Minisymposium 18: Seed Biology

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Abs # M1801: Chaperonin cpn20 and cpn60 of exceptionally long-living fruits of Nelumbo nucifera: thermostability and peptide identities

Presenter:	Shen-Miller, Jane       Contact Presenter
Authors	Shen-Miller, Jane  (A) (B)  Xie, Yongming  (C)   Lindner, Petra  (D)   Wooding, Kerry W (C)   Zhang, Heidi  (C)   Loo, Joseph A (C)   Rachel, Reinhard  (D)
Affiliations:	(A): Inst Geophysics Planetary Physics-Ctr Stdy Evol Orig Life, UCLA (B): Dept Ecol, Evol, Biol, UCLA (C): Dept Chem Biochem, UCLA (D): Microbiol Inst, Univ Regensburg, Germany

The genome of Nelumbo nucifera (sacred lotus), 0.24 pg (235 Mb), is one of the smallest known in plants (sequence not studied). (N. nucifera has no relation to the nitrogen-fixing legume Lotus japonicus or the hallucinogen-producing buckthorn Zizyphus lotus.) The exceptional long-term viability of Nelumbo fruits, up to 1300 yr, makes this plant a model organism for identification of genetic determinants involved in health and viability. Of 13 fruits (var. China antique) 200 to 1300 yr-old tested for germination, 85% proved viable. Ohga in 1927 reported 100% germination of China antique after treating them in "steam" at 120^oC for 7 days. One factor that may have contributed to such longevity is an ability to maintain high amounts of durable protein under stressful conditions (total protein content of China antique cotyledons, 6-22%, compares favorably with 8-21% of beans, cereals, and corn). In a 549 yr-old Nelumbo fruit (a ¹⁴C-age determined by accelerator MS; judged viable), 100% of the total protein both of the embryo axis and of cotyledons maintained fluidity after being heated for 10 min at 50^oC. The total content and hardiness of the axis proteins in this old fruit were equal to or greater than those of modern controls. Though the protein content of the cotyledons of the 549 yr-old fruit was only about one-quarter that of the controls, all such proteins remained soluble at 80^o, and 76% at 110^oC. We here identify one type of the hardy proteins in modern China antique seeds (Western blot, negative-staining TEM), the duo protein-folding chaperonins cpn20 and cpn60 (stable to 110^o and 50^oC, respectively), and document the peptide sequences of cpn20 (nano LC-tandem MS). Chaperonins have a "rescue" role that restores functionality to malformed or damaged proteins. Another heat-stable protein, one that co-travels with cpn20 on a 1-D SDS-PAGE gel (~25 kD), shares sequence homologies with 1-Cys-peroxiredoxin (a thiol-specific antioxidant functioning in the protection of germinating seeds from reactive oxygen during early imbibition). Existence of a mobile-loop motif in one of the Nelumbo cpn20 peptide sequences (GLLL) places this chaperonin with the IUPs (intrinsically unstructured proteins, inherently flexible) that are assumed to have critical cellular functions. We have available a supply of Nelumbo fruits, old as well as modern, that can be shared with other researchers for useful investigations.