The activities of sucrolytic enzymes (soluble invertase, cell wall invertase and sucrose synthase) were determined in Easter lily (Lilium longiflorum Thunb.) floral organs during flower development. Although all three enzymes were present in every flower organ of L. longiflorum, soluble invertase was the predominant enzyme in all flower organs except in stigma where cell wall invertase dominated. Soluble invertase activity was highly correlated with dry weight gain in most of the flower organs. However, hexose content had different degrees of correlation with soluble invertase activity in various flower organs indicating different turnover rates of hexoses.; Soluble invertase resolved to three isoforms on a DEAE-Sephacel column, and designated as invertase I, II, or III according to the order of elution. Invertase I was mainly expressed in the anther, whereas the other two forms were expressed in all flower organs. The fluctuation of invertase activity in the anther during flower development was primarily due to changes in invertase I activity. Each isoform was an acid invertase, with pH optima between 4.0 and 5.0. The isoform I, II and III had sucrose {dollar}Ksb{lcub}rm m{rcub}{dollar} values of 1.0, 6.4 and 6.6 mM, and temperature optima of 40, 50 and 45{dollar}spcirc{dollar}C, respectively. Invertase III was the most thermostable, followed by invertase II and I. Invertases II and III hydrolyzed raffinose and stachyose at a lower rate than invertase I.; Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified invertase I gave a single band at 78 kD, whereas invertase II and III gave three bands at 54, 52 and 24 kD. Two-dimensional electrophoresis profiles, cross-reactivity with heterologous invertase antibodies, and the deglycosylation pattern with peptide-N-glycosidase F of invertase I were substantially different from the other two isoforms which shared similar properties.; Genomic DNA was extracted, and southern blot analysis was performed using heterologous cDNA probes from maize, carrot and tomato to detect invertase genes in L. longiflorum. None of these probes was hybridized to L. longiflorum genomic DNA indicating the distinct nature of invertase genes in L. longiflorum. Isolation of cDNA or genomic clones from L. longiflorum, is therefore, necessary for invertase gene expression studies. |