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Biologia

Análisis cinemático de la división celular y la expansión: La cuantificación de la base celular del crecimiento y el desarrollo en las zonas de muestreo<em> Zea mays</em> Las hojas

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

Análisis del crecimiento depende de un conjunto de herramientas que se utilizan comúnmente por los científicos para describir la planta de genotipo determinado las diferencias de crecimiento y / o respuestas fenotípicas a factores ambientales. Incluyen mediciones de tamaño y peso de la planta entera o un órgano y los cálculos de las tasas de crecimiento para explorar los mecanismos subyacentes de crecimiento. el crecimiento de órganos se determina por la división celular y la expansión a nivel celular. Por lo tanto, incluyendo la cuantificación de estos dos procesos en el crecimiento de los análisis es clave para entender las diferencias en el crecimiento conjunto de órganos 1. En consecuencia, es fundamental contar con una metodología adecuada para determinar los parámetros de crecimiento celular que es relativamente fácil de usar por los laboratorios no especializados.

Análisis cinemático ya se ha establecido como un enfoque que proporciona un marco de gran alcance para el desarrollo de modelos de crecimiento de órganos 2. La técnica ha sido optimizado para sistemas lineales,como las raíces de Arabidopsis thaliana y las hojas monocotiledóneas, sino también para sistemas no lineales, tales como hojas dicotiledóneas 3. Hoy en día, esta metodología se utiliza cada vez para estudiar cómo genéticos, hormonales, de desarrollo, y los factores ambientales influyen en la división celular y la expansión en varios órganos (Tabla 1). Por otra parte, también proporciona un marco para vincular los procesos celulares a sus regulaciones bioquímicos, moleculares y fisiológicos subyacentes (tabla 2), aunque las limitaciones pueden ser impuestas por el tamaño del órgano y la organización espacial de las técnicas que requieren una mayor cantidad de material vegetal (por ejemplo, metabolitos mediciones, proteómica, etc.).

Hojas monocotiledóneas, tales como el maíz (Zea mays) hoja, representan sistemas lineales en los que las células se mueven desde la base de la hoja hacia la punta, pasando secuencialmente a través de la zona de meristemas y la elongación para llegar a la madurazona. Esto hace que sea un sistema modelo ideal para los estudios cuantitativos de los patrones espaciales de crecimiento del 4. Por otra parte, las hojas del maíz tienen grandes zonas de crecimiento (meristemas y zona de elongación que abarcan varios centímetros 5) y ofrezcan posibilidades para estudios en otros niveles de organización. Esto permite la investigación de las (supuestas) mecanismos reguladores que controlan la división celular y la expansión, cuantificado por análisis cinemático a través de una serie de técnicas moleculares, las mediciones fisiológicas, y métodos de biología celular (Tabla 2).

A continuación, ofrecemos un protocolo para la realización de un análisis cinemático en las hojas de monocotiledóneas. En primer lugar, explicamos cómo llevar a cabo un análisis adecuado de tanto la división celular y elongación celular como una función de la posición a lo largo del eje de la hoja y la forma de calcular los parámetros cinemáticos. En segundo lugar, también muestran cómo se puede utilizar como base para el diseño de muestreo. A continuación, se discuten dos casos: de alta resolución de un muestreod centró toma de muestras, lo que permite la interpretación de datos mejorada y el ahorro de tiempo / dinero, respectivamente.

Tabla 1. Resumen de los análisis cinemático métodos para la cuantificación de la división celular y la expansión en varios órganos.

Organo referencia
hojas de monocotiledóneas 16, 20, 21, 22
puntas de las raíces 2, 23, 24, 25, 26, 27, 28, 29
hojas dicotiledóneas 21, 30, 31
disparar meristemo apical 32

Tabla 1. Resumen de los análisis cinemático métodos para la cuantificación de la división celular y la expansión en varios órganos.

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

Tabla 2. Relación entre procesos celulares cuantificados por el análisis cinemático para su regulación a nivel molecular. Las referencias a diversos estudios que relacionan la cuantificación de los procesos celulares con los resultados de los ensayos bioquímicos y moleculares en distintas especies y órganos. Endotransglucosylase xiloglucano (XET), malondialdehído (MDA), quinasas dependientes de ciclina (CDK). Haga clic aquí para ver una versión más grande de esta tabla.

Protocol

NOTA: La siguiente protocolo para el análisis cinemático sólo es válida para las hojas durante el crecimiento de estado estacionario. Esto implica una velocidad de alargamiento de la hoja estable y los patrones espaciales de longitud de la célula y la expansión en una hoja durante un período de varios días 6. 1. Crecimiento de las Plantas y mediciones de la hoja Elongación Rate (LER) Elija una hoja en el crecimiento de estado estacionario y una etapa d…

Representative Results

Aquí, se muestra una comparación entre las plantas bien regadas (control, el contenido de agua del suelo 54%, (SWC)) y plantas sometidas a condiciones de estrés hídrico (sequía, el 34% de CSA) en términos de su crecimiento de las hojas. Todas las plantas se cultivaron en una cámara de crecimiento bajo condiciones controladas (16 horas día / 8 horas por la noche, 25 ° C / 18 ° C día / noche, 300-400 μEm -2 s -1 de radiación fotosintéticamente activa (PA…

Discussion

Un análisis cinemático completo en las hojas de maíz permite la determinación de la base celular de crecimiento de la hoja y permite el diseño de estrategias de muestreo eficientes. Aunque el protocolo es relativamente sencillo, algunos se recomienda precaución en los siguientes pasos críticos: (1) Es importante separar las hojas más jóvenes cerrados (paso 2.3) sin dañar el meristemo, ya determinación de la longitud de meristemas (paso 3) requiere la completa meristemo de estar presente. Un poco de práctica …

Declarações

The authors have nothing to disclose.

Acknowledgements

Este trabajo fue apoyado por una beca de doctorado de la Universidad de Amberes a VA; una beca de doctorado de la Fundación de Ciencias de Flandes (FWO, 11ZI916N) de KS; subvenciones para proyectos de la FWO (G0D0514N); una beca de investigación actividad de investigación concertada (GOA), "Un enfoque de Biología de Sistemas de la hoja de la morfogénesis" del Consejo de Investigación de la Universidad de Amberes; y la atracción Interuniversitario polacos (IUAP VII / 29, MARS), "El maíz y Arabidopsis raíces y brotes de crecimiento" de la Oficina Federal de Bélgica Ciencia Política (BELSPO) para GTSB Han Asard, Bulelani L. Sizani y Hamada Abdelgawad contribuyeron 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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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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