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Biologia

Kinematische analyse van de celdeling en Expansion: Het kwantificeren van de cellulaire basis van groei en Sampling Developmental Zones in<em> Zea Mays</em> Leaves

Published: December 02, 2016
doi:
10.3791/54887
, ,
1Department of Biology,University of Antwerp

Summary

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.

Abstract

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.

Introduction

Groei analyse is afhankelijk van een set van tools die vaak worden gebruikt door planten wetenschappers om genotype bepaald groei verschillen en / of fenotypische reacties op milieu-factoren te beschrijven. Ze beschikken over de grootte en het gewicht metingen van de hele plant of een orgaan en berekeningen van groeicijfers om de onderliggende mechanismen van de groei te verkennen. Orgaangroei wordt bepaald door celdeling en expansie op cellulair niveau. Daarmee ook de kwantificering van deze twee processen in groeianalyses is essentieel voor het begrijpen van verschillen in groei 1 whole-orgaan. Bijgevolg is het cruciaal om een ​​geschikte methodologie celgroei parameters die relatief eenvoudig te gebruiken door niet-gespecialiseerde laboratoria te bepalen.

Kinematische analyse reeds ingevoerd als een benadering die een krachtige kader voor de ontwikkeling van orgaangroei modellen 2. De techniek is geoptimaliseerd voor lineaire systemen,zoals Arabidopsis thaliana wortels en bladeren monocotyle, maar ook voor niet-lineaire systemen, zoals tweezaadlobbige bladeren 3. Tegenwoordig wordt deze methode steeds vaker gebruikt om te onderzoeken hoe genetische, hormonale, ontwikkelings- en milieufactoren invloed celdeling en groei in diverse organen (tabel 1). Bovendien biedt een raamwerk om cellulaire processen te verbinden met de onderliggende biochemische, moleculaire en fysiologische voorschriften (tabel 2), hoewel beperkingen door orgaangrootte en ruimtelijke organisatie kan worden opgelegd technieken die grotere hoeveelheden plantenmateriaal vereisen (bijv metaboliet metingen, proteomics, etc.).

Eenzaadlobbige bladeren, zoals maïs (Zea mays) bladeren vertegenwoordigen lineaire systemen waarbij cellen zich van de basis van het blad naar de punt, achtereenvolgens door het meristeem en elongatiezone de rijpe bereikenzone. Dit maakt het een ideaal modelsysteem voor kwantitatieve studies van de ruimtelijke patronen van de groei 4. Bovendien, maïs bladeren hebben grote groei zones (meristeem en rek zone verspreid over enkele centimeters 5) en bieden mogelijkheden om studies bij andere organisatieniveaus. Dit maakt het mogelijk voor het onderzoek naar de (vermeende) regulerende mechanismen die celdeling en expansie, gekwantificeerd door kinematische analyse door middel van een reeks van moleculaire technieken, fysiologische metingen, en celbiologie benaderingen (tabel 2).

Hier bieden we een protocol voor het uitvoeren van een kinematische analyse in eenzaadlobbige bladeren. Eerst wordt uitgelegd hoe een goede analyse van zowel celdeling en elongatie voeren als functie van positie langs de vleugelas en hoe kinematische parameters berekenen. Ten tweede hebben wij laten ook zien hoe deze kan worden gebruikt als basis voor de steekproef. Hier bespreken we twee gevallen: high-resolution sampling eend gerichte steekproeven, waardoor betere interpretatie van gegevens en de besparing van tijd / geld, respectievelijk.

Tabel 1. Overzicht van kinematische analysemethoden voor kwantificeren van celdeling en groei in diverse organen.

orgaan referentie
eenzaadlobbige bladeren 16, 20, 21, 22
Root tips 2, 23, 24, 25, 26, 27, 28, 29
tweezaadlobbige bladeren 21, 30, 31
schieten topmeristeem 32

Tabel 1. Overzicht van kinematische analysemethoden voor kwantificeren van celdeling en groei in diverse organen.

<p class="jove_content" fo:keep-together.within-page = "1"> figuur 3

Tabel 2. Verband tussen cellulaire processen gekwantificeerd door de kinematische analyse om de regulering op moleculair niveau. Verwijzingen naar verscheidene studies die de kwantificering van cellulaire processen om resultaten van biochemische en moleculaire assays verschillende soorten en organen. Xyloglucan endotransglucosylase (XET), malondialdehyde (MDA), cycline-afhankelijke kinasen (CDK). Klik hier om een grotere versie van deze tabel te bekijken.

Protocol

LET OP: De volgende protocol voor kinematische analyse is alleen geldig voor bladeren tijdens de groei van steady-state. Dit impliceert een stabiele blad reksnelheid en ruimtelijke patronen van cellengte en expansie in een blad gedurende enkele dagen 6. 1. Plant Groei en Metingen van Leaf Rek Rate (LER) Kies een blad in de groei steady-state en een ontwikkelingsstadium van belang. NB: Er is een verschil tussen de groei van steady-state en repetitieve gro…

Representative Results

Hier tonen we een vergelijking tussen goed bewaterd planten (controle, 54% bodem water inhoud, (SWC)) en planten blootgesteld aan droogtestress omstandigheden (droogte, 34% SWC) in termen van hun bladgroei. Alle planten werden gekweekt in een groeikamer onder gecontroleerde omstandigheden (16 uur dag / 8 uur nacht, 25 ° C / 18 ° C dag / nacht, 300-400 μEm -2 -1 sec fotosynthetisch actieve straling (PAR). De droogte werden opgericht door inhouding water totdat de …

Discussion

Een volledige analyse van kinematische maïsblaadjes maakt de bepaling van de cellulaire basis van bladgroei en maakt het ontwerp van efficiënte bemonsteringsstrategieën. Hoewel het protocol is relatief eenvoudig is enige voorzichtigheid bij de volgende belangrijke stappen: (1) Het is belangrijk dat de jongere, afgesloten bladeren (stap 2,3) los te maken zonder beschadiging van de meristeem, aangezien meristeem lengte bepalen (stap 3) vereist een totale meristeem aanwezig te zijn. Wat oefening op voorhand nodig zou zi…

Declarações

The authors have nothing to disclose.

Acknowledgements

Dit werk werd ondersteund door een PhD-beurs van de Universiteit van Antwerpen naar VA; een PhD beurs van de Vlaamse Science Foundation (FWO, 11ZI916N) naar KS; projectsubsidies van het FWO (G0D0514N); een gezamenlijke onderzoeksactiviteiten (GOA) onderzoek subsidie, "een systeembiologische benadering van bladmorfogenese" uit het onderzoek van de Raad van de Universiteit van Antwerpen; en de interuniversitaire attractiepolen (IUAP VII / 29, MARS), "maïs en Arabidopsis Root en Schiet groei" van het Federaal Wetenschapsbeleid (BELSPO) naar GTSB Han Asard, Bulelani L. Sizani en Hamada Abdelgawad allemaal bijgedragen tot de video .

Materials

Pots Any Any 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.
Planting substrate Any Any We use potting medium (Jiffy, The Netherlands), but other substrates can be used, depending on treatment/study.
Ruler Any Any An extension ruler that covers at least 1,5 meters is needed to measure the final leaf length of the plants.
Seeds  Any NA Seeds can be ordered from a breeder.
Scalpel Any Any 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. 
15 ml falcon tubes Any Any The 15 ml falcon tubes are used for storing samples used for cell length measurements during sample clearing with absolute ethanol and lactic acid.
Eppendorf tubes Any Any The eppendorf tubes are used for storing samples used for meristem length measurements in ethanol:acetic acid 3:1 (v:v) solution.
Gloves Any Any Latex gloves, which protect against corrosive reagents.
Acetic acid Any Any CAUTION: Corrosive to metals, category 1 Skin corrosion, categories 1A,1B,1C Serious eye damage, category 1; Flammable liquids, categories 1,2,3
Absolute ethanol Any Any CAUTION: Hazardous in case of skin contact (irritant), of eye contact (irritant), of inhalation. Slightly hazardous in case of skin contact (permeator), of ingestion
Lactic acid >98% Any Any CAUTION: Corrosive to metals, category 1 Skin corrosion, categories 1A,1B,1C Serious eye damage, category 1
Sodium chloride (NaCl) Any Any
Ethylenediaminetetraacetic acid (EDTA) Any Any CAUTION: Acute toxicity (oral, dermal, inhalation), category 4 Skin irritation, category 2 Eye irritation, category 2 Skin sensitisation, category 1 Specific Target Organ Toxicity – Single exposure, category 3
Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) Any Any This material can be an irritant, contact with eyes and skin should be avoided. Inhalation of dust may be irritating to the respiratory tract.
4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) Any Any Cell permeable fluorescent minor groove-binding probe for DNA. Causes skin irritation. May cause an allergic skin reaction. May cause respiratory irritation.
Ice Any NA The DAPI solution has to be kept on ice.
Fluorescent microscope AxioScope A1, Axiocam ICm1 from Zeiss or other Any fluorescent microscope can be used for determining meristem length.
Microscopic slide Any Any
Cover glass Any Any
Tweezers Any Any 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. 
Image-analysis software Axiovision (Release 4.8) from Zeiss NA 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.
Microscope equipped with DIC AxioScope A1, Axiocam ICm1 from Zeiss or other Any  microscope, equipped with differential interference contrast (DIC) can be used to measure cell lengths.
R statistical analysis software R Foundation for Statistical Computing NA Open source; Could be downloaded at https://www.r-project.org/
R script NA NA We use the kernel smoothing function locpoly of the Kern Smooth package (Wand MP, Jones MC.  Kernel Smoothing: Chapman & Hall/CRC (1995)). The script is available for Mac and Windows upon inquire with the corresponding author. We have versions for Mac and Windows.
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Tags

Keywords: Cell DivisionCell ExpansionLeaf GrowthKinematic AnalysisDevelopmental ZonesZea MaysMonocotsGrowth RegulationSamplingVisual Demonstration
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Sprangers, K., Avramova, V., Beemster, G. T. S. Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves. J. Vis. Exp. (118), e54887, doi:10.3791/54887 (2016).
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