RNA-seq data analysis of shoot apical meristem cell types revealed Kip related protein 3 (KRP3) as one of the early marker for endoreduplication in differentiated cells Monika Mahajan* and Ram Kishor Yadav
Regional Research Station, PAU, Bathinda, Punjab, India
IISER, Mohali, Punjab, India
ABSTRACT The shoot apical meristem (SAM) of higher plants is organised into cell layers (L1–L3) with stem cells mainly reside in the central zone, differentiating cells in the peripheral zone and rib zone forms the stem cell niche that helps in maintaining the stem cell number in the central zone. Cells in the meristem remain in the dedifferentiated state and begin to differentiate in response to the underlying cell signals and led to the formation of differentiated tissues. Various cell cycle regulators (cyclins and CDKs) are responsible for maintaining the meristematic state of stem cells and its differentiation into peripheral cells. These regulators are expressed either in the mitotically activated cells, endoreduplicated cells or in both. In order to have a better insight of SAM cell cycle regulation, the celltype specific fluorescent reporter lines that marks different layers of SAM were generated. The cells from different layers were sorted separately by Florescent Activated Cell Sorter (FACS, BD Aria Fusion) and were used for ploidy and RNAseq analysis. Inspite of diploid nature of shoot, the differences in cell cycle phases was observed amongst the different cell types of SAM. And that difference is very well correlated with the differential expression of cell cycle regulatory transcripts in SAM cell types observed through whole transcriptomic analysis. The significant differential expression of cell cycle inhibitor kip related protein (KRP3) was observed in transient differentiating cells whose overexpression led to endoreduplicated shoot, sepals, petals. The above study has concluded that lack of KRP3 (cell cycle inhibitor) expression in stem cell region might be responsible for stem cells meristematic nature.
METHODOLOGY & RESULTS
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Fig. 1: Shoot apical meristem (SAM) cell types specific promoter reporters. 3D view of SAM labelled with FM4-64 (red) pCLV3::mGFP-ER (A; stem cells); pHMG::H2B-YFP (B; L1 layer); pHDG4::H2B-YFP (C; L2 layer), pFIL::ds-Red (D; differentiated cells), pWUS::mCherry (E; stem cell niche),
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pHMG::H 2B-YFP G RNA seq
Fig 4: Cell cycle regulated transcription factors profiling generated through RNA seq analysis. Heatmap showing the expression pattern of cell cycle regulated genes in CLV (stem cells) vs A
GFP Expressing cells
Confocal image of pHMG::H2BYFP
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Fig. 2: Sorting of florescent labelled cell type specific promoter reporters using Florescent Activated Cell Sorter (FACS; A-E), confocal image of sorted cells (F) which is further used ForRNA seq analysis Control E
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pKRP3::H2B-YFP
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Fig5:pKRP3::H2B-YFP expression in shoot apical meristem. The expression is clearly observed in differentiated cells with no expression in stem cells
Fig. 3: Cell cycle analysis of SAM cell types. pCLV3::mGFP-ER (A); pHMG::H2B-YFP (B); pHDG4::H2B-YFP (C), pFIL::ds-Red (D), pWUS::mCherry (E). The histogram showing the percentage of cells in G0/G1, S and G2/M phase of cell cycle (G)
35S::KRP3 G I
Fig 6: Phenotype of KRP3 overexpressing transgenic lines.OE line is showing the small flowers with wrinkled petals and sepals (A,B).KRP3 OE shoot and control stained with propidium iodide (100 µg/ml) showing the difference in shoot size (C-F). DNA ploidy in sepals and petals of control and OE lines (G). (G). KRP3 OE line showed endoreduplicated petals and sepals with higher ploidies