Comparative proteomic study reveals the involvement of diurnal cycle in cell division, enlargement, and starch accumulation in developing endosperm of Oryza sativa.

The development and starch accumulation of cereal endosperms rely on the sugar supply of leaves, which is subject to diurnal cycles, and the endosperm itself also experiences a light/dark switch. However, revealing how the cereal endosperm responds to diurnal input remains a major challenge. We used comparative proteomic approaches to probe diurnally affected processes in rice endosperm (Oryza sativa) 10 days after flowering under 12-h light/12-h dark. Starch granules in rice endosperm showed a growth ring structure under a normal light/dark cycle but not under constant light. Sucrose showed a high level in light and low level in dark. Two-dimensional (2-D) differential in-gel electrophoresis-based proteomic analysis revealed 101 protein spots diurnally changed and 91 identities, which were involved in diverse processes with preferred distribution in stress response, protein synthesis/destination and metabolism. Proteins involved in cell division showed high expression in light and those in cell enlargement and cell wall synthesis high in dark, while starch synthesis proteins were light-downregulated and dark-upregulated. Redox homeostasis-associated proteins showed in-phase peaks under light and dark. These data demonstrate diurnal input-regulated diverse cellular and metabolic processes in rice endosperm, and coordination among these processes is essential for development and starch accumulation with diurnal input.

Integrated proteomic and cytological study of rice endosperms at the storage phase.

The endosperm at the storage phase undergoes a series of coordinated cellular and metabolic events, including starchy endosperm cell death, starch synthesis, and starch granule packaging, which leads to efficient accumulation of starch. However, the mechanism underlying the interconnections remains unknown. We used integrated proteomic and cytological approaches to probe the interconnections in rice (Oryza sativa) endosperm at the storage phase from 12 to 18 days after flowering (DAF). Starch granule packaging was completed first in the inner part of endosperm at 15 DAF and spread to almost the entire endosperm at 18 DAF. Programmed starchy endosperm cell death occurred after the starch granule packaging. Endogenous H(2)O(2) was detectable in the inner part of endosperm at 12 DAF and the region beyond the inner part at 15 DAF, with an H(2)O(2) burst at 15 DAF. Proteomics analysis with 2-D fluorescent difference gel electrophoresis and matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry revealed 317 proteins, including almost all known antioxidants, differentially expressed throughout the 3 stages of the developmental phase. More than two-thirds of the 317 proteins were potential thioredoxin targets, with a preferential skew toward central carbon metabolism, alcoholic fermentation, starch metabolism, amino acid metabolism, and protein synthesis or folding. These proteins implicated in starch synthesis and gluconeogenesis were upregulated, whereas those involved in anabolism of biomacromolecules such as proteins, lipids, and cell wall components were downregulated, with upregulated expression of proteins involved in catabolism of these biomacromolecules, which suggests remobilization of nutrients for starch synthesis. These data suggested important roles of the H(2)O(2)-antioxidant interface in coordinating starch accumulation, programmed cell death of starchy endosperm, and remobilization of nutrients during the cell death.

Dynamic proteomic analysis reveals a switch between central carbon metabolism and alcoholic fermentation in rice filling grains.

Accumulation of reserve materials in filling grains involves the coordination of different metabolic and cellular processes, and understanding the molecular mechanisms underlying the interconnections remains a major challenge for proteomics. Rice (Oryza sativa) is an excellent model for studying grain filling because of its importance as a staple food and the available genome sequence database. Our observations showed that embryo differentiation and endosperm cellularization in developing rice seeds were completed approximately 6 d after flowering (DAF); thereafter, the immature seeds mainly underwent cell enlargement and reached the size of mature seeds at 12 DAF. Grain filling began at 6 DAF and lasted until 20 DAF. Dynamic proteomic analyses revealed 396 protein spots differentially expressed throughout eight sequential developmental stages from 6 to 20 DAF and determined 345 identities. These proteins were involved in different cellular and metabolic processes with a prominently functional skew toward metabolism (45%) and protein synthesis/destination (20%). Expression analyses of protein groups associated with different functional categories/subcategories showed that substantially up-regulated proteins were involved in starch synthesis and alcoholic fermentation, whereas the down-regulated proteins in the process were involved in central carbon metabolism and most of the other functional categories/subcategories such as cell growth/division, protein synthesis, proteolysis, and signal transduction. The coordinated changes were consistent with the transition from cell growth and differentiation to starch synthesis and clearly indicated that a switch from central carbon metabolism to alcoholic fermentation may be important for starch synthesis and accumulation in the developmental process.