Image analysis of stress-induced lignin deposition in Arabidopsis thaliana using the macro program LigninJ for ImageJ software.

In vascular plants, lignin is deposited during morphogenesis but also under stress conditions. Assessing the degree of stress-induced lignin deposition is complicated because it occurs locally and irregularly in plant tissues. In this study, we developed a macro program, LigninJ, for the open-source software ImageJ to automatically and efficiently determine areas and levels of lignification after Wiesner (phloroglucinol-HCl) staining. We used the CIELAB color space for detection of red color following the Wiesner reaction. In addition, LigninJ has a function for adjusting the background level and its white balance to reduce biases that are inherent to individual color images. Furthermore, LigninJ can be used for batch analyses of multiple images, taking about 2 s per image. In this study, we analyzed wound-induced lignin deposition in cotyledons of the Arabidopsis thaliana ecotypes Landsberg erecta and Columbia and assessed ectopic lignin depositions in roots of lignescence (lig) mutants of Arabidopsis. Our results confirmed that this method is efficient for evaluating the degree of stress-induced lignin deposition.