In vivo cytological and chemical analysis of Casparian strips using stimulated Raman scattering microscopy

The Casparian strip, a barrier to the apoplastic movement of solutes from the cortex to the stele, is essential for the exclusion of salts, selective nutrient uptake, and many other processes. To date, extensive studies have focused on the physiological functions of endodermal Casparian strips. However, the chemical deposition nature of Casparian strips, as well as its relevance with respect to diffusion barrier functions, remains to be further elucidated. Here, we revealed three developmental stages of Casparian strips in maize primary roots using a traditional fluorescent staining method. Apoplastic permeability tests demonstrated that the barrier function of Casparian strips is largely related to their developmental stage and the pattern of lignin and suberin deposits. Fourier transform infrared (FTIR) analysis showed that the Casparian strips from the roots exhibited significant absorption bands characteristic of lignin and suberin, implying that the Casparian strips in maize primary roots consist largely of lignin and suberin. Furthermore, we developed a new method for label-free, in vivo structural, and biochemical analysis of Casparian strips based on stimulated Raman scattering (SRS) microscopy. Using SRS microscopy, we found that lignin and suberin accumulate simultaneously during the Casparian strip formation process. Based on these results, we propose a potential application of SRS for the chemical composition analysis of plant Casparian strips in situ.