Cell size: a key determinant of meristematic potential in plant protoplasts

Abstract

Metabolic pathway reconstruction and gene edits for native natural product synthesis in single plant cells are considered to be less complicated when compared to the production of non-native metabolites. Being an efficient eukaryotic system, plants encompass suitable post-translational modifications. However, slow cell division rate and heterogeneous nature is an impediment for consistent product retrieval from plant cells. Plant cell synchrony can be attained in cultures developed in vitro. Isolated plant protoplasts capable of division, can potentially enhance the unimpaired yield of target bioactives, similar to microbes and unicellular eukaryotes. Evidence from yeast experiments suggests that 'critical cell size' and division rates for enhancement machinery, primarily depend on culture conditions and nutrient availability. The cell size control mechanisms in Arabidopsis shoot apical meristem is analogous to yeast notably, fission yeast. If protoplasts isolated from plants are subjected to cell size studies and cell cycle progression in culture, it will answer the underlying molecular mechanisms such as, unicellular to multicellular transition states, longevity, senescence, 'cell-size resetting' during organogenesis, and adaptation to external cues.

Keywords: CRISPR; Cell cycle; Cell size; Plant protoplasts; TOR signalling.

Fig. 1

Transmuting plant protoplasts A Selection of a plant host. B Protoplast isolation from the selected host. C Yield, viability check and diversity in protoplast-derived plant cell sub-populations. Since the protoplast pool has a heterogeneous population, it is crucial to segregate them using fluorescent activated cell sorters. D Cell culture of the protoplast sub-populations to track their doubling time and division rates. Cell sizer experiments are carried out to track the growth phases based on nutrient dependency. Cell size regulators are identified to track the transition from each phase. E The cell regulators are orchestrated to induce multiple fission in protoplast population to bring in homogeneity in cell size and cell physiology. F The regulators are modulated to induce division at the scotophase, utilising the energy trapped during the light phase. Shortlisting the best sub-population of protoplast with fastest division rate and the TOR signaling mechanisms. G Biosynthetic gene clusters of desired products are identified in the shortlisted protoplast-derived plant cell sub-populations. The significant transcription regulators, tailoring and scaffolding enzymes are identified. H Biosynthetic cascade rewiring with CRISPR/Cas9 to eliminate genes that have negating effects. I Mathematical modelling studies to validate the gene edits and protoplast sizing. J Validating the biosynthetic pathway reconstructions using functional assays to improve product yield. K The proliferation of engineered protoplast lines. L Metabolite cartography using sensitive, robust analytical workflows and cross-validating the identification of the target bioactive. M Bioreactor scale-up of recombinant bio preparations. N Development of transgenics and transplastomics from engineered plant protoplast-derived cell-lines