Metabolomic and Proteomic Profiles Reveal the Dynamics of Primary Metabolism during Seed Development of Lotus (Nelumbo nucifera)

Abstract

Sacred lotus (Nelumbo nucifera) belongs to the Nelumbonaceae family. Its seeds are widely consumed in Asian countries as snacks or even medicine. Besides the market value, lotus seed also plays a crucial role in the lotus life cycle. Consequently, it is essential to gain a comprehensive understanding of the development of lotus seed. During its development, lotus seed undergoes cell division, expansion, reserve accumulation, desiccation, and maturation phases. We observed morphological and biochemical changes from 10 to 25 days after pollination (DAP) which corresponded to the reserve synthesis and accumulation phase. The volume of the seed expanded until 20 DAP with the color of the seed coat changing from yellow-green to dark green and gradually fading again. Starch and protein rapidly accumulated from 15 to 20 DAP. To further reveal metabolic adaptation, primary metabolites and proteins profiles were obtained using mass spectrometry based platforms. Metabolites and enzymes involved in sugar metabolism, glycolysis, TCA cycle and amino acid metabolism showed sequential dynamics enabling the clear separation of the different metabolic states during lotus seed development. The integration of the data revealed a highly significant metabolic switch at 15 DAP going through a transition of metabolically highly active tissue to the preparation of storage tissue. The results provide a reference data set for the evaluation of primary metabolism during lotus seed development.

Keywords: GC-TOF-MS; LC-Orbitrap-MS; Nelumbo nucifera; mass spectrometry; primary metabolism; seed development.

FIGURE 1

Morphology and histology of developing lotus seeds. (A) Lotus seed at different developmental stages. Histological observation of polysaccharides (B) and protein accumulation (C) during lotus seed development. The polysaccharides and proteins were stained with PAS and Commassie blue method, respectively. The rulers indicate 1 cm in (A) whereas 100 μm in (B,C).

FIGURE 2

Principal component analysis (PCA) of metabolomic (A) and proteomic datasets (B) during lotus seed development. The normalized metabolite dataset (Supplementary Table S1) and NSAFs of all the identified proteins (Supplementary Table S3 sheet “Combined”) were log 10 transformed and used for PCA. The NSAFs of those proteins that identified with both in-gel and in-solution procedures were averaged for PCA.

FIGURE 3

Bi-clustering analysis of annotated primary metabolites (A). Clustering analysis of the dynamics of sugars (B), amino acids (C), and organic acids (D) during lotus seed development.

FIGURE 4

Quantitative analysis of the proteomic data. (A) Venn diagram showing the overlap of proteins identified in samples using in-gel and in-solution digestion methods; (B) Clustering analysis of the identified proteins.

FIGURE 5

Functional categorization of the differentially displayed proteins (A,B) sub-functional categorization of the metabolism related proteins.

FIGURE 6

Primary metabolism dynamics during lotus seed development. Metabolites (black) and proteins (red) involved in sugar metabolism, glycolysis, TCA cycle and amino acid metabolism were mapped on their metabolism pathways. The relative ratios of normalized metabolite peak areas were colored with blue-white-red color bar corresponding to the values from the minimum (Min) to median (Med) to maximum (Max). The relative ratios of the protein NSAFs were colored with green-white-red color bar corresponding to the ratios from 0 to 0.25 to 1. All the protein candidates that annotated as the same enzyme were list here in consecutive rows. Four squares from left to right indicate samples of 10, 15, 20, and 25 DAP, respectively. Lowercase characters in the squares indicate significant levels according to Duncan’s test (p < 0.05). Abbreviations: RS, raffinose synthase; Susy, sucrose synthase; AGPase, ADP glucose pyrophosphorylase; StarchP, starch phosphorylase; PGlcM, phosphoglucomutase (plastid); GPI, glucose-6-phosphate isomerase (plastid); PFK, phosphofructokinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 1,3-BPG, 1,3-Bisphosphoglyceric acid; PGK, 3-phosphoglycerate kinase; PGM, phosphoglycerate mutase; PK, pyruvate kinase; PDC, pyruvate dehydrogenase complex; CS, citrate synthase; IDH, isocitrate dehydrogenase; SCS, succinyl coenzyme A synthetase; SDH, succinate dehydrogenase; MDH, malate dehydrogenase; GAD, glutamic acid decarboxylase; GABA, 4-aminobutanoic acid (γ-Aminobutyric acid); AspAT, aspartate aminotransferase; ALAT, alanine aminotransferase; AGT, alanine-glyoxylate aminotransferase; ASADH, aspartate semialdehyde dehydrogenase; TAL, threonine ammonia-lyase; 3PGDH, 3-phosphoglycerate dehydrogenase; OASTL, O-acetylserine(thiol)lyase; DAHP synthase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.