Original Article
Reducing properties, energy efficiency and carbohydrate metabolism in hyperhydric and normal carnation shoots cultured in vitro: a hypoxia stress?

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Abstract

Hyperhydricity is considered as a physiological disorder that can be induced by different stressing conditions. In the present work we have studied the metabolic and energetic states of hyperhydric carnation shoots. We have evaluated the hypothesis that hypoxia stress is the main factor affecting the metabolism of hyperhydric leaves. Our results indicate a low level of ATP in hyperhydric tissues, but only slight modifications in pyridine nucleotide contents. Concurrently, the glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49) activity in hyperhydric leaves was increased but glucokinase (GK; EC 2.7.1.2) activity was unchanged. We have observed that the metabolism of pyruvate was altered in hyperhydric tissues by the induction of pyruvate synthesis via NADP-dependent malic enzyme (EC 1.1.1.40). The enzymes of the fermentative metabolism pyruvate decarboxylase (PDC; EC 4.1.1.1) and alcohol dehydrogenase (ADH; EC 1.1.1.1) were highly increased in hyperhydric leaves. Sucrose metabolism was modified in hyperhydric leaves with a high increase in the activity of both synthesis and catabolic enzymes. The analysis of the sucrose, glucose and fructose contents indicated that all of these sugars were accumulated in hyperhydric leaves. However, the pinitol content was drastically decreased in hyperhydric leaves. We consider that these results suggest that hyperhydric leaves of carnation have adapted to hypoxia stress conditions by the induction of the oxidative pentose phosphate and fermentative pathways.

Introduction

Vitrification was first used as a term in conventional plant tissue culture to describe a morphological response of plant tissues to stresses [12] and it has recently been redefined as hyperhydricity [9]. The visual symptoms were water-soaked, thick, elongated, wrinkled, curled, brittle and translucent leaves, shoots with shorter internodes and a morphological appearance indicating excess water content [8], [28]. The affected plants appear glassy and have reduced or retarded growth, a bushy habit and thickened and malformed stems and leaves with hypertrophy of cortical and pith parenchyma cells [26], [36]. Hyperhydricity is a major problem in the tissue culture industry since it can affect shoot multiplication and culture vigor and can impede the successful transfer of micropropagated plants to in vivo conditions. Up to 60% of affected plants fail to acclimatize [38], thereby limiting the application of in vitro techniques for mass propagation.

To date, there is scarce information about the physiological state of hyperhydric shoots [13], [14], [15], [28], [44], [45], [46]. Biochemically, the activity of several enzymes is altered in hyperhydric leaves. Recently, Kevers et al. [28] have revised the physiological state of hyperhydric tissues. The principal conclusions are based mainly on work studying the biochemical and physiological states of hyperhydric shoots of Prunus avium, a woody plant [13], [14], [15]. Based on these studies, they have proposed the application of the state-change concept to the phenomenon of hyperhydricity. We consider that this hypothesis was well developed and could explain how hyperhydricity was induced in those conditions, but also that it needs to be validated in different species. As they proposed, hyperhydric tissues could be subjected to limiting-oxygen conditions imposed by the accumulation of water in the apoplast of the hyperhydric shoots, thus reducing the rate of oxygen diffusion to the cells.

The aim of the present work was to determine whether hyperhydric carnation shoots presented a biochemical and physiological metabolism altered during hyperhydricity development, and whether some of this alteration can be induced by a hypoxia stress. The activities of the enzymes implicated in the energy metabolism under hypoxia stress and sucrose metabolism were studied in both types of shoots.

Section snippets

ATP and pyridine nucleotide content

ATP content in hyperhydric leaves of all varieties was reduced for both periods of cultured (14 and 28 days) compared to the control (Fig. 1).

After 14 days of culture, the content of oxidized and reduced pyridin nucleotides were similar in hyperhydric leaves as in the control in all varieties, except for Killer, the NADH content was decreased (Fig. 2B) and NAPDH was increased compared with the control (Fig. 2D). Similarly, at the end of culture (28 days) the content remained stable, except for

Respiratory metabolism

In previous work, we have observed that hyperhydric leaves of carnation were affected by oxidative stress [44], [45]. The induction of hyperhydricity could be related to different stressing factors such as wounding, high relative humidity in the culture vessels and softening of the medium [28]. All of these stresses can act to induce an oxidative stress. Different workers have previously observed that hyperhydric tissues accumulate water in the apoplast [19], [20], [27], creating a water layer

Plant material and shoot micropropagation

Mother-shoots of three Dianthus 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). Shoots were subcultured every 5 weeks in a new

Acknowledgments

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

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