Phosphoproteomics unveils stable energy supply as key to flooding tolerance in Kandelia candel

The mangrove Kandelia candel (L.) Druce experiences daily flooding cycles. To explore the molecular mechanism underlying the physiological adaptation of K. candel to flooding, the potential role of protein phosphorylation in flooding responses was investigated by a large-scale quantitative phosphoproteomic analysis using isobaric tag for relative and absolute quantitation. Total 2141 unique phosphopeptides and 2603 non-redundant phosphorylation sites were identified from 1516 phosphoproteins in K. candel leaves. In addition to known phosphorylation motifs, three new motifs [GSP], [GxxSP] and [RSxS] were discovered. The phosphorylation levels of 96 differentially expressed phosphoproteins, including those involved in pyruvate metabolism and energy production, were identified in response to flooding. The physiological parameters and transcriptional levels relevant to flooding responses including photosynthesis, pyruvate metabolism, and ROS production were investigated and all were found to be robust under flooding conditions. The consistent results of the phosphoproteomic, physiological analyses and transcriptional levels reinforce each other to demonstrate that K. candel adapts to flooding through maintaining sufficient photosynthesis activities, achieving effective anaerobic respiration and increasing pentose phosphate pathway flux. Protein phosphorylation is likely to play a major role in the regulation of these pathways which together contribute to stable energy supply that enhances flooding tolerance in K. candel.

Flooding stress is one of the major environmental stresses. The woody mangrove Kandelia candel experiences daily flooding cycles in its natural habitat. Protein phosphorylation is a crucial regulatory mechanism in plants' responses to both biotic and abiotic stresses. To analyze phosphorylation levels in critical enzymes involved in key metabolic pathways, we employed phosphoproteomic approach to dissect the adaptive mechanism of K. candel to flooding conditions. To our knowledge, this is the first large-scale quantitative phosphoproteomic analyses of K. candel's flooding responses. Multiplex iTRAQ-based quantitative proteomic and Nano-LC-MS/MS methods were used to construct the phosphorproteome. Our results indicate that K. candel is able to acquire stable energy supply under flooding by maintaining sufficient photosynthesis activities, enhancing effective anaerobic respiration and increasing pentose phosphate pathway (PPP) flux. The protein phosphorylation found in photosynthesis, anaerobic respiration and PPP is likely to play important roles in the flooding tolerance of K. candel.