We present a novel measurement setup for monitoring changes in leaf water status using nondestructive terahertz time-domain spectroscopy (
THz-TDS). Previous studies on a variety of plants showed the principal applicability of
THz-TDS. In such setups, decreasing leaf water content directly correlates with increasing THz transmission. Our new system allows for continuous, nondestructive monitoring of the water status of multiple individual plants each at the same constant leaf position. It overcomes previous drawbacks, which were mainly due to the necessity of relocating the plants. Using needles of silver fir (
Abies alba) seedlings as test subjects, we show that the transmission varies along the main axis of a single needle due to a variation in thickness. Therefore, the relocation of plants during the measuring period, which was necessary in the previous
THz-TDS setups, should be avoided. Furthermore, we show a highly significant correlation between gravimetric water content and respective THz transmission. By monitoring the relative change in transmission, we were able to narrow down the permanent wilting point of the seedlings. Thus, we established groups of plants with well-defined levels of water stress that could not be detected visually. This opens up the possibility for a broad range of genetic and physiological experiments.Climate change simulations predict an increase in the occurrence of drought events in the Mediterranean area and in central Europe due to smaller amounts of precipitation, especially during summer periods (
IPCC, 2007). With the exception of the boreal zone, this leads to an increase in drought risks for every region on the European continent (
Iglesias et al., 2007). Water availability is very important for a variety of plant species. Trees and crops play major roles regarding ecosystem stability and food supply. Forest trees are keystone elements in shaping long-term, regional ecosystem composition and stability and are, like most forest species, highly vulnerable to increases in drought severity (
Breshears et al., 2005;
Choat et al., 2012). Drought-induced forest die-offs thereby directly reduce ecosystem services such as carbon sequestration and timber supply (
Allen et al., 2010). Further research is clearly necessary to elucidate the physiological traits and responses of plants regarding their water status.European silver fir (
Abies alba) is an important forest tree species of ecological and economic relevance. This study is embedded in the European project LinkTree, “linking genetic variability with ecological responses to environmental changes: forest trees as model systems.” Our group is concerned with the identification of genes involved in the water stress response of silver fir. This species is of special interest because of its lower water-use efficiency compared with other conifer species (
Guehl and Aussenac, 1987;
Guehl et al., 1991).For this purpose, monitoring plant water status without inducing other forms of stress is instrumental in order to apply well-defined levels of water stress. Obtaining information regarding the water status of a plant is highly problematic without using invasive and destructive methods that usually only allow a retrospective assessment. These include commonly established methods, such as the gravimetric water content and pressure chamber techniques, most notably Scholander’s pressure bomb (
Scholander et al., 1965).Chlorophyll fluorescence, stomatal conductance, and visual assessment are examples of nondestructive and noninvasive measurement techniques. The former two only provide indirect information about the plant stress status and, therefore, the water content via photosynthetic activity (
Lichtenthaler and Rinderle, 1988;
Tardieu and Davies, 1993). The latter is difficult to standardize and highly dependent on the morphology of the studied plant species. Conifers especially are challenging subjects for visually assessing drought stress. Due to their needle morphology, it is nearly impossible to detect early signs of dehydration.Measurement techniques using electromagnetic radiation in the terahertz (
THz) regime have shown promising results, due to the nondestructive nature and high sensitivity of
THz waves to water. With
THz waves, we refer to frequencies in the electromagnetic spectrum between 0.1 and 1
THz, corresponding to wavelengths between 3 and 0.3 mm, which are located between infrared light (thermal radiation) and microwave radiation (used in common wireless data communication systems). In the last decade, terahertz time-domain spectroscopy (
THz-TDS) has proven to be a very strong and accurate tool for characterizing and imaging various materials (for review, see
Jepsen et al., 2011). Crucial for our study is the remarkably high absorption coefficient of water in this part of the electromagnetic spectrum. Thus, it is a robust technique hardly affected by physiological concentrations of soluble substances. Using transmission geometry, the resulting absorption by plant tissues directly reflects the quantity of water molecules.Furthermore,
THz-TDS does not suffer from the disadvantages of other radiation-based techniques. These are mainly focused on the infrared or microwave spectrum but either lack the sensitivity for small changes in leaf water status or are affected by the plant’s inorganic salt content, leading to significant disturbances (
Ulaby and Jedlicka, 1984). Moreover, the applicability of emitting microwave radiation is limited to minimal wavelengths of approximately 2.5 mm. The Abbe diffraction limit, therefore, restricts the minimum diameter of a measurable object to approximately 1.25 mm. In order to measure small leaves, such as coniferous needles, electromagnetic radiation with shorter wavelengths is necessary.Although presenting a useful alternative,
THz-TDS was not feasible until recently, due to the difficulty of generating and detecting electromagnetic radiation with wavelengths in the
THz spectrum. Despite its promising applicability in plant sciences, until now this relatively novel method relied exclusively on measurement setups that allowed only a single measurement per alternating plant (
Hadjiloucas et al., 1999;
Jördens et al., 2009;
Breitenstein et al., 2012;
Castro-Camus et al., 2013;
Gente et al., 2013). For the purpose of continuously monitoring multiple plants, these setups are only of limited use, since the plants must be relocated for every measurement. This results in two problems: (1) an increase in possible disturbances (e.g. mechanical), influencing the plant’s stress response, and (2) the necessity to precisely target the same measurement spot on every analyzed plant at every consecutive measurement. The latter is of crucial importance for the exact monitoring of any individual plant’s water status because, as we will show in this study, the transmission varies substantially across the area of plant leaf tissue.We present a novel measurement procedure that overcomes the drawbacks of previously proposed methods. Our approach enables us to precisely monitor changes in the water content of multiple plants simultaneously.In the course of this study, three different experiments were performed. The profile measurement and the rehydration experiment were preliminary investigations to examine the influences of needle and tissue thickness and to define a nonlethal stress level. The main experiment established groups of plants with comparable levels of water stress.
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