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

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 ce....

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)

1. 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 groei, die soortgelijke ruimtelijke patronen houdt op achtereenvolgende bladeren op dezelfde as. Tijdens de vroege stadia van zaailingsgroei opeenvolgend....

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 jui.......

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 zijn........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pots	Any	Any	We use pots with the following measures, but can be different depending on the treatment/study: bottom diameter: 11 cm, 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 inquiry with the corresponding author.