Quantitative Proteomic Analysis Reveals Novel Insights into Intracellular Silicate Stress-Responsive Mechanisms in the Diatom Skeletonema dohrnii

Abstract

Diatoms are a successful group of marine phytoplankton that often thrives under adverse environmental stress conditions. Members of the Skeletonema genus are ecologically important which may subsist during silicate stress and form a dense bloom following higher silicate concentration. However, our understanding of diatoms' underlying molecular mechanism involved in these intracellular silicate stress-responses are limited. Here an iTRAQ-based proteomic method was coupled with multiple physiological techniques to explore distinct cellular responses associated with oxidative stress in the diatom Skeletonema dohrnii to the silicate limitation. In total, 1768 proteins were detected; 594 proteins were identified as differentially expressed (greater than a two-fold change; p < 0.05). In Si-limited cells, downregulated proteins were mainly related to photosynthesis metabolism, light-harvesting complex, and oxidative phosphorylation, corresponding to inducing oxidative stress, and ROS accumulation. None of these responses were identified in Si-limited cells; in comparing with other literature, Si-stress cells showed that ATP-limited diatoms are unable to rely on photosynthesis, which will break down and reshuffle carbon metabolism to compensate for photosynthetic carbon fixation losses. Our findings have a good correlation with earlier reports and provides a new molecular level insight into the systematic intracellular responses employed by diatoms in response to silicate stress in the marine environment.

Keywords: abiotic stress; carbon fixation; diatom; iTRAQ; photosynthesis; proteomics; silicate limitation.

Figure 1

Cell density, net primary productivity, and photosynthetic efficiency in Si-limited and Si-added samples of S. dohrnii. Cell density (A), net primary productivity (B) and photochemical quantum yield of PS II (FV/FM) (C). The circle in Figure A shows the duration of sample collection for iTRAQ proteomics. The error bars represent the standard errors from triplicate measurements.

Figure 2

(A) Bar plot of Gene Ontology analysis; the different color shows different GO category. (B) The diagram shows the bar plot of COG analysis x-axis displays the COG term; y-axis displays the corresponding protein count illustrating the protein number of different functions.

Figure 3

Statistics of pathway enrichment factor analysis of differentially expressed proteins comparing of Si-limited vs. Si-added conditions.

Figure 4

A schematic diagram of the electron transfer pathway in oxygenic photosynthesis. During the reaction, the reducing equivalents generated at PSII and transferred to plastoquinone then cytochrome, plastocyanin, PSI, Fd, and ferredoxin NADP+. Electron transfer/flow around PSI is mediated by PGRL1 and NAD2. Reduction in the electron flow around PSII, cytochrome, and PSI resulting in a deficiency in ATP, which is compensated by different mechanisms depending on the demand for ATP. For example, (1) A mitochondrial respiration-based first compensating mechanism likely involving malate/oxaloacetate shuttle, that reduced energy from the chloroplasts to cytosol, and eventually mitochondria. After that, the reducing energy in the mitochondria are converted as ATP by electron transport, which would return to the chloroplast by the ATP translocator (2), the probable occurrence of oxygen photoreduction at the level of NADPH with the help of FLV proteins, which allows ATP biosynthesis via pseudo-cyclic phosphorylation (3). When mechanism (1) and (3) are highly activated, the plastoquinone becomes more reduced, resulting in STT7 kinase activation, therefore, altering LHC phosphorylation and state transition (4). The higher decrease in PSI acceptors would induce the Mehler reaction by direct O₂ photoreduction through reduced Fd, therefore making H₂O₂ (5), and ROS accumulation then induces the AOX and affects the PSI protein complex and decreases the PSII/PSI ratio. Furthermore, downregulation of PSII, cytochrome, and PSI proteins show it makes fewer reducing equivalents flow across a chloroplast and mitochondria, thus resulting in lower ATP production and impact on PSII and PSI, and the LHCs of S. dohrnii.

Figure 5

Representation of the changes in proteins abundance associated with carbon metabolism in S. dohrnii at the onset of silicate limitation. Increases are shown in red arrows. Boxes show where carbon skeletons from amino acid feed into the pathway.

Figure 6

Identified energy and carbon metabolomic pathways and processes of S. dohrnii under Si-limited-Si-added condition. All red arrows indicate up-regulated proteins and processes and green shows downregulation proteins and processes. PS: photosystem, Lhc: light-harvesting complex, OEM: oxygen-evolving complex, Cyt: cytochrome, Fd: ferredoxin; FNR: ferredoxin-NADP+, Fd-NiR: ferredoxin nitrite reductase; TCA: tricarboxylic acid, RUBO: RuBisCO; ROS: reactive oxygen species, PHK: phosphoglycerate kinase; TRI: triosephosphate isomerase; PYTD: pyruvate dehydrogenase; CYTS: citrate synthase; MAL: malate/lactate dehydrogenase; CARP: carbamoyl-phosphate synthase; ARGS: argininosuccinate synthase; ARG: arginase, complex I; NADH ubiquinone reductase, complex III; cytochrome c1.