1 Pheophytin Formation with Senescence in Stored Japanese Bunching 1 Onion 2 3 Prasajith Kapila Dissanayake 4 Faculty of Agricultural sciences 5 Sabaragamuwa University of Sri Lanka 6 Belihuloya, Sri Lanka 7 8 Naoki Yamauchi, Masayoshi Shigyo and 9 Shuhei Tanaka 10 Faculty of Agriculture, Yamaguchi 11 University, 1677-1 Yoshida, 12 Yamaguchi 753-8515, Japan 13 14 Keywords: Chlorophyll degradation, Pheophytin a, Japanese Bunching Onion, 15 plastoglobules 16 17 Abstract 18 The shelf life of Japanese bunching onion (Allium fistulosum L.; JBO genome 19 FF) leaves diminished with yellowing as a consequence of chlorophyll (Chl) degradation, 20 which seems to be the main factor in yellowing of stored JBO leaves. The aim of this 21 study was to investigate pheophytin (Phy) formation as a Chl derivative during Chl 22 degradation in stored JBO leaves. A series of alien monosomic addition lines (AMALs, 23 FF+1A to FF+8A) of JBO, with extra chromosomes from shallot (A. cepa L., 24 Aggregatum group; genome AA) was used as supportive materials to study the 25 formation of Chl a derivatives and the activity of Chl-degrading enzymes during 26 storage. Chl contents in FF+3A and FF+5A decreased greatly during storage at 25°C, 27 whereas in FF+4A the reduction in Chl content was the lowest compared to the control, 28 FF. In JBO leaves, during Chl degradation, the presence of Phy a was prominent as Chl 29 derivatives, which was not recorded in other horticultural crops. Moreover, the 30 activities of Chl-degrading enzymes, especially Mg-dechelation, also progressively 31 increased during storage at 25 o C. The formation of Phy a in stored JBO leaves was 32 further investigated. By incubating the reaction mixture of crude enzyme extract and 33 Chl a, Phy a was formed by Mg-dechelating action. Especial significant was the greater 34 formation of Phy a in FF+3A than FF and FF+4A. Electron microscopic observation 35 elucidated the formation of plastoglobules (Pgs) in chloroplasts and its movement from 36 chloroplast to vacuole in JBO. A large number of Pgs in vacuole of FF+3A might relate 37 to the high Phy a formation with Chl degradation. These findings suggest that Chl a 38 could be degraded in part, through Phy a, and the vacuole along with the chloroplast 39 might be important sites for Chl degradation in JBO during storage. 40 41 INTRODUCTION 42 Yellowing is the main factor in quality deterioration of stored Japanese bunching 43 onion (JBO, Allium fistulosum L.; FF). Our previous studies showed that Chl degradation is 44 the reason for yellowing in JBO leaves during storage (Dissanayake et al., 2008a). 45 Chl degradation is considered to be started by hydrolysis of Chl a into chlorophyllide 46 (Chlide) a and phytol, catalysed by chlorophyllase (Chlase), (Amir-Shapira et al., 1987) and 47 then, as the second step, elimination of Mg from Chlide a to produce pheophorbide (Pheide) 48