but the upstream 463 bp did not hit any reported sequence
The sequence obtained from this product was 96% identical to PmrbcII

Summary

Introduction

Ribulose bisphosphate carboxylase/oxygenase (RuBisCO) is the first major enzyme responsible for photosynthetic fixation of inorganic carbon and is critical in sustaining the ecosystem and in carbon biogeochemical cycling. There are three classes of RuBisCO, forms I, II and III, all catalyzing the primary CO2 fixation reaction [1,2]. Most common in eukaryotic algae is the form I RuBisCO, which contains eight large subunits (RbcL) and eight small subunits (RbcS), both typically encoded in the plastid genome except in land plant and chlorophyte algae in which RbcS is encoded in the nucleus. Form III RuBisCO has only been found in Archaea so far, and seems to be a dimer of the large subunit [7]. RuBisCO-like proteins, categorized as form IV RuBisCO, have been found in some bacteria, Archaea and algae [1,2,8], but unlike typical RuBisCO they do not catalyze carbon fixation. Recent phylogenetic analyses show that all the four forms of RuBisCO may have evolved from a common ancestor [1]

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