Results

Cell Collapse Occurred in Specific Locations in the Root Cortex

Aerenchyma formation was examined in primary seminal roots from seeds either presoaked or not in water for 24 h before germinating on wet paper towels. Roots were divided into different sections. The soaking treatment stimulated the rate of cavity formation in roots. Little porosity was observed toward the tip. Cell collapse occurred in the central cortical cells, and this collapse then expanded radially to include peripheral cells.

Rice root has synchronous and distinctively oriented cortical cell files. Furthermore, the number of cortical cells of rice is less than in species such as maize and Phragmites aus-tralis, which also show cortical cell death. In roots of 70 day old rice plants, the highly developed aerenchyma is present in the cortex. However, not all the cortical cells collapse; the outermost or the innermost cells remain intact. The remains of the collapsed cortical cells form "forks" or "spokes of a wheel". To determine where cells first collapsed, the cortical cells were numbered radially, 1 to 7 from the inside. Using young seminal roots (20 mm long, derived from unsoaked seeds), the position of the first cells which underwent lysis in the radial files was determined in 80 different cross sections. Cells at position five located near the center of the cortex showed the highest incidence of early lysis.

Observation of Cell Death Using Dye Probes

We analyzed the frequency and location of cells staining with neutral red, which reddens visibly at low pH. The oldest regions of the root contained the most red cells. The first cells to stain with neutral red (section II) were at position 5 in the cortex. Thus, acidification was apparent in the mid-region of the cortex.

It is assumed that Evans blue can only be taken up by cells where plasma membranes were damaged. Thus, tissues excised from section III were treated with 2.5% Evans blue. Cells staining with this dye appeared most frequently in the central cell cortex. Cells stained with Evans blue in older tissue extended radially away from the mid-region of the cortex. The cell positions that stained most readily with Evans blue were also those staining strongly with neutral red. Although there were differences in the numbers of cortical cells and patterns of root growth, similar results using neutral red and Evans blue were obtained with seminal roots of maize. Rice roots, however, have some advantages over maize because of distinctive cell positions allowing accurate observation of position-related cell death.

Cell to Cell Movement in Cortex

Once cell collapse has started, neighboring cells become stained with Evans blue, suggesting, perhaps, radial movement of a solute associated with cell death. To understand the cell to cell plasmodesmal connection in root cortical parenchyma, we injected Lucifer yellow (LY) and FITC conjugated dextran (F-dextran) of different molecular weights into root cortical cells. We used cortical tissues which had been treated with Evans blue for such microinjection experiments. In cells which did not stain with Evans blue, there was a distinctive molecular exclusion limit between 9.3-19.6 kDa in the cortical cells. Regardless of the root position, intercellular movement of 4.4 kDa F-dextran was also observed.

In every case tested, we saw no evidence of leakage of injected probes from unstained cells. We found that about 50% of the cells that stained with Evans blue showed leakage of injected substances, which may have been caused by the loss of membrane integrity. Amounts of molecules in neighboring cells declined compared with those in injected cells.

More cells (67%) showed radial transfer of F-dextran of 9.3 kDa than with 4.4 kDa (27%) in unstained cells. These results indicated that movement of 9.3 kDa molecules was predominantly in the radial direction, similar to the radial pattern of cell death.

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