A rapid freezing method to determine tissue layer thickness in drought-stressed leaves

Plants have been affected by water stress ever since they settled on dry land. In severe and persisting drought, plant leaves are wilting. However, a documentation at the anatomical level of the minute changes that occur before wilting is challenging. On the other hand, understanding the anatomical alteration in plant leaves with respect to water stress provides a stronger basis to study molecular and submolecular processes through which plants enhance drought tolerance. In this work, we applied an affordable method to visualise mesophyll layers of Arabidopsis thaliana cell lines without preparation steps that would alter the volume of the cells. We rapidly plunge-froze the leaves in liquid nitrogen, cut them while in the N₂ bath, and immediately imaged the mesophyll cross sections in a scanning electron microscope. We applied a reduction of watering from 60 to 40 to 20 mL per day and investigated two time points, 7 and 12 days, respectively. Interestingly, the overall thickness of leaves increased in water stress conditions. Our results showed that the palisade and spongy layers behaved differently under varying watering regimes. Moreover, the results showed that this method can be used to image leaf sections after drought stress without the risk of artefacts or swelling caused by contact to liquids as during chemical fixation.