Name: kinematic analysis of cell division and expansion: quantifying the cellular basis of growth and sampling developmental zones in zea mays leaves
Uploaded: 2016-12-02T11:00:00
Description: Watch this Scientific Journal Video about Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves at JoVE.com

This work was supported by a PhD fellowship from the University of Antwerp to V.A.; a PhD fellowship from the Flemish Science Foundation (FWO, 11ZI916N) to K.S.; project grants from the FWO (G0D0514N); a concerted research activity (GOA) research grant, &#34;A Systems Biology Approach of Leaf Morphogenesis&#34; from the research council of the University of Antwerp; and the Interuniversity Attraction Poles (IUAP VII/29, MARS), &#34;Maize and Arabidopsis Root and Shoot Growth&#34; from the Belgian Federal Science Policy Office (BELSPO) to G.T.S.B. Han Asard, Bulelani L. Sizani and Hamada AbdElgawad all contributed to the video.

NOTE: The following protocol for kinematic analysis is only valid for leaves during steady-state growth. This implies a stable leaf elongation rate and spatial patterns of cell length and expansion in a leaf during a period of several days6.
1. Plant Growth and Measurements of Leaf Elongation Rate (LER)
<ol>
	<li>Choose a leaf in steady-state growth and a developmental stage of interest. 
	NOTE: There is a difference between steady-state growth and repetitive growth,...

A full kinematic analysis on maize leaves enables the determination of the cellular basis of leaf growth and allows for the design of efficient sampling strategies. Although the protocol is relatively straightforward, some caution is recommended in the following critical steps: (1) It is important to detach the younger, enclosed leaves (step 2.3) without damaging the meristem, since meristem length determination (step 3) requires the complete meristem to be present. Some practice beforehand might be needed. (2) Meristema...

Growth analysis depends on a set of tools that are commonly used by plant scientists to describe genotype determined growth differences and/or phenotypic responses to environmental factors. They include size and weight measurements of the whole plant or an organ and calculations of growth rates to explore the underlying mechanisms of growth. Organ growth is determined by cell division and expansion at the cellular level. Therefore, including the quantification of these two processes in growth analyses is key to understanding differences in whole-organ growth1. Consequently, it is crucial to have an appropriate methodology to determine cellular growth parameters that is relatively easy to use by non-specialized laboratories.
Kinematic analysis has already been established as an approach providing a powerful framework for the development of organ growth models2. The technique has been optimized for linear systems, such as Arabidopsis thaliana roots and monocotyledonous leaves, but also for non-linear systems, such as dicotyledonous leaves3. Nowadays, this methodology is increasingly being used to study how genetic, hormonal, developmental, and environmental factors influence cell division and expansion in various organs (Table 1). Moreover, it also provides a framework to link cellular processes to their underlying biochemical, molecular, and physiological regulations (Table 2), although limitations can be imposed by organ size and spatial organization for techniques that require higher amounts of plant material (e.g., metabolite measurements, proteomics, etc.).
Monocotyledonous leaves, such as the maize (Zea mays) leaf, represent linear systems in which cells move from the base of the leaf towards the tip, sequentially passing through the meristem and elongation zone to reach the mature zone. This makes it an ideal model system for quantitative studies of the spatial patterns of growth4. Moreover, maize leaves have large growth zones (meristem and elongation zone spanning several centimeters5) and provide possibilities for studies at other organizational levels. This allows for the investigation of the (putative) regulatory mechanisms controlling cell division and expansion, quantified by kinematic analysis through a range of molecular techniques, physiological measurements, and cell biology approaches (Table 2).
Here, we provide a protocol for performing a kinematic analysis in monocot leaves. First, we explain how to conduct a proper analysis of both cell division and cell elongation as a function of position along the leaf axis and how to calculate kinematic parameters. Secondly, we also show how this can be used as a basis for sampling design. Here, we discuss two cases: high-resolution sampling and focused sampling, enabling improved data interpretation and the saving of time/money, respectively.
Table 1. Overview of kinematic analyses methods for quantification of cell division and expansion in various organs.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>organ</td>
			<td>reference</td>
		</tr>
		<tr>
			<td>monocotyledonous leaves</td>
			<td>16, 20, 21, 22</td>
		</tr>
		<tr>
			<td>root tips</td>
			<td>2, 23, 24, 25, 26, 27, 28, 29</td>
		</tr>
		<tr>
			<td>dicotyledonous leaves</td>
			<td>21, 30, 31</td>
		</tr>
		<tr>
			<td>shoot apical meristem</td>
			<td>32</td>
		</tr>
	</tbody>
</table>
Table 1. Overview of kinematic analyses methods for quantification of cell division and expansion in various organs.
<img alt="Figure 3" src="/files/ftp_upload/54887/54887table2.jpg" />
Table 2. Link between cellular processes quantified by the kinematic analysis to their regulation at the molecular level. References to various studies linking the quantification of cellular processes to results from biochemical and molecular assays in various species and organs. Xyloglucan endotransglucosylase (XET), malondialdehyde (MDA), cyclin-dependent kinases (CDK). <a href="http://cloudflare.jove.com/files/ftp_upload/54887/54887table2large.jpg" target="_blank">Please click here to view a larger version of this table.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1670">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Pots</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">We use pots with the following measueres, but can be different depending on the treatment/study : bottom diameter: 11cm, opening diameter: 15 cm, height: 12 cm. We grow one maize plant per pot.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Planting substrate</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td>We use potting medium (Jiffy, The Netherlands), but other substrates can be used, depending on treatment/study.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ruler</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td>An extension ruler that covers at least 1,5 meters is needed to measure the final leaf length of the plants.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Seeds&#160;</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">NA</td>
			<td>Seeds can be ordered from a breeder.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Scalpel</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">The scalpel is used during leaf harvesting to detach the leaf of interest from its surrounding leaves and right after harvesting to cut a proper sample for cell length and meristem length measurements.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">15 ml falcon tubes</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">The 15 ml falcon tubes are used for storing samples used for cell length measurements during sample clearing with absolute ethanol and lactic acid.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Eppendorf tubes</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">The eppendorf tubes are used for storing samples used for meristem length measurements in ethanol:acetic acid 3:1 (v:v) solution.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Gloves</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td>Latex gloves, which protect against corrosive reagents.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Acetic acid</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">CAUTION: Corrosive to metals, category 1 Skin corrosion, categories 1A,1B,1C Serious eye damage, category 1; Flammable liquids, categories 1,2,3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Absolute ethanol</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">CAUTION: Hazardous in case of skin contact (irritant), of eye contact (irritant), of inhalation. Slightly hazardous in case of skin contact (permeator), of ingestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Lactic acid &#62;98%</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td>CAUTION: Corrosive to metals, category 1 Skin corrosion, categories 1A,1B,1C Serious eye damage, category 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium chloride (NaCl)</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">CAUTION: Acute toxicity (oral, dermal, inhalation), category 4 Skin irritation, category 2 Eye irritation, category 2 Skin sensitisation, category 1 Specific Target Organ Toxicity &#8211; Single exposure, category 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">This material can be an irritant, contact with eyes and skin should be avoided. Inhalation of dust may be irritating to the respiratory tract.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">4&#8242;,6-Diamidine-2&#8242;-phenylindole dihydrochloride (DAPI)</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">Cell permeable fluorescent minor groove-binding probe for DNA. Causes skin irritation. May cause an allergic skin reaction. May cause respiratory irritation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ice</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">NA</td>
			<td>The DAPI solution has to be kept on ice.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fluorescent microscope</td>
			<td>AxioScope A1, Axiocam ICm1 from Zeiss or other</td>
			<td style="width:209px;"></td>
			<td>Any fluorescent microscope can be used for determining meristem length.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microscopic slide</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cover glass</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Tweezers</td>
			<td style="width:333px;">Any</td>
			<td style="width:209px;">Any</td>
			<td style="width:813px;">Tweezers are needed for unfolding the rolled maize leaf right after harvesting in order to cut a proper sample for cell length and meristem length measurements.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Image-analysis software</td>
			<td>Axiovision (Release 4.8) from Zeiss</td>
			<td style="width:209px;">NA</td>
			<td style="width:813px;">The software can be downloaded at: http://www.zeiss.com/microscopy/en_de/downloads/axiovision.html. Other softwares such as ImageJ (https://imagej.nih.gov/ij/) could be used as well.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microscope equipped with DIC</td>
			<td>AxioScope A1, Axiocam ICm1 from Zeiss or other</td>
			<td style="width:209px;"></td>
			<td>Any&#160; microscope, equipped with differential interference contrast (DIC) can be used to measure cell lengths.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">R statistical analysis software</td>
			<td>R Foundation for Statistical Computing</td>
			<td style="width:209px;">NA</td>
			<td>Open source; Could be downloaded at https://www.r-project.org/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:315px;">R script</td>
			<td style="width:333px;">NA</td>
			<td style="width:209px;">NA</td>
			<td style="width:813px;">We use the kernel smoothing function locpoly of the Kern Smooth package (Wand MP, Jones MC.&#160; Kernel Smoothing: Chapman &#38; Hall/CRC (1995)). The script is available for Mac and Windows upon inquire with the corresponding author. We have versions for Mac and Windows.</td>
		</tr>
	</tbody>
</table>

kinematic analysis of cell division and expansion: quantifying the cellular basis of growth and sampling developmental zones in zea mays leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

Here, we show a comparison between well-watered plants (control, 54% soil water content, (SWC)) and plants subjected to drought stress conditions (drought, 34% SWC) in terms of their leaf growth. All plants were grown in a growth chamber under controlled conditions (16 hr day/8 hr night, 25 &#176;C/18 &#176;C day/night, 300-400 &#181;Em-2sec-1 photosynthetically active radiation (PAR). The drought conditions were established by withholding water until the correct SWC...

Watch this Scientific Journal Video about Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves at JoVE.com

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

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.

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