Sub-cellular location of H2O2, peroxidases and pectin epitopes in control and hyperhydric shoots of carnation

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Abstract

In carnation shoots (Dianthus caryophyllus cv. Killer), hyperhydricity was induced in in vitro culture using a low agar concentration. Using transmission electron microscopy, cytochemical techniques and immunolocation of JIM5 and JIM7 pectin epitopes, we followed the sub-cellular modifications of cell walls in relation to peroxidase activity and hydrogen peroxide accumulation during hyperhydricity induction. Peroxidase activity revealed a significant induction of the stomatal and epidermal cells as well as of the intercellular spaces of hyperhydric leaves. Similarly, hydrogen peroxide accumulated in the epidermal cell walls and the intercellular spaces of hyperhydric leaves. Immunolocation of an epitope recognised by the JIM5 antibody revealed the main unesterified nature of the cell walls. Such an epitope was located in the epidermal cell walls as well as in the corners of cell junctions in control leaves. However, hyperhydric leaves showed a total reduction of JIM5 labelling in the corners of cell junctions and a significant reduction of the intercellular spaces and the middle lamella. Highly-methylsterified pectin, recognised by the JIM7 antibody, was present to a slight extent in cell walls in control and hyperhydric leaves. We propose that the altered anatomy observed in hyperhydric carnation leaves could be regulated by the concomitant actions of pectin methyl esterases and free radicals, modifying the structure of the pectin and polysaccharides of the cell walls.

Introduction

Hyperhydricity is one of the main abnormalities observed in plant micropropagation. Hyperhydricity is a major problem in the tissue culture industry since it can affect shoot multiplication and culture vigour and can impede the successful transfer of micropropagated plants to in vivo conditions.

For the majority of hyperhydric tissues, common physiological characteristics can be described: they contain less chlorophyll and have hypoliginification and a lower dry weight. However, other cellular markers are affected differently depending on the plant material origin and species. In general, hyperhydric tissues are hypolignified and this fact has been correlated with low acid peroxidase and low phenylalanine ammonia lyase activities (Kevers and Gaspar, 1985, Olmos et al., 1997, Saher et al., 2004). Hyperhydricity is considered to be a physiological abnormality induced by an excess uptake of water by plant tissues cultured in vitro. The accumulation of water in the intercellular spaces creates a poor diffusion of oxygen from the atmosphere to the parenchymatic cells (Gaspar et al., 2002, Saher et al., 2005a). We previously observed that hyperhydric leaves of different varieties of carnation suffer oxidative stress (Saher et al., 2004). Under these conditions, a high production of hydrogen peroxide is induced and the hyperhydric tissues try to reduce the damage via an induction of the sub-cellular antioxidant systems, by increasing the activity of detoxifying enzymes like catalase and peroxidases (Franck et al., 1995, Chen and Ziv, 2001, Gaspar et al., 2002, Saher et al., 2004, Saher et al., 2005b, Chakrabarty et al., 2006, Dewir et al., 2006).

Recently, we observed that hyperhydric shoots of carnation showed an alteration of their pectin content, correlated with a significant increase of the activity of the pectin methyl esterases that can regulate the physical structure of cell walls. This would decrease the wall pressure, thus facilitating the cellular expansion of the hyperhydric tissues (Olmos and Hellin, 1998). Therefore, water uptake from the apoplast can induce the hyperhydric state (Saher et al., 2005c).

The aim of the present work is to study the possible correlations between the structural modification of the cell wall pectins, the sub-cellular co-location of hydrogen peroxide, peroxidases and the anatomical modifications observed in hyperhydric shoots of carnation plants cultured in vitro.

Section snippets

Plant material and shoot micropropagation

Mother-shoots of Dianthus caryophyllus, cultivar Killer, 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). Shoots were sub-cultured every 5 weeks in a new medium. Cultures were

Leaf structure and peroxidase activity localisation

Hyperhydric leaves of carnation shoots were thicker and the mesophyll and epidermal cells much bigger, with larger intercellular spaces, than in control leaves (Fig. 1A and B, Table 1). The stomata were also significantly larger in hyperhydric leaves than in control leaves (Table 1 and Fig. 1G and H). However, the stomatal density was reduced significantly in hyperhydric leaves compared with the control (Table 1).

Peroxidase staining was mainly located in the vascular bundles and epidermal cells

Discussion

Our morphological results show that the hyperhydric leaves had an altered anatomy that we have demonstrated previously in hyperhydric leaves of regenerated plants (Olmos and Hellin, 1998). The larger intercellular spaces in hyperhydric leaves seem to be water-filled, reducing the diffusion of air and probably inducing hypoxia and an oxidative stress in the mesophyll tissues (Gribble et al., 1998, Gaspar et al., 2002, Saher et al., 2004, Saher et al., 2005a).

The sub-cellular localisation of the

Acknowledgments

The authors wish to thank Dr. David Walker for correction of the written English in the manuscript.

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