Summary

Analisi cinematica di divisione cellulare e di espansione: Quantificare la base cellulare della crescita e dello sviluppo di campionamento zone in<em> Zea mays</em> Foglie

Published: December 02, 2016
doi:

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

analisi della crescita dipende da una serie di strumenti che vengono comunemente utilizzati dagli scienziati vegetali per descrivere genotipo determinato differenze di crescita e / o risposte fenotipiche a fattori ambientali. Essi comprendono dimensione e peso misurazioni di tutta la pianta o di un organo e calcoli dei tassi di crescita per esplorare i meccanismi alla base della crescita. Crescita degli organi è determinata dalla divisione cellulare e l'espansione a livello cellulare. Pertanto, compresa la quantificazione di questi due processi in crescita analizza è la chiave per comprendere le differenze nella crescita dell'intero organo-1. Pertanto, è fondamentale disporre di un metodo adeguato per determinare i parametri di crescita cellulare che è relativamente facile da usare da laboratori non specializzati.

Analisi cinematica è già stato stabilito come un approccio che fornisce un quadro potente per lo sviluppo di modelli di crescita di organi 2. La tecnica è stata ottimizzata per sistemi lineari,come radici Arabidopsis thaliana e foglie monocotiledoni, ma anche per i sistemi non lineari, come foglie dicotiledoni 3. Al giorno d'oggi, questa metodologia è sempre più utilizzato per studiare come genetici, ormonali, dello sviluppo e fattori ambientali influenzano la divisione cellulare e di espansione in vari organi (Tabella 1). Inoltre, fornisce anche un quadro di collegare processi cellulari ai loro regolamenti biochimici, molecolari e fisiologici sottostanti (Tabella 2), anche se le limitazioni possono essere imposte in base alle dimensioni degli organi e l'organizzazione spaziale per tecniche che richiedono una maggiore quantità di materiale vegetale (ad esempio, di metaboliti misurazioni, proteomica, etc.).

Foglie monocotyledonous, come il mais (Zea mays) foglie, rappresentano sistemi lineari in cui le cellule si spostano dalla base della foglia verso la punta, sequenzialmente passando attraverso il meristema e l'allungamento zona per raggiungere la maturitàzona. Questo lo rende un sistema modello ideale per studi quantitativi dei modelli spaziali di crescita del 4. Inoltre, foglie di mais hanno zone di forte crescita (meristema e zona di allungamento che abbracciano diversi centimetri 5) e forniscono possibilità per gli studi ad altri livelli organizzativi. Questo permette la ricerca dei (presunti) meccanismi regolatori che controllano la divisione cellulare e di espansione, quantificato da analisi cinematica attraverso una serie di tecniche molecolari, misurazioni fisiologiche, e approcci di biologia cellulare (Tabella 2).

Qui, forniamo un protocollo per l'esecuzione di una analisi cinematica in foglie monocot. In primo luogo, spieghiamo come condurre una corretta analisi sia di divisione cellulare e l'allungamento delle cellule in funzione della posizione lungo l'asse foglia e come calcolare i parametri cinematici. In secondo luogo, mostriamo anche come questo può essere utilizzato come base per il disegno di campionamento. Qui, discutiamo due casi: ad alta risoluzione di campionamento di und focalizzata campionamento, consentendo una migliore interpretazione dei dati e il risparmio di tempo / denaro, rispettivamente.

Tabella 1. Panoramica di cinematica analizza i metodi per la quantificazione della divisione cellulare e di espansione in vari organi.

organo riferimento
foglie monocotiledoni 16, 20, 21, 22
apici radicali 2, 23, 24, 25, 26, 27, 28, 29
foglie dicotiledoni 21, 30, 31
sparare meristema apicale 32

Tabella 1. Panoramica di cinematica analizza i metodi per la quantificazione della divisione cellulare e di espansione in vari organi.

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

Tabella 2. Collegamento tra processi cellulari quantificati dall'analisi cinematica per la loro regolamentazione a livello molecolare. I riferimenti ai vari studi che collegano la quantificazione dei processi cellulari ai risultati di saggi biochimici e molecolari di varie specie e organi. Endotransglucosylase Xyloglucan (XET), malondialdeide (MDA), chinasi ciclina-dipendenti (CDK). Clicca qui per vedere una versione più grande di questa tabella.

Protocol

NOTA: il seguente protocollo per l'analisi cinematica è valida solo per le foglie durante la crescita di stato stazionario. Questo implica un tasso foglio di allungamento stabile e modelli spaziali di largo cellulare e l'espansione in una foglia durante un periodo di diversi giorni 6. 1. crescita delle piante e misure di Leaf Allungamento Rate (LER) Scegli una foglia nella crescita di stato stazionario e uno stadio di sviluppo di interesse. NOTA…

Representative Results

Qui, si mostra un confronto tra le piante ben irrigate (controllo, contenuto idrico del suolo 54%, (SWC)) e le piante sottoposte a condizioni di stress siccità (siccità, 34% SWC) in termini di crescita delle foglie. Tutte le piante sono state coltivate in una camera di crescita in condizioni controllate (16 ore al giorno / notte 8 ore, 25 ° C / 18 ° C giorno / notte, 300-400 μEm -2 sec -1 fotosinteticamente radiazione attiva (PAR). Le condizioni di siccità son…

Discussion

Una completa analisi cinematica sulle foglie di mais consente la determinazione della base cellulare della crescita delle foglie e consente la progettazione di strategie di campionamento efficienti. Anche se il protocollo è relativamente semplice, una certa cautela è raccomandata nei seguenti passaggi critici: (1) E 'importante staccare le foglie più giovani chiusi (punto 2.3) senza danneggiare il meristema, dal momento che la determinazione lunghezza meristema (fase 3) richiede la completa meristem di essere pre…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Questo lavoro è stato sostenuto da una borsa di dottorato di ricerca presso l'Università di Anversa a VA; una borsa di dottorato di ricerca dal fiammingo Science Foundation (FWO, 11ZI916N) di KS; sovvenzioni per progetti dal FWO (G0D0514N); una borsa di attività di ricerca concertata (GOA) la ricerca, "Un Systems Biology Approach di Leaf morfogenesi" dal Consiglio di ricerca dell'Università di Anversa; e il Interuniversitario di attrazione Poli (IUAP VII / 29, MARS), "mais e Arabidopsis radice e sparare crescita" dal Science Policy Ufficio federale belga (BELSPO) per GTSB Han Asard, Bulelani L. Sizani e Hamada AbdElgawad tutti hanno contribuito al 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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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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