MorphoGraphX Cell Size and Nuclear Volume Measurement

For diploid and tetraploid plants, sepals expressing pAR169 (ML1:mCitrine-RCI2A) and pAR229 (ML1:H2B-TFP) were imaged using a 20× water objective (NA = 1.0) on a Zeiss 710 confocal microscope. mCitrine was excited with a 514-nm laser, and emission was collected from 519 to 649 nm. TFP was excited with a 458-nm laser, and emission was collected from 463 to 509 nm. Three to nine tiles were used to capture whole sepals. Stacks were optimized to 1:1:1 X:Y:Z resolution. Channels were split in FIJI.

The TFP channel (marking nuclei) was processed in MorphoGraphX as follows: (1) brighten darken (2.0); (2) Gaussian blur (x,y,z sigma = 0.7); (3) binarize stack (threshold 10,000); (4) hand-correct stack: use Pixel Editor tool to remove noise and any misrepresented nuclei; (5) generate marching cubes 3D mesh (cube size = 1); (6) clear segmentation from mesh, seed each nucleus individually, and run watershed segmentation. These processes generate three-dimensional representations of individual nuclei.

The mCitrine channel (marking plasma membranes) was processed in MorphoGraphX as follows: (1) Gaussian blur 2× (x,y,z sigma = 1); (2) edge detect (threshold = 8000); (3) fill holes 1 to 3×; (4) generate marching cubes surface (cube size = 15); (5) delete adaxial (flat) face of mesh; (6) alternately smooth and subdivide mesh until at least 500,000 vertices exist and delete bad normals; (7) project plasma membrane signal onto surface (minimum distance 8, maximum distance 12; adjust for best signal); (8) smooth mesh signal (passes: 3); (9) run autosegmentation; (10) hand-correct original segmentation to match plasma membrane signal; overlay nuclear channel for additional positional information. These processes generate two-dimensional representations of cell area that take into account the sepal’s curvature. Cross sections of sepal epidermal cells were created using the MorphoGraphX function Clip2, and cross-sectional areas were measured in FIJI.

Nuclei and cells were manually paired using the Lineage Tracking tool. Nuclear volumes were generated using Heat Map (Heat Map type: volume; signal average; global coordinates). 2D cell areas were generated using Heat Map (Heat Map type: area; signal average; global coordinates). Nuclear and cell size measurements were paired using the Parents spreadsheet generated by Lineage Tracking.

Octoploid sepal images were processed in the same way, but nuclei were marked by pAR180 (ML1:H2B-mGFP) and cell walls were stained with propidium iodide. Samples were excited with a 488-nm laser. GFP emission was collected from 493 to 556 nm. PI emission was collected from 566 to 649 nm.

For crwn1 plants, PI was used to stain cell walls. TFP was excited with a 458-nm laser, and emission was collected from 463 to 523 nm. PI was excited with a 561-nm laser, and emission was collected from 566 to 649 nm.