Analysis Of Lignin And The Activity And Isozymogram Of POD On Juice Sacs Of Citrus Maxima

Juice sac granulation occurring in pummelo fruits [Citrus maxima (Burm.) Merr.] has a great effect on its eating quality and commodity value, and the mechanism underlying juice sac granulation remains unresolved. Previous studies have shown that lignin metabolism is closely associated with the process of juice sac granulation. Hence, in this report, lignin from different pummelo tissues and cultivars was analyzed, and the activities and isoenzymes of POD associated with monolignols oxidation were detected for providing more useful information for the subsequent studies on the mechanism of juice sac granulation.Firstly, a method suitable for isolating lignin from pummelo tissues was established. Acetylated lignin from different pummelo tissues and cultivars were analyzed by UV-Vis spectroscopy and HSQC NMR. The results showed that β-O-4’ substructures were predominant in all the tested tissues, followed by β-5’ substructures that were associated with the abundances of guaiacyl units. β-β’ Substructures were only observed in the syringyl-rich tissue. Lignin of granulated juice sacs was characterized with an extremely high abundance of guaiacyl units (91.13-96.82%), and was different in lignin composition from other tissues, including leaves, stems and segment membranes. The abnormally accumulated lignin in granulated juice sac was difficult for degradation and mainly polymerized from coniferyl alcohol. There was no significant difference in the types of lignin among different cultivars, but their guaiacyl/syringyl ratio showed great difference. It appeared that the mechanism of juice sac granulation might be the same among different cultivars, although they could have very different degrees of juice sac granulation.Before studying the POD correlated with coniferyl alcohol oxidation, pH 8.0,100 mM Tris-HCl and pH 8.0,100 mM Tris-HCl containing 0.1% Triton X-100 and 1 M NaCl were established as the optimal extraction buffer for soluble and ion-bound POD, respectively. The whole activities of soluble and ion-bound POD were both falling during growth and development process, and the activity of soluble POD was much higher than ion-bound POD, which showed significant difference between them. The soluble POD activities between juice sacs close to the stele and away from the stele had significant difference. And its alkaline POD activity in juice sacs close to the stele was higher than that in juice sacs away from the stele, as showed in isozymogram. This indicated the possible correlation between soluble POD and juice sac granulation. The correlation between ion-bound POD and juice sac granulation could not be found from its enzyme activity, but several specific alkaline POD of it were detected on granulation stages. Consequently, ion-bound POD might be associated with juice sac granulation more closely. In addition, although GA treatment might have some effects on the activities and isozymogram of soluble and ion-bound POD, its real role in the process of juice sac granulation remains to be tested in the subsequent studies.