Chromosome doubling was induced in vitro in a diploid hybrid of Rosa rugosa Thunb. using oryzalin as the spindle inhibitor. Nodal sections, 2 mm long, were exposed to 2.5 or 5 microM oryzalin and 10 mm nodal sections were exposed to 5 microM oryzalin for 0 (controls), 6, 12, 24 and 48 h. The ploidy of the emergent shoots was determined by flow cytometry. The frequency of tetraploid and mixoploid leaves that developed from 2 mm nodal sections exposed to 5 microM oryzalin peaked at 12 h exposure, when 35% of the leaves were tetraploid, but fell after longer exposures. Fewer tetraploid and mixoploid leaves were found when 2 mm nodes were exposed to 2.5 microM oryzalin for 6 and 12 h, indicating that it took longer for a spindle inhibiting concentration of oryzalin to build up in the meristem. However, the frequencies of tetraploid and mixoploid leaves continued to rise after 12 h and were highest at 48 h, when 44% were tetraploid. In treatments with 5 microM oryzalin, the frequencies of tetraploid and mixoploid leaves were lower, at equivalent exposure times, in 10 mm nodes than 2 mm nodes. This suggests that oryzalin diffused to the meristem mainly via the cut surfaces and that access via the epidermis and cuticle was impeded.

Summary

SummaryTypically, nuclear-encoded chloroplast proteins are synthesized as precursors and require proteolytic processing upon import before their assembly into functional complexes within the organelle. A cDNA encoding a chloroplast processing enzyme (CPE), which was originally identified as a protease that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP), was introduced into the tobacco genome in an antisense orientation to investigate the role of the enzyme in vivo. The presence of the antisense-CPE gene resulted in chlorotic leaves, and retarded shoot and root growth. The introduction of the antisense-CPE gene disrupted the normal pattern of plastid division. Chloroplast numbers in cotyledon and first leaf cells were reduced 25% compared to the control plants. Chloroplasts contained fewer thylakoids and large starch grains, the latter an indication of a change in carbon flux. CPE levels and activity were significantly lower in stromal extracts in the transgenic plants. Interestingly, in vitro import of precursor proteins was defective. Most of the preLHCP remained on the exterior of the organelle, and only a small fraction of preRBCA was imported, suggesting that a change in CPE levels can influence translocation across the envelope. Our in vivo results support the conclusion that CPE plays a critical role during chloroplast biogenesis, and that the pleiotropic effects of CPE down-regulation reflect its function as a general stromal processing peptidase as part of the import machinery. Furthermore, these findings indicate the importance of regulating the expression of components of the import machinery for normal plant development.

No abstract