Histochemical Techniques in Plant Science: More Than Meets the Eye Vaishali Yadav 1 , Namira Arif 1 , Vijay Pratap Singh 2 , Gea Guerriero 3 , Roberto Berni 4 , Suhas Shinde 5 , Gaurav Raturi 6 , Rupesh Deshmukh 6 , Luisa M. Sandalio 7, *, Devendra Kumar Chauhan 1, * and Durgesh Kumar Tripathi 8, * 1 D D Pant Interdisciplinary Research Laboratory, Department of Botany, University of Allahabad, Prayagraj 211002, India 2 Plant Physiology Laboratory, Department of Botany, C.M.P. Degree College, A Constituent Post Graduate College of University of Allahabad, Prayagraj 211002, India 3 Environmental Research and Innovation Department, Luxembourg Institute of Science and Technology, Hautcharage, Luxembourg 4 TERRA Teaching and Research Center, Gembloux Agro-Bio Tech, University of Liège, Gembloux 5030, Belgium 5 Department of Biology and Gus R. Douglass Institute, West Virginia State University, Institute, WV 25112, USA 6 Department of Agri-Biotechnology, National Agri-Food Biotechnology Institute (NABI), Mohali, India 7 Department of Biochemistry, Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, Granada 18008, Spain 8 Amity Institute of Organic Agriculture, Amity University Uttar Pradesh, I 2 Block, 5th Floor, AUUP Campus Sector-125, Noida 201313, India *Corresponding authors: L. M. Sandalio, E-mail, luisamaria.sandalio@eez.csic.es; D. K. Chauhan, E-mail, dkchauhanau@yahoo.com; D. K. Tripathi, dktripa- thiau@gmail.com. (Received 31 May 2020; Accepted 31 January 2021) Histochemistry is an essential analytical tool interfacing extensively with plant science. The literature is indeed con- stellated with examples showing its use to decipher specific physiological and developmental processes, as well as to study plant cell structures. Plant cell structures are translu- cent unless they are stained. Histochemistry allows the identification and localization, at the cellular level, of biomolecules and organelles in different types of cells and tissues, based on the use of specific staining reactions and imaging. Histochemical techniques are also widely used for the in vivo localization of promoters in specific tissues, as well as to identify specific cell wall components such as lignin and polysaccharides. Histochemistry also enables the study of plant reactions to environmental constraints, e.g. the production of reactive oxygen species (ROS) can be traced by applying histochemical staining techniques. The possibility of detecting ROS and localizing them at the cellular level is vital in establishing the mechanisms involved in the sensitivity and tolerance to different stress conditions in plants. This review comprehensively highlights the additional value of histochemistry as a complementary tech- nique to high-throughput approaches for the study of the plant response to environmental constraints. Moreover, here we have provided an extensive survey of the available plant histochemical staining methods used for the localiza- tion of metals, minerals, secondary metabolites, cell wall components, and the detection of ROS production in plant cells. The use of recent technological advances like CRISPR/ Cas9-based genome-editing for histological application is also addressed. This review also surveys the available literature data on histochemical techniques used to study the response of plants to abiotic stresses and to identify the effects at the tissue and cell levels. Keywords: Biogenic minerals • Cell wall • Histochemical staining • Imaging • Reactive oxygen species • Secondary metabolites. Introduction Histochemical staining has become a widely used technique in the field of plant science for the localization of proteins, enzym- atic and promoter activities, elements, oxidative stress markers, minerals and cell wall components in plant cells/tissues (Al Bitar et al. 2002, Romero-Puertas et al. 2004, Law and Exley 2011, Prabhu et al. 2011, Hassan and El-Awadi 2013, Giuliani et al. 2018, de Ávila et al. 2019). Histochemical methods are also beneficial for the in situ quantification of several metabolites, allowing to determine the place of their respective production or action (Dubey and Trivedi 2012). Histochemistry allows in situ analysis of specific components while retaining the 3D spa- tial information of the cell/tissue by using particular staining and photographic recording under microscopes, such as light, electron, stereoscopic, fluorescent and confocal microscope (Hassan and El-Awadi 2013). The histochemical detection is based on the simple principle that when a tissue or cell is exposed to a staining solution, it will react specifically by form- ing a colored insoluble end product detectable by microscopy (Ruifrok and Johnston 2001). Different microscopes can be used depending on the specific characteristic of the compound. For instance, light microscopy is used to image the birefringence of Plant Cell Physiol. doi:https://doi.org/10.1093/pcp/pcab022, available online at https://academic.oup.com/pcp © The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com Review 62(10): 1509–1527 Advance Access publication on 1 2021, 7 February Downloaded from https://academic.oup.com/pcp/article/62/10/1509/6140781 by guest on 11 February 2023