Enhancement of Non-photochemical Quenching as an Adaptive Strategy under Phosphorus Deprivation in the Dinoflagellate Karlodinium veneficum

Abstract

Intensified water column stratification due to global warming has the potential to decrease nutrient availability while increasing excess light for the photosynthesis of phytoplankton in the euphotic zone, which together will increase the need for photoprotective strategies such as non-photochemical quenching (NPQ). We investigated whether NPQ is enhanced and how it is regulated molecularly under phosphorus (P) deprivation in the dinoflagellate Karlodinium veneficum. We grew K. veneficum under P-replete and P-depleted conditions, monitored their growth rates and chlorophyll fluorescence, and conducted gene expression and comparative proteomic analyses. The results were used to characterize NPQ modulation and associated gene expression dynamics under P deprivation. We found that NPQ in K. veneficum was elevated significantly under P deprivation. Accordingly, the abundances of three light-harvesting complex stress-related proteins increased under P-depleted condition. Besides, many proteins related to genetic information flow were down-regulated while many proteins related to energy production and conversion were up-regulated under P deprivation. Taken together, our results indicate that K. veneficum cells respond to P deprivation by reconfiguring the metabolic landscape and up-tuning NPQ to increase the capacity to dissipate excess light energy and maintain the fluency of energy flow, which provides a new perspective about what adaptive strategy dinoflagellates have evolved to cope with P deprivation.

Keywords: dinoflagellates; energy flow; metabolic machinery reconfiguration; non-photochemical quenching; phosphorus deprivation.

FIGURE 1

Dynamics of NPQ (non-photochemical quenching) under contrasting light and P (phosphorus) conditions in Karlodinium veneficum. The dark-adapted algal cells were exposed to actinic light for 10 min to induce the NPQ and then the actinic light was turned off for another 10 min. Fm’ was measured at the end of each minute. (A) Induction and relaxation of NPQ of K. veneficum cells exposed to different actinic light intensities. Open triangles, actinic light of 50 μmol photons m^-2 s^-1; open squares, actinic light of 200 μmol photons m^-2 s^-1; closed squares, actinic light of 300 μmol photons m^-2 s^-1; closed triangles, actinic light of 600 μmol photons m^-2 s^-1. (B) Induction and relaxation of NPQ of K. veneficum cells grown under +P and –P conditions and an actinic light intensity of 700 μmol photons m^-2 s^-1. Closed circles, P-replete condition; open circles, P-depleted condition. Data shown are means ± SD (error bars) from the triplicated measurements.

FIGURE 2

Growth curves (A), dissolved inorganic phosphate (DIP) concentration change (B), Fv/Fm (C), and NPQ capacity (D) of K. veneficum cells grown under +P and –P conditions. Closed circles, P-replete condition; open circles, P-depleted condition. Data shown are means ± SD (error bars) from the triplicated cultures. Asterisks represent that significant differences were detected (p < 0.05) between +P and –P conditions.

FIGURE 3

Transcript abundances of lhcx1 (A), lhcx2 (B), lhcx3 (C), lhcx4 (D), lhcx5 (E), and phot2 (F) genes normalized to calmodulin in K. veneficum cells grown under +P and –P conditions (the same batch cultures as Figure 2). Closed circles, P-replete condition; open circles, P-depleted condition. Shown are means ± SD (error bars) from the triplicated cultures. Asterisks represent that significant differences were detected (p < 0.05) between +P and –P conditions.

FIGURE 4

Growth curves (A), transcript abundances of lhcx1 compared to calmodulin(B), transcript abundances of lhcx2, 3, 4, 5, and phot2 normalized to 5 ng RNA (C) and normalized to calmodulin(D) of K. veneficum cells grown under different light intensities. The samples for gene expression analysis were collected on the 6th day. Shown are means ± SD (error bars) from the triplicated cultures. Asterisks represent that the condition is significantly different from the other two conditions.

FIGURE 5

Distribution of up-regulated (empty bar) and down-regulated (solid bar) proteins under –P condition compared to +P condition based on iTRAQ comparative proteomic analysis in K. veneficum. The proteins for which we could not identify a COG functional category were excluded in this analysis.

FIGURE 6

Relative abundances of LHCX and PHOT2 proteins in K. veneficum grown under –P (empty bar) versus +P (solid bar) conditions based on the iTRAQ comparative proteomic analysis. The expression levels of each protein under +P condition were set as one.

FIGURE 7

Schematic representation of the NPQ enhancement and the metabolic machinery reconfiguration in K. veneficum under P deprivation inferred from the iTRAQ comparative proteomic analysis. Glycolysis (red arrows), tricarboxylic acid (TCA) cycle (orange arrows), lipid metabolism (purple arrows), pyrophosphate hydrolysis (gray arrows), NPQ and chloroplast avoidance movement were strengthened while photochemistry, genetic information flow (e.g., DNA replication, transcription, translation, and post-translation, blue arrows) and phosphonate metabolism (light blue arrows) were weakened under P deprivation in K. veneficum. Representative up-regulated or down-regulated proteins under P deprivation are indicated in white font with a green or blue background, respectively. LHCs, light harvesting protein complexes; LHCX, light-harvesting complex stress-related family proteins; PHOT2, Phototropin-2; ALDO, fructose-bisphosphate aldolase; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde phosphate dehydrogenase; PGK, phosphoglycerate kinase, ENO: enolase; GPDH, Glycerol-3-phosphate dehydrogenase; snRNP, small nuclear ribonucleoprotein.