Flowering plants are all around us and use pollen to reproduce. Pollen, from the male part of a plant (stamen) is transported in a variety of ways to the sticky surface (stigma) of the female part of a plant (pistil). In many species of flowering plants, pollen granules arrive at the stigma dehydrated and dormant. When pollen sticks to the stigma, it sucks up water to rehydrate and produces a long tube for reproduction. The pollen tube pushes through the stigma, reaching inside the pistil to fertilize an ovule and form a seed. Of course, the process is a little more complicated than this. As the pollen rehydrates, water rushes in and swells the granule, causing an imbalance in the ratio of ions and water found inside and outside the granule (osmotic stress). To successfully produce a seed, pollen must have a mechanism to detect and reduce this stress.
Dr. Elizabeth Haswell, Associate Professor in the Department of Biology at Washington University in St. Louis, and members of the Haswell lab, including graduate student Eric Hamilton and undergraduate Andrew Katims, turned to bacteria for insight about this mechanism and recently discovered how pollen handles the stress. E. coli bacteria use a mechanosensitive channel of small conductance (MscS) to create a connection between the interior of the cell and the outside world through the cell membrane by sensing and responding to physical or mechanical forces. During times of cell swelling brought on by external environmental stressors like rain, the MscS channel senses osmotic stress, opens, and allows ions to exit. As ions flow out through the channel, water also flows out of the cell through other mechanisms. Bacteria, fungi, archaea, and plants are all known to have MscS-like (MSL) proteins, but in most cases their physiological roles are unknown.
Dr. Haswell noted, “We recently discovered a related, but distinct, role for MSL proteins in pollen.” Instead of using mechanosensitive channels to detect stress brought on by changes in the environment, the Haswell team discovered that plants use these channels to sense and reduce osmotic stress during reproduction.
As described in an article published in Science, the Haswell lab used various techniques, including in vivo confocal imaging of fluorescent MscS-like 8 (MSL8) protein fusions, to show that MSL8 is located in the membrane of pollen. Next, studies were conducted to determine if MSL8 could produce a mechanosensitive ion channel. The researchers also showed that MSL8 protein produced in Xenopus laevis (African clawed frog) oocytes generated currents like those in a small-conductance mechanosensitive ion channel (MscS).
To determine if the MSL8 protein functions as a mechanosensitive ion channel during rehydration of pollen granules, the Haswell lab isolated two mutants of the MSL8 protein (msl8-1 and msl8-4). The msl8-1 mutant produced reduced levels of MSL8 transcripts and the msl8-4 mutant generated no detectable levels of MSL8 transcripts. The researchers rehydrated pollens containing MSL8 and pollens containing the mutants in distilled water for two hours and looked for an effect on the viability of pollen. Pollen containing natural levels of MSL8 transcripts was 83-95% viable, but for pollen with a reduced level of transcripts (msl8-1) viability diminished to 46% and for pollen lacking detectable transcripts (msl8-4) viability was only 21-38%. Thus, the Haswell lab concluded MSL8 protein is a mechanosensitive channel important for the survivability of pollen during rehydration.
MSL8 plays another important role in germination. In vitro germination assays showed pollen containing the msl8-4 mutant form of MSL8 burst 26% of the time, compared to only a 3% bursting rate in natural pollen with normal MSL8 protein. Bursting in msl8 mutant pollen may be due to the inability to modulate osmotic stress during pollen tube production. Also, overexpression of fluorescent MSL8-YFP protein in pollen inhibited the rate of germination to only 4-39% of natural pollen. As Dr. Haswell noted, “MSL8 negatively regulates pollen germination, but is required for cellular integrity during germination and tube growth.”
This study shows the important role of mechanosensitive ion channels in plants and the delicate balance achieved by MSL8 channels during pollen rehydration and germination. In addition, Dr. Haswell and her students have provided evidence of a homolog of the prokaryotic E. coli MscS channel that suits the needs of a significantly more complex organism. Dr. Haswell noted “Pollen development is a stage of plant development that is highly sensitive to environmental stress, and a better understanding of how pollen grains handle water stress may help mitigate the effects of climate change.”
In an effort to share their work with broader audiences, the Haswell lab has created a YouTube whiteboard animation depicting their discovery of the MSL8 mechanosensitive channel and its regulation of osmotic forces during pollen rehydration and tube creation.
This work is partially funded by the Division of Molecular and Cellular Biosciences, Award #MCB-1253103.