Synthesis of dehydrin-like proteins in Quercus ilex L. and Quercus cerris L. seedlings subjected to water stress and infection with Phytophthora cinnamomi

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Abstract

The synthesis of dehydrin-like proteins in 24-month-old Quercus cerris and Q. ilex seedlings subjected to water stress and/or infection with Phytophthora cinnamomi, was investigated. An estimated 50 kDa dehydrin-like protein was observed in leaves of symptomless Q. ilex (drought tolerant) in control, as well as in seedlings infected with P. cinnamomi, throughout the experiment. Water stress, with a midday stem water potential value lower than −2.7 MPa (up to 8 weeks), increased band intensity. Two new polypeptides with a molecular weight of about 60 and 63 kDa, respectively, were detected in Q. ilex, under enhanced water stress conditions (≤−4.0 MPa). No dehydrin-like proteins were observed in leaves of symptomless Q. cerris (drought susceptible) neither in control nor in infected treatments. With a midday stem water potential lower than −3.8 MPa (up to 7 weeks), water stress induced the synthesis of a protein with an approx. molecular weight of 43 kDa, while a band of about 36.5 kDa was detected for both water stress and infection treatments. These results suggest the involvement of dehydrin-like proteins in the response of Q. ilex and Q. cerris seedlings to different stresses.

Introduction

Plants are exposed in nature to a number of stresses. Drought, salt, high or low temperature, and infectious plant diseases induce physiological, biochemical and molecular modifications that reflect gene expression. Although plant response (i.e. decrease in growth, yield, leaf and root area; accumulation and translocation of nutrients and water along the whole plant) depends on type, severity of stress, and plant sensitivity, it may be assumed that all plants have encoded capability for stress perception, signalling and response. Some of these responses may occur in a few seconds, others may require minutes or hours [1].

The most prevalent stresses have in common an effect on plant water relations [2], [3]. When plants are exposed to an imposed stress, a decrease in water potential occurs to reflect osmotic adjustment at cellular levels, and a number of processes are modified. Changes in enzyme activity and gene expression, different synthesis or accumulation of osmolytes (i.e. amino acid and sugars) and proteins within the cells are only a few examples of protective mechanisms necessary to prevent cellular damage and any alteration in structure and function of cellular components [4], [3]. Among the biochemical plant adaptations, an important role is held by late-embryogenesis abundant (LEA) proteins, encoded by a small, multigene family [5].

Dehydrin (DHN) proteins, also known as LEA D-11 [6], are characterized by a highly conserved and usually repeated lysine-rich 15 amino acid consensus—EKKGIMDKIKEKLPG—, referred to for convenience as a K segment. K segments are soluble at high temperature, rich in polar and charged amino acids, tend to be glycine-rich and free of amino acids cysteine and tryptophan, and show an apparent lack of a defined structure under several conditions [7], [8]. Dehydrins have been found and purified from several herbaceous and woody plants under water stress, low temperature and ABA treatments [9], [10], [11], [12]. Homology between the poly(A)+RNA extracted from recalcitrant seeds and a cDNA probe of the cotton dehydrins is reported by Finch-Savage et al. [13]. Close [14] has hypothesized that dehydrins are involved in maintaining the stability of macromolecules, namely proteins and lipids, avoiding their denaturation during tissue dehydration. Although the majority of immunolocalization studies of dehydrin-like proteins have reported a cytosolic and nuclear distribution [15], [16], [17], Sarnighausen et al. [18] showed that a 24 kDa protein, immunologically related to dehydrins, represents the major component of the CaCl2-extractable fraction from wood proteins in Cornus sericea L.

Turkey oak (Quercus cerris L.) and holm oak (Q. ilex L.) are two species affected by oak decline, a disease with complex etiology whose main causes can be considered in the interaction between fungi, defoliating insects, and frequent soil drought conditions to induce, in the short or long term, whole plant death [19], [20], [21]. Among the several fungal species involved in oak decline [19], [20], [21], [22], [23], attention is focused at present on the role of the oomycete Phytophthora cinnamomi Rands as a casual factor, especially in relation to water deficit conditions [24], [25], [26]. A negative correlation between both rate and length of root lesions due to P. cinnamomi infections and the relative bark water content (RWC) was shown in 45-year-old Q. rubra L. plants [27]. Consistent wilting and root rot was reported in 24-month-old Q. ilex seedlings, suggesting that soil drought conditions might play an important role in the P. cinnamomi pathogenicity [28]. Maurel et al. [29] observed a synergistic effect between root infection and water stress in decreasing leaf nitrogen content, as well as a strong aerial biomass reduction in plants subjected to infection; on the contrary, root lesions due to P. cinnamomi were more severe in Q. ilex under flooding [30].

The aim of this study was to investigate whether water stress and root rot caused by P. cinnamomi infection induce the synthesis of dehydrin-like proteins in leaves of Q. ilex (drought tolerant) and Q. cerris (drought susceptible) seedlings. Variations in water potential were evaluated by measuring the midday stem water potential (MSWP). The experimental approach tended to be closer to nursery conditions, where a higher disease incidence under drought is often observed, and to act as a model to identify a parameter for an early diagnosis of oak decline in forest stands.

Section snippets

Plants and fungal inoculation

In September 2001, 112-year-old Q. cerris and Q. ilex seedlings (provenance Alto Tevere, central Italy), were transplanted to 2.5 l plastic pots filled with a 2:1 turf:sand mixture and transferred to a growth chamber under 10/14 h (light/dark) photoperiod, temperature 17/10 °C, 10.000/0 lux and 70% constant relative humidity. Environmental conditions from February to May 2002 were adjusted in the chamber to 14/10 h photoperiod, temperature 26/17 °C, 15.000/0 lux and 70% constant relative humidity.

Above and below-ground symptoms

Slight chlorosis and wilting were visible on a few Q. ilex and Q. cerris seedlings leaves in NI–WS and I–WS treatments, starting 9 weeks from the beginning of the experiment. No symptoms were observed in either NI–I (control) and I–I seedlings for both species. After harvesting, 11 weeks from the beginning of the experiment, the root system of both species, under water stress, showed taproot necrosis and absence of lateral roots. Inoculated Q. ilex displayed more root necroses than Q. cerris

Discussion

This paper is the first to report the synthesis of dehydrin-like proteins in two oak species, Q. ilex and Q. cerris, when subjected to water stress and infection with P. cinnamomi.

During the 11 weeks of experiment, decrease of MSWP was similar in both oak species, in spite of their different ecological features. From 0 to 7 days, a rapid decrease of MSWP was detected in Q. ilex seedlings, probably due to the saturated soil conditions provided at the beginning of the experiment, to enhance the

Acknowledgements

The authors thank Dr Gian Paolo Barzanti, CRA -Istituto Sperimentale per la Zoologia Agraria, MiPAF, Florence, Italy, for providing the Phytophthora cinnamomi isolate used in this study; Prof. Dr Ken Shackel, Dept. of Pomology, University of California-Davis, USA, for his precious suggestion concerning the water potential assessment. This research was supported by CNR-National Research Council of Italy ‘Physiological parameters to an early diagnose an abiotic stress on asymptomatic plants’

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