10–12 Furthermore, multiple proteins participating in the same event may have distinct vulnerabilities to protein degradation over the duration of an experiment. 9 Individual mRNA molecules can correspond to numerous proteins that interact each other and might not behave similarly in a single plant cell. 8 It is unclear whether different gene expression cassettes produce similar levels of mRNA molecules in the same plant tissue. This fine-tuning is necessary because the regulation of gene expression is strictly controlled at both the transcription and translation steps. Rational fine-tuning of multiple transgene expression approaches in plants is critical but is one of the drawbacks of such biomolecule delivery methods.

3,7 However, many issues prevent extensive use of this technique, such as an inability to deliver multiple exogenous gene expression cassettes into plant cells, unequal expression of multiple transgenes, delays in interacting protein function, and differences in the stability of the recombinant proteins prior to execution of their related functions. 6 Protein–protein interactions can be subsequently elucidated by observing the fluorescence signals from the restructured fluorescent proteins under a fluorescence microscope a few days after transformation. 4,5 Expression cassettes of the two fusion proteins can be transiently introduced into plant cells through conventional plant transformation. 1–4 In protein–protein interaction studies using BiFC, two proteins thought to potentially interact with each other are fused to two nonfluorescent fragments of a fluorescent protein. The bimolecular fluorescence complementation (BiFC) assay is one powerful tool for visualizing interactions between two proteins during a biological process. How these interacting biomolecules participate in specific biological reactions is intriguing, because protein interactions are highly complex and dynamic. Introduction Plant cells create functional proteins that synergistically interact with each other to function in particular metabolic processes and in cellular mechanism.

We present a cutting-edge technique for codelivery of multiple biomolecules into plant cells that can be used for elucidation of functional correlations and for metabolic engineering. We demonstrate that multiple biomolecule/CPP cargos can be simultaneously internalized by a particular plant cell, albeit with different efficiencies. Here, we employed the engineered CPP KH 9-BP100 as a carrier to deliver multiple biomolecules into plant cells and performed a bimolecular fluorescence complementation assay to assess the simultaneous introduction of multiple biomolecules. Cell-penetrating peptides (CPPs) have demonstrated remarkable abilities to introduce diverse biomolecules into various plant species. However, the currently existing biomolecule delivery methods face difficulties in delivering multiple components into plant cells, mediating transgene expression, and maintaining the stability of the numerous components and lead to delays in biomolecular function. Exogenous codelivery of multiple biomolecules is an essential step for elucidation of the biological significance of these molecules and enables various biotechnological applications in plants. Plant cells contain groups of biomolecules that participate together in a particular biological process.

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