Diel growth cycle of isolated leaf discs analyzed with a novel, high-throughput three-dimensional imaging method is identical to that of intact leaves.

Dicot leaves grow with pronounced diel (24-h) cycles that are controlled by a complex network of factors. It is an open question to what extent leaf growth dynamics are controlled by long-range or by local signals. To address this question, we established a stereoscopic imaging system, GROWSCREEN 3D, which quantifies surface growth of isolated leaf discs floating on nutrient solution in wells of microtiter plates. A total of 458 leaf discs of tobacco (Nicotiana tabacum) were cut at different developmental stages, incubated, and analyzed for their relative growth rates. The camera system was automatically displaced across the array of leaf discs; visualization and camera displacement took about 12 s for each leaf disc, resulting in a time interval of 1.5 h for consecutive size analyses. Leaf discs showed a comparable diel leaf growth cycle as intact leaves but weaker peak growth activity. Hence, it can be concluded that the timing of leaf growth is regulated by local rather than by systemic control processes. This conclusion was supported by results from leaf discs of Arabidopsis (Arabidopsis thaliana) Landsberg erecta wild-type plants and starch-free1 mutants. At night, utilization of transitory starch leads to increased growth of Landsberg erecta wild-type discs compared with starch-free1 discs. Moreover, the decrease of leaf disc growth when exposed to different concentrations of glyphosate showed an immediate dose-dependent response. Our results demonstrate that a dynamic leaf disc growth analysis as we present it here is a promising approach to uncover the effects of internal and external cues on dicot leaf development.