Original article
Pectin methyl esterases and pectins in normal and hyperhydric shoots of carnation cultured in vitro

https://doi.org/10.1016/j.plaphy.2005.01.017Get rights and content

Abstract

Control and hyperhydric micropropagated plantlets from three carnation cultivars have been used to study their pectin composition and the activity of pectin methyl esterases (PMEs; EC 3.1.1.11). Pectins are a highly heterogeneous group of polymers that contribute to cell adhesion, cell wall architecture, and cell wall mechanical strength. Pectins control cell wall porosity and cell wall ionic status and are implicated in intercellular space development. The degree of esterification of pectins is controlled by the activity of cell wall PMEs; their different actions can affect the properties of the cell wall, which have been considered important with respect to controlling the development of hyperhydricity. The total pectins of hyperhydric leaves of the three varieties were significantly reduced in comparison with controls. The pectate fraction was significantly increased in hyperhydric leaves of all varieties while soluble pectins and protopectins were significantly lower. The PME activity of hyperhydric leaves was higher (4–10 times) compared to controls of the three varieties. Isoelectric focusing of PME isozymes revealed the presence of three isoforms; neutral PME activity was the major isozyme in control and hyperhydric leaves of the three varieties, whilst a decrease in the activity of the acidic isoforms was observed in hyperhydric leaves. The different PME activities could regulate some of the structural changes related to hyperhydricity in micropropagated carnation plants.

Introduction

The technique of plant micropropagation has been widely used for commercial production of plants. However, for shoots cultured in vitro, a malformation problem called hyperhydricity often appears. One of the most affected plants is carnation (Dianthus caryophyllus), as well as all the species of the family Caryophylliceae [1]. Hyperhydric plants display abnormal physiological and anatomical characteristics [4], [12]. Hyperhydric leaves of carnation are composed typically of hypertrophic cells and large intercellular spaces [17] and the vascular system in hyperhydric leaves is not properly lignified [3], [9], [18], [22].

Cell wall properties and composition can be considered one of the most important factors controlling the development of the anomalous morphology in hyperhydric tissues. Less lignin, associated with low lignin biosynthesis and decreased lignification of the vascular system, has been frequently considered one of the possible causes of hyperhydricity [1]. However, hyperhydricity is a complex response that implicates physiological, biochemical and anatomical modifications [10], [12], [14], [17].

Different workers have shown modifications in the cell wall constituents mainly cellulose and lignin [9], [10], [14], [18] and their mechanical properties [9], [13]. However, little is known about the role of pectin and pectin methyl esterases (PMEs) in hyperhydric tissues [15].

Pectins represent about 35% of the dry weight of dicot cell walls. Pectins are a highly heterogeneous group of polymers that includes homogalacturonans and ramnogalactorunans I and II [19]. They contribute both to cell adhesion, via their gel-like properties, and to cell wall architecture or cell wall mechanical strength. Pectins control cell wall porosity and ionic status and are implicated in the intercellular space development [8]. The degree of esterification of pectins is generally thought to be controlled in muro by the activity of cell wall PMEs. It is thought that homogalactorunans are highly methyl-esterified when exported into cell walls and are subsequently de-esterified by the action of PMEs in the cell wall [16]. The different actions of PMEs can affect the properties of the cell walls.

The aim of the present work was to compare PMEs and pectins in hyperhydric and normal carnation shoots of three different varieties, during the hyperhydric state at the end of the culture cycle. We consider that studies of cell wall pectins and PMEs can help to elucidate the metabolic processes induced by the physiological state of hyperhydricity.

Section snippets

Results

The pectin fractions of control and hyperhydric leaves of the three varieties of carnation are shown in Table 1. The total pectins of hyperhydric leaves of all varieties were significantly reduced in comparison with controls. However, the analysis of the different fractions of pectins showed that the pectate fraction was significantly increased in hyperhydric leaves of all varieties whereas soluble pectins were significantly reduced. This fact affected the ratio of soluble pectins to pectates:

Discussion

Pectin composition and mechanical properties of pectins have been previously analyzed in hyperhydric carnation plants [9], [11]. These authors observed that hyperhydric shoots of carnation growing in liquid media showed lower levels of uronic acids, a reduced calcium concentration and higher neutral sugar contents compared to normal leaves. The hypothesis of these authors was that the plastic properties observed in hyperhydric leaves could be explained by the different compositions of pectin,

Plant material

Mother-shoots of three D. caryophyllus cultivars (Oslo, Killer and Alister) were provided by Barber and Blanc S.A.E. (Puerto Lumbreras, Murcia). Shoots were multiplied on MS-based medium, pH 5.8 ± 0.1 supplemented with 2% (w/v) sucrose and solidified with 0.85% (w/v) agar without plant growth regulators (Control shoots). Hyperhydricity was induced by transferring the shoots to the same medium containing 0.58% (w/v) agar (Hyperhydric shoots). Cultures were maintained under a 16/8 h light/dark

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