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Hyperspectral imaging is a promising tool for non-destructive phenotyping of plant physiological traits, which has been transferred from remote to proximal sensing applications, and from manual laboratory setups to automated plant phenotyping platforms. Due to the higher resolution in proximal sensing, illumination variation and plant geometry result in increased non-biological variation in plant spectra that may mask subtle biological differences. Here, a better understanding of spectral measurements for proximal sensing and their application to study drought, developmental and diurnal responses was acquired in a drought case study of maize grown in a greenhouse phenotyping platform with a hyperspectral imaging setup. The use of brightness classification to reduce the illumination-induced non-biological variation is demonstrated, and allowed the detection of diurnal, developmental and early drought-induced changes in maize reflectance and physiology. Diurnal changes in transpiration rate and vapor pressure deficit were significantly correlated with red and red-edge reflectance. Drought-induced changes in effective quantum yield and water potential were accurately predicted using partial least squares regression and the newly developed Water Potential Index 2, respectively. The prediction accuracy of hyperspectral indices and partial least squares regression were similar, as long as a strong relationship between the physiological trait and reflectance was present. This demonstrates that current hyperspectral processing approaches can be used in automated plant phenotyping platforms to monitor physiological traits with a high temporal resolution.

Jasmonate-mediated regulation of VOC emission has been extensively investigated in higher plants, however, only little is known about VOC production and its regulation in ferns. Here, we investigate whether the emission of VOCs from bracken fern Pteridium aquilinum is triggered by herbivory and if so - whether it is regulated by the octadecanoid signaling pathway. Interestingly, feeding of both generalist (Spodoptera littoralis) and specialist (Strongylogaster multifasciata) herbivores as well as application of singular and continuous mechanical wounding of fronds induced only very low levels of VOC emission. In contrast, treatment with jasmonic acid (JA) led to the emission of a blend of VOCs that was mainly comprised of terpenoids. Likewise, treatment with the JA precursor 12-oxo-phytodienoic acid (OPDA) and α-linolenic acid also induced VOC emission, albeit to a lower intesity than the JA treatment. Accumulation of endogenous JA was low in mechanically wounded fronds and these levels were unaffected by the application of oral secretions from both generalist or specialist herbivores. The emission of terpenoids upon JA treatment could be blocked with fosmidomycin and mevinolin, which are inhibitors of the MEP- and MVA pathways, respectively. These results indicate that similar to higher plants, terpenoid VOCs are produced via these pathways in bracken fern and that these pathways are JA-responsive. However, the very low amounts of terpenoids released after herbivory or mechanical damage are in stark contrast to what is known from higher plants. We speculate that S. multifasciata and S. littoralis feeding apparently did not induce the threshold levels of JA required for activating the MEP and MVA pathways and the subsequent volatile emission in bracken fern.