Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching

Abstract

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms.

Zeaxanthin Binds to Light-Harvesting Complex Stress-Related Protein to Enhance Nonphotochemical Quenching in Physcomitrella patens

“…High zeaxanthin dependence of NPQ amplitude was reported in diatoms (Coesel et al, 2008;Nymark et al, 2009;Bailleul et al, 2010). While NPQ is decreased in a LHCSR knockdown genotype (lhcx1), clear evidence that LHCSR is the only agent for NPQ is lacking in these algae (Lavaud et al, 2012).…”

Growth form defines physiological photoprotective capacity in intertidal benthic diatoms

“…In EPM-M, the discrepancy between E50 NPQ and E k is more pronounced than in non-motile forms: while there is no significant difference in E k between both growth forms, E50 NPQ is significantly higher in the motile growth forms. This suggests a weaker relationship between NPQ development and photochemistry in the latter group, with slower NPQ development with increasing E. Remarkably, E50 DT does not significantly differ between both growth forms, and the significantly higher initial induction of DT synthesis (n DT ) but not NPQ (n NPQ ) in the motile group, together with the fact that some representatives of this group show DT synthesis without NPQ, suggests that either DT is less or not involved in NPQ development, or that the light-dependent built-up of the transthylakoidal proton gradient (which is involved in both the activation of the DD de-epoxidase and the molecular control of NPQ) and the onset of NPQ are uncoupled (Lavaud et al, 2012;Lavaud and Lepetit, 2013). Our observations thus suggest that in contrast to the non-motile group, Photoprotection in intertidal benthic diatoms A Barnett et al EPM-M species rely more on a behavioural response (motility) and/or involve DT in other photoprotective processes such as the prevention of lipid peroxidation by reactive oxygen species (Lepetit et al, 2010).…”

High Light Acclimation in the Secondary Plastids Containing Diatom Phaeodactylum tricornutum is Triggered by the Redox State of the Plastoquinone Pool

“…While the role of VDE is clear, the function of VDL/VDR remains speculative. Studies on P. tricornutum mutants containing a reduced amount of VDE indicate that these alternative deepoxidases are involved in Dd-to-Dt conversion (Lavaud et al, 2012). It is possible that during DTT inhibition, VDR and VDL perform the Dd deepoxidation reaction, as they might be less sensitive to DTT than VDE.…”

“…The de novo synthesis of Dd+Dt does not necessarily lead to higher NPQ Schumann et al, 2007), but it can enhance the antioxidant activity within the thylakoid membranes (Lepetit et al, 2010). Nevertheless, reduced expression of the gene encoding the Dd deepoxidase clearly leads to both lower Dt synthesis and qE (Lavaud et al, 2012). Overexpression or underexpression of LHCX1 promotes higher or lower qE, respectively, and the up-regulation of LHCX2 and LHCX3 might confer more efficient photoprotection (Bailleul et al, 2010).…”