Features of fluorescent protein application to study the root system development of cucurbits (Cucurbitaceae)

Modern studies of detailed processes in plant development would not be possible without using a wide range of fluorescent proteins (R. Day and M. Davidson, 2009; D. Chudakov et al., 2010). However, application of fluorescent proteins is restricted due to problems with their visualization in plant tissues. Plants are difficult objects for microscopic studies: even the most advanced methods have significant limitations regarding the depth of light penetration due to the scattering and absorption of light by cell walls. Therefore, to study the distribution of reporter fluorescent proteins in large organs typical for most plants, it is necessary to fix the plant material and prepare thick histological sections with a vibrating-blade microtome. Chemicals traditionally used for fixing, dehydrating and embedding of plant tissues samples lead to changes in the structure of fluorescent proteins and, as a result, often to the loss of their fluorescence. Therefore, it is important to optimize the protocols for fixing plant tissues, preparing sections and studying the distribution of fluorescent proteins by laser scanning confocal microscopy. In this work, we have proposed for the first time an integrated approach to fixation of tissues of transgenic plants and preparation of sections in the course of the study of patterns of cellular response to auxin and expression of transcription factors using laser scanning confocal microscopy. Our aim was to sum up modern approaches to the application of an effective universal technique for visualization of tissue and cellular patterns of the fluorescent reporter proteins distribution on sections of large non-model plants. The first step for using fluorescent proteins in plants is the generation of genetic constructs that carry the promoter of the gene of interest fused to a reporter gene encoding a fluorescent protein. In this case, a transformation protocol has to be available for the selected plant species. We have described the use of the Gateway® cloning technology for the construction of vectors for plant transformation that meet modern experimental requirements. In order to study auxin localization in vivo we developed a series of vectors with genes encoding various fluorescent proteins (eGFP, tdTomato, mRuby3) under the control of the auxin-sensitive DR5 promoter (E. Ilina et al., 2012). We now demonstrate the advantage of nuclear-targeted fluorescent proteins (mNeonGreen-H2B, tdTomato-H2B, mRuby3-H2B), as well as the possibility of their application for additional visualization of cell nuclei in combination with highly specific cell wall staining using SCRI Renaissance2200. An effective method for constructing vectors to study cell-specific expression patterns of developmental regulators is presented using the transcription factor genes GATA24 (A. Kiryushkin et al., 2019) and LBD16 in some Cucurbitaceae species as examples. We also applied an expression cassette, pAtUBQ10::DsRED1 (E. Limpens et al., 2004), carrying a gene encoding red fluorescent protein under the control of a constitutive promoter, to demonstrate the advantages of the use of fluorescent proteins in screening for transgenic vs. wild type roots. A new method of fixation and clearing of plant tissues containing reporter fluorescent proteins and preparation of sections is presented for the example of transgenic roots of Cucurbitaceae. The advantage of using melted agarose compared to embedding media for orienting plant parts during the preparation of samples was demonstrated. The increase of the photostability of fluorescent proteins in sections due to the use of clearing reagent ClearSee (D. Kurihara et al., 2015) as a mounting medium is demonstrated. Altogether, we present a combination of the advantages that are provided by application of a complex of several modern methodical approaches of sample preparation and laser scanning confocal microscopy that offers significant advantages for studying of the developmental biology of large non-model plants.