The overall goals of this analysis, are to determine the cellular basis of maize leaf growth, and to use these data to investigate molecular growth regulatory mechanisms by performing developmental, stage specific sampling strategies. This method can help to address a key question in plant developmental biology. How do cell division and cell expansion contribute to differences in whole organ growth?
This approach allows the investigation and spacial mapping of biochemical molecular and physiological processes, underlying the growth of the whole plant organ. Though this system can provide insight into maize leaf growth, the technique can be used for other systems. Including other grass species, and organs.
Visual demonstration of these methods is critical. At the harvesting, sample bi-blation and measurementive steps are difficult to perform without that demonstration. Demonstrating the procedure, will be Katrien Sprangers, and Viktoriya Avramova.
Two PHD students from my laboratory. To perform a full chenematic analysis of leaf growth in monocots, grow at least 15 plants for each treatment and genotype, under controlled conditions in a growth room. When a leaf of interest appears, use a ruler to measure the length of the leaf, that is the length from the soil to the tip of the leaf, daily until the leaf is fully expanded.
At the developmental stage of interest, cut the above ground part of the plant from at least five representative plants as close to the roots as possible, to keep the meristematic region intact. Then, starting from the outer leaves, gently unroll all of the leaves up to the leaf of interest, one by one. Removing a few extra millimeters from the base to detach the leaves as necessary.
Next, remove the apex and the small leaves enclosed by the leaf of interest, and cut a three centimeter segment from the leaf, starting from the base on one side of the mid vein. It is crucial to cut the leaf right where it is connected to the stem. As cutting to high, would lead to an under estimation of the size of the meristem.
Store the segment in a 1.5 milliliter test tube, filled with three two one volume by volume absolute ethanol acetic acid solution, at four degrees Celsius for 24 hours up to several months. From the other side of the vein, cut an 11 centimeter segment from the base, and store this segment in a 15 milliliter tube filled with absolute ethanol, at four degrees Celsius for at least six hours to remove the pigments. After a second 24 hour round in absolute ethanol at four degrees Celsius, replace the ethanol with pure lactic acid, and store the segment at four degrees Celsius for another 24 hours, or until further analysis.
To measure the meristem, first transfer the three centimeter leaf segment into freshly prepared rinsing buffer for 20 minutes. At the end of the incubation, transfer the segment into a container of Dapi staining solution, for two to five minutes on ice in the dark. Then, quickly mount the tissue onto a glass microscope slide, and cover the segment with a cover glass.
Confirm the fluoresces of the epidermal cells under a microscope, using UV fluorescence. Then, mount the sample in a drop of rinsing buffer on a new microscope slide with a new glass cover. Using a 20 times magnification, return the segment to the microscope, and scroll through the sample to locate proliferative mitotic figures.
Avoid any formative cell division of the developing stemmata, and then define where the most distal mitotic figure is located. Using and image analysis software program, measure the full length of the image frame. In this example, 645 micrometer.
Then count the number of frames that cover the full meristem length from the most distal mitotic figure to the leaf base, and multiply this number by the length of one frame to obtain the full meristem length. To obtain a cell length profile, transfer the 11 centimeter segment from the lactic acid, to the bench, and use a scalpel to cut the tissue into 10 one centimeter segments. Mount the resulting leaf segments onto a single microscope slide, and a small drop of lactic acid.
Placing all of the pieces with the same side up. And then, transfer the slide under a microscope equipped with differential interference contrast optics. Beginning with the segment nearest to the leaf base, use an appropriate image analysis software program to measure the length of at least 20 replicate epidermal cells and files directly adjacent to the stemmatal files, to ensure the consistent selection of the same cell type at equally spaced positions along each of the segments.
Take are to wright down the corresponding position for each measurement throughout the leaf. Then determine the average cell length at each millimeter along the leaf axis using a local polynomial smoothing procedure implemented in R script that is available as supplementary file one. Be careful not to over smooth.
The smoothing should remove the noise in the celling data, but not affect the overall shape of the curve. Here, a comparison between well watered plant, and plants subjected to drought stress conditions in terms of their leaf growth are show. The final leaf length of the control plants, was 40 percent lower than that of the drought stressed plants, due to a lower leaf elongation rate.
The leaf elongation rate of the drought stressed plants, is 73 percent lower than that of well watered controls, due to a reduced cell production. Which in tern, is mainly caused by a reduced cell division rate. Kinematic analysis provides a map of the leaf growth zone localization, allowing sampling with a subzonal resolution for molecular and biochemical analysis.
For example, MDA quantification along the growth zone of the maize leaf in plant subjected to well watered control and drought conditions, demonstrates a significant increase in this product of lipid breakdown throughout the growth zone in response to water withholding conditions. Due to leaf shortening in response to the drought, developmental zones did not correspond in the control and in the stressed plants in this experiment. Using the cell length profile, however, the length of the developmental zones can be determined, allowing the MDA levels in the individual zones to be compared.
Once mastered, a single sample can be processed in four hours, if the technique is performed properly. While attempting this procedure, it is important to remember to keep the meristem intact during it's harvesting. Following this procedure, other techniques can be applied, such as the determination of molecular and biochemical parameters, to study their involvement in growth regulation.
After watching this video, you should have a good understanding of how to quantify cell division and cell expansion in growing maize leaf, determine the size of the different growth zones, and interpret molecular and biochemical data accordingly. Don't forget that working with lactic and acetic acids, and EDTA can be hazardous. Therefore, precautions such as wearing gloves, and cleaning the microscope each stage after use, should always be taken when performing this procedure.
