Dataset Information

Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies.

ABSTRACT:

Background

Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue.

Results

Here, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima). In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence) or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence) are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days.

Conclusions

The simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.

SUBMITTER: Pitzschke A

PROVIDER: S-EPMC3478982 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The family Brassicaceae is a source of important crop species, including Brassica napus (oilseed rape), Brassica oleracea, and B. rapa, that is used globally for oil production or as a food source (e.g., pak choi or turnip). However, despite advances in recent years, including genome sequencing, a lack of established tools tailored to the study of Brassica crop species has impeded efforts to understand their molecular processes in greater detail. Here, we describe the use of a simple Agrobacterium-mediated transient expression system adapted to B. rapa and B. napus that could facilitate study of molecular and biochemical events in these species. We also demonstrate the use of this method to characterize the N-degron pathway of protein degradation in B. rapa. The N-degron pathway is a subset of the ubiquitin-proteasome system and represents a mechanism through which proteins may be targeted for degradation based on the identity of their N-terminal amino acid residue. Interestingly, N-degron-mediated processes in plants have been implicated in the regulation of traits with potential agronomic importance, including the responses to pathogens and to abiotic stresses such as flooding tolerance. The stability of transiently expressed N-degron reporter proteins in B. rapa indicates that its N-degron pathway is highly conserved with that of Arabidopsis thaliana. These findings highlight the utility of Agrobacterium-mediated transient expression in B. rapa and B. napus and establish a framework to investigate the N-degron pathway and its roles in regulating agronomical traits in these species. Significance statement:We describe an Agrobacterium-mediated transient expression system applicable to Brassica crops and demonstrate its utility by identifying the destabilizing residues of the N-degron pathway in B. rapa. As the N-degron pathway functions as an integrator of environmental signals, this study could facilitate efforts to improve the robustness of Brassica crops.