Organelle Visualization With Multicolored Fluorescent Markers in Bamboo

Abstract

Bamboo is an important model plant to study the molecular mechanisms of rapid shoot growth and flowering once in a lifetime. However, bamboo research about protein functional characterization is largely lagged behind, mainly due to the lack of gene transformation platforms. In this study, a protoplast transient gene expression system in moso bamboo has been first established. Using this reliable and efficient system, we have generated a set of multicolored fluorescent markers based on the targeting sequences from endogenous proteins, which have been validated by their comparative localization with Arabidopsis organelle markers, in a combination with pharmaceutical treatments. Moreover, we further demonstrated the power of this multicolor marker set for rapid, combinatorial analysis of the subcellular localization of uncharacterized proteins, which may play potential functions in moso bamboo flowering and fast growth of shoots. Finally, this protoplast transient gene expression system has been elucidated for functional analysis in protein–protein interaction by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. Taken together, in combination with the set of moso bamboo organelle markers, the protoplast transient gene expression system could be used for subcellular localization and functional study of unknown proteins in bamboo and will definitely promote rapid progress in diverse areas of research in bamboo plants.

Tracking organelle activities through efficient and stable root genetic transformation system in woody plants

“…Currently, investigation of subcellular protein localization in most crops primarily relies on heterologous transient expression systems, such as onion epidermal cells, Arabidopsis leaf protoplasts, rice leaf protoplasts, or tobacco leaves. However, due to the potentially limited conservation in protein sequences and regulatory factors governing protein trafficking within these heterologous expression systems, there is a risk of engendering spurious subcellular localization results in such approaches [ 27 – 29 ]. In this study, we infiltrated various organelle markers labeled with GFP or RFP, including those for ER ( Fig.…”

Identification and Analysis of Expression Patterns of the Caleosin Genes in Hickory (Carya cathayensis Sarg.)

“…However, as Arabidopsis is not an oil-producing plant, further research is needed to investigate the network mechanism of CcCLO1 in lipid accumulation, specifically in Chinese hickory or pecan. Additionally, ectopic expression of proteins in different plant species may result in mis-localization [44], necessitating enhancements to the transient transformation system for hickory. Currently, we are employing the procedures outlined in our previous study on bamboo transient transformation for the manipulation of hickory callus.…”

Protoplast: A Valuable Toolbox to Investigate Plant Stress Perception and Response

“…Besides the functional role of proteins in gene regulation, TEA can also provide information on their subcellular locations and dynamics (i.e., their mobility) into the cell when protoplasts are expressing both the studied protein fused with a fluorescent one, such as YFP, GFP, CFP, or mCherry, and a fluorescent marker specific of a cellular compartment. To that end, several markers have been developed to mark specifically plant organelles ( Nelson et al, 2007 ; Zhang et al, 2021 ), and their diversity for the different organelles has been recently reviewed ( Zhu et al, 2020 ). These information may help to elucidate protein function ( Nelson et al, 2007 ), since TFs are expected to be found in the nucleus ( Asai et al, 2002 ; Sheikh et al, 2016 ; Moon et al, 2019 ), protein receptors in the PM ( Li et al, 2017 ; Liu et al, 2017 ; Pham et al, 2020 ), and proteins with a more versatile function can be found both in the cytosol and in cellular organelles ( Boudsocq et al, 2010 ).…”