Rethinking temperature effects on leaf growth, gene expression and metabolism: Diel variation matters.

Plants have evolved to grow under prominently fluctuating environmental conditions. In experiments under controlled conditions, temperature is often set to artificial, binary regimes with constant values at day and at night. This study investigated how such a diel (24 hr) temperature regime affects leaf growth, carbohydrate metabolism and gene expression, compared to a temperature regime with a field-like gradual increase and decline throughout 24 hr. Soybean (Glycine max) was grown under two contrasting diel temperature treatments. Leaf growth was measured in high temporal resolution. Periodical measurements were performed of carbohydrate concentrations, carbon isotopes as well as the transcriptome by RNA sequencing. Leaf growth activity peaked at different times under the two treatments, which cannot be explained intuitively. Under field-like temperature conditions, leaf growth followed temperature and peaked in the afternoon, whereas in the binary temperature regime, growth increased at night and decreased during daytime. Differential gene expression data suggest that a synchronization of cell division activity seems to be evoked in the binary temperature regime. Overall, the results show that the coordination of a wide range of metabolic processes is markedly affected by the diel variation of temperature, which emphasizes the importance of realistic environmental settings in controlled condition experiments.

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.

Diel growth patterns of young soybean (Glycine max) leaflets are synchronous throughout different positions on a plant.

Leaf growth is controlled by various internal and external factors. Leaves of dicotyledonous plants show pronounced diel (24 h) growth patterns that are controlled by the circadian clock. To date, it is still uncertain whether diel leaf growth patterns remain constant throughout the development of a plant. In this study, we followed growth from the primary leaves to leaflets of the seventh trifoliate leaf of soybean (Glycine max) on the same plants with a recently developed imaging-based method under controlled conditions and at a high temporal resolution. We found that all leaflets displayed a consistent diel growth pattern with maximum growth towards the end of the night. In some leaves, growth maxima occurred somewhat later - at dawn - as long as the leaves were still in a very early developmental stage. Yet, overall, diel growth patterns of leaves from different positions within the canopy were highly synchronous. Therefore, the diel growth pattern of any leaf at a given point in time is representative for the overall diel growth pattern of the plant leaf canopy and a deviation from the normal diel growth pattern can indicate that the plant is currently facing stress.

Terrestrial 3D laser scanning to track the increase in canopy height of both monocot and dicot crop species under field conditions.

BACKGROUND: Plant growth is a good indicator of crop performance and can be measured by different methods and on different spatial and temporal scales. In this study, we measured the canopy height growth of maize (Zea mays), soybean (Glycine max) and wheat (Triticum aestivum) under field conditions by terrestrial laser scanning (TLS). We tested the hypotheses whether such measurements are capable to elucidate (1) differences in architecture that exist between genotypes; (2) genotypic differences between canopy height growth during the season and (3) short-term growth fluctuations (within 24 h), which could e.g. indicate responses to rapidly fluctuating environmental conditions. The canopies were scanned with a commercially available 3D laser scanner and canopy height growth over time was analyzed with a novel and simple approach using spherical targets with fixed positions during the whole season. This way, a high precision of the measurement was obtained allowing for comparison of canopy parameters (e.g. canopy height growth) at subsequent time points. RESULTS: Three filtering approaches for canopy height calculation from TLS were evaluated and the most suitable approach was used for the subsequent analyses. For wheat, high coefficients of determination (R(2)) of the linear regression between manually measured and TLS-derived canopy height were achieved. The temporal resolution that can be achieved with our approach depends on the scanned crop. For maize, a temporal resolution of several hours can be achieved, whereas soybean is ideally scanned only once per day, after leaves have reached their most horizontal orientation. Additionally, we could show for maize that plant architectural traits are potentially detectable with our method. CONCLUSIONS: The TLS approach presented here allows for measuring canopy height growth of different crops under field conditions with a high temporal resolution, depending on crop species. This method will enable advances in automated phenotyping for breeding and precision agriculture applications. In future studies, the TLS method can be readily applied to detect the effects of plant stresses such as drought, limited nutrient availability or compacted soil on different genotypes or on spatial variance in fields.

The ETH field phenotyping platform FIP: a cable-suspended multi-sensor system.

Crop phenotyping is a major bottleneck in current plant research. Field-based high-throughput phenotyping platforms are an important prerequisite to advance crop breeding. We developed a cable-suspended field phenotyping platform covering an area of ~1ha. The system operates from 2 to 5m above the canopy, enabling a high image resolution. It can carry payloads of up to 12kg and can be operated under adverse weather conditions. This ensures regular measurements throughout the growing period even during cold, windy and moist conditions. Multiple sensors capture the reflectance spectrum, temperature, height or architecture of the canopy. Monitoring from early development to maturity at high temporal resolution allows the determination of dynamic traits and their correlation to environmental conditions throughout the entire season. We demonstrate the capabilities of the system with respect to monitoring canopy cover, canopy height and traits related to thermal and multi-spectral imaging by selected examples from winter wheat, maize and soybean. The system is discussed in the context of other, recently established field phenotyping approaches; such as ground-operating or aerial vehicles, which impose traffic on the field or require a higher distance to the canopy.

Diel leaf growth of soybean: a novel method to analyze two-dimensional leaf expansion in high temporal resolution based on a marker tracking approach (Martrack Leaf).

BACKGROUND: We present a novel method for quantitative analysis of dicot leaf expansion at high temporal resolution. Image sequences of growing leaves were assessed using a marker tracking algorithm. An important feature of the method is the attachment of dark beads that serve as artificial landmarks to the leaf margin. The beads are mechanically constricted to the focal plane of a camera. Leaf expansion is approximated by the increase in area of the polygon defined by the centers of mass of the beads surrounding the leaf. Fluctuating illumination conditions often pose serious problems for tracking natural structures of a leaf; this problem is circumvented here by the use of the beads. RESULTS: The new method has been used to assess leaf growth in environmental situations with different illumination conditions that are typical in agricultural and biological experiments: Constant illumination via fluorescent light tubes in a climate chamber, a mix of natural and artificial illumination in a greenhouse and natural illumination of the situation on typical summer days in the field. Typical features of diel (24h) soybean leaf growth patterns were revealed in all three conditions, thereby demonstrating the general applicability of the method. Algorithms are provided to the entire community interested in using such approaches. CONCLUSIONS: The implementation Martrack Leaf presented here is a robust method to investigate diel leaf growth rhythms both under natural and artificial illumination conditions. It will be beneficial for the further elucidation of genotype x environment x management interactions affecting leaf growth processes.