Hydrogen peroxide is involved in the acclimation of the Mediterranean shrub, Cistus albidus L., to summer drought

Abstract

This study evaluated the possible role of hydrogen peroxide (H(2)O(2)) in the acclimation of a Mediterranean shrub, Cistus albidus L., to summer drought growing under Mediterranean field conditions. For this purpose, changes in H(2)O(2) concentrations and localization throughout a year were analysed. H(2)O(2) changes in response to environmental conditions in parallel with changes in abscisic acid (ABA) and oxidative stress markers, together with lignin accumulation, xylem and sclerenchyma differentiation, and leaf area were also investigated. During the summer drought, leaf H(2)O(2) concentrations increased 11-fold, reaching values of 10 micromol g(-1) dry weight (DW). This increase occurred mainly in mesophyll cell walls, xylem vessels, and sclerenchyma cells in the differentiation stage. An increase in ABA levels preceded that of H(2)O(2), but both peaked at the same time in conditions of prolonged stress. C. albidus plants tolerated high concentrations of H(2)O(2) because of its localization in the apoplast of mesophyll cells, xylem vessels, and in differentiating sclerenchyma cells. The increase in ABA, and consequently of H(2)O(2), in plants subjected to drought stress might induce a 3.5-fold increase in ascorbic acid (AA), which maintained and even decreased its oxidative status, thus protecting plants from oxidative damage. After recovery from drought following late-summer and autumn rainfall, a decrease in ABA, H(2)O(2), and AA to their basal levels (approximately 60 pmol g(-1) DW, approximately 1 micromol g(-1) DW, and approximately 20 micromol g(-1) DW) was observed.

Fig. 1.

Environmental conditions. Top: monthly maximum diurnal photosynthetic photon flux density PPFD (filled circles); maximum (Tair, solid line) and minimum diurnal (Tair, dashed solid line) air temperature. Midddle: maximum (Tsoil, solid line) and minimum (Tsoil, dashed solid line) soil temperatures. Bottom: maximum diurnal vapour pressure deficit (VPD, solid line), precipitation (vertical bars), and soil water content at 0–20 cm (filled circles) and 40–60 cm (open circles) deep during the measurement period, autumn 2004 to autumn 2005.

Fig. 2.

Variations in plant water status, ABA, and H₂O₂ in C. albidus plants. Top: relative water content (filled circles, RWC, %) and hydration (open circles, H, g H₂O g⁻¹ DW). Middle: endogenous ABA content (pmol g⁻¹ DW). Bottom: endogenous H₂O₂ content (μmol g⁻¹ DW). For the experiment, leaves situated at 5–15 cm from the apex were collected at midday (at maximum diurnal incident PFD) on a clear sunny day once a month. Values followed by the same letter in the same column are not significantly different at P <0.05 according to Duncan's multiple range test. Data are the means ±SE, n=6.

Fig. 3.

Oxidative stress markers in C. albidus plants. Fluctuations of ascorbic acid (filled circles, AA, μmol g⁻¹ DW), dehydroascorbate (open circles, DHA, μmol g⁻¹ DW), ascorbate oxidative status (DHA/AA+DHA), malondialdehyde (MDA, nmol g⁻¹ DW), and maximum efficiency of PSII (F_v/F_m). For the experiment, leaves situated at 5–15 cm from the apex were collected at midday (at maximum diurnal incident PFD) on a clear sunny day once a month. Values followed by the same letter in the same column are not significantly different at P <0.05 according to Duncan's multiple range test. Data are the means ±SE, n=4.

Fig. 4.

Ultrastructural localization of H₂O₂ in leaf cells of C. albidus with CeCl₃ staining and transmission electron microscopy. (A) Mesophyll cells of well-watered plants (December) did not show any H₂O₂ accumulation (bar = 1 μm), and (B) xylem vessels in the same period also did not show any accumulation (bar = 5 μm). (C) Mesophyll cells at the onset of drought (June) showed faint and patchy spots located at the outer part the cell walls, facing intercellular spaces (bar = 2 μm). (D, F) Xylem vessel spiral thickenings and sclerenchyma showed continuous deposits of cerium hydroperoxides at the onset of drought and after 1 month of stress (July, bar = 2 μm). (E) Mesophyll cells in July showed continuous deposits of H₂O₂, also at the outer part of cell walls and facing intercellular spaces, as well as an intact ultrastructure (bar = 2 μm). Arrowheads show electron-dense deposits of cerium perhydroxides. Chl, chloroplast; Cw, cell wall; Sc, sclerenchyma; St, spiral thickening; Xv, xylem vessel.

Fig. 5.

Involvement of H₂O₂ in sclerenchyma formation. (A) Cell with thick walls surrounded by hydrogen peroxide localized at the plasmalemma (bar = 1 μm). (B) Cell death: hydrogen peroxide is located at the disrupted plasmalemma, and surrounding unstructured chloroplasts (bar = 1 μm). (C) Diferentiated sclerenchyma (bar = 2 μm). Cw, cell wall; Sc, sclerenchyma.

Fig. 6.

Lignin accumulation and leaf area in C. albidus plants. Fluctuation on lignin formation (mg g⁻¹ DW). Leaf area (cm⁻²). For the experiment, leaves from the first 15 cm from the apex were collected at midday (at maximum diurnal incident PFD) on a clear sunny day once a month. Values followed by the same letter in the same column are not significantly different at P <0.05 according to Duncan's multiple range test. Data are the means ±SE, n=4.