Figure 1. Effect of sense- and antisense-ßGlu I transformation on the time courses of ßGlu I expression and testa rupture during tobacco seed after-ripening.
(A) ßGlu enzyme activities in sense-ßGlu I (TKSG7), antisense-ßGlu I (TKAG4), and 'empty vector' control seeds (TCIB1) during after-ripening; DAH = days after harvest; mature seeds were harvested at appr. DAP40 (i.e. fresh seeds at DAH0).
(B) Kinetics of testa rupture of fresh and after-ripened (> 6 months of air-dry storage) TCIB1, TKSG7, and TKAG4 seeds. Note that over-expression of ßGlu I in the covering layers of TKSG7 seeds caused promoted testa rupture already in the fresh state and that antisense-ßGlu I transformation prevented the after-ripening-mediated promotion of testa rupture of TKAG4 seeds.
(C) Promotion of GlbGus seed testa rupture during after-ripening. Time courses of testa rupture of GlbGus seeds at various DAH. The curves and corresponding DAH numbers are indicated by rainbow colors. TR50 is the time needed to reach 50 % testa rupture. Note that these TR50 values are presented in Fig. 2D and that endosperm rupture is equally promoted by after-ripening.
Figure 2. Time-course analyses of tobacco after-ripening investigated by air-dry storage of GlbGus seeds.
(A) ßGlu and Chn enzyme activities measured in the same protein extracts by equally sensitive and specific radiometric assays. Further analyses of regions 1 and 2 are presented in Fig. 3.
(B) Gus enzyme activities measured in the same protein extracts. Seed moisture content expressed as % (w/w) H2O per FW.
(C) Relative levels of ßGlu and Gus mRNAs determined by semi-quantitative RT-PCR with 18S rRNA as internal standard. Note: The same RNA samples at several DAH were analysed by TaqMan real-time RT-PCR presented in Fig. 3E
(D) Times in h for 50 % testa rupture (TR50; Fig. 1C) or endosperm rupture as determined from kinetic analyses of imbibed seed populations. Note that a decrease in these values represents a promotion of testa or endosperm rupture.