Non-destructive measurement of soybean leaf thickness via X-ray computed tomography allows the study of diel leaf growth rhythms in the third dimension

Present-day high-resolution leaf growth measurements provide exciting insights into diel (24-h) leaf growth rhythms and their control by the circadian clock, which match photosynthesis with oscillating environmental conditions. However, these methods are based on measurements of leaf area or elongation and neglect diel changes of leaf thickness. In contrast, the influence of various environmental stress factors to which leaves are exposed to during growth on the final leaf thickness has been studied extensively. Yet, these studies cannot elucidate how variation in leaf area and thickness are simultaneously regulated and influenced on smaller time scales. Only few methods are available to measure the thickness of young, growing leaves non-destructively. Therefore, we evaluated X-ray computed tomography to simultaneously and non-invasively record diel changes and growth of leaf thickness and area. Using conventional imaging and X-ray computed tomography leaf area, thickness and volume growth of young soybean leaves were simultaneously and non-destructively monitored at three cardinal time points during night and day for a period of 80 h under non-stressful growth conditions. Reference thickness measurements on paperboards were in good agreement to CT measurements. Comparison of CT with leaf mass data further proved the consistency of our method. Exploratory analysis showed that measurements were accurate enough for recording and analyzing relative diel changes of leaf thickness, which were considerably different to those of leaf area. Relative growth rates of leaf area were consistently positive and highest during ‘nights’, while diel changes in thickness fluctuated more and were temporarily negative, particularly during ‘evenings’. The method is suitable for non-invasive, accurate monitoring of diel variation in leaf volume. Moreover, our results indicate that diel rhythms of leaf area and thickness show some similarity but are not tightly coupled. These differences could be due to both intrinsic control mechanisms and different sensitivities to environmental factors.