Systemic Signaling and Acclimation in Response to Excess Excitation Energy in Arabidopsis

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Science  23 Apr 1999:
Vol. 284, Issue 5414, pp. 654-657
DOI: 10.1126/science.284.5414.654

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Land plants are sessile and have developed sophisticated mechanisms that allow for both immediate and acclimatory responses to changing environments. Partial exposure of low light–adaptedArabidopsis plants to excess light results in a systemic acclimation to excess excitation energy and consequent photooxidative stress in unexposed leaves. Thus, plants possess a mechanism to communicate excess excitation energy systemically, allowing them to mount a defense against further episodes of such stress. Systemic redox changes in the proximity of photosystem II, hydrogen peroxide, and the induction of antioxidant defenses are key determinants of this mechanism of systemic acquired acclimation.

Large increases in light intensity for a short period can be beneficial for photosynthetic yields in low light (LL)–adapted plants (1). However, if these conditions persist, an imbalance can be created such that the energy absorbed through the light-harvesting complex is in excess of that which can be dissipated or transduced by photosystem II (PSII). This imbalance [excess excitation energy (EEE)] can be generated by excess light (EL) or chilling or both and can be strongly enhanced by a combination with other factors such as rapid and large increases in temperature and limitations in nutritional and H2O status (1–8).

Dissipation of such EEE is an immediate response that occurs through heat irradiation (2, 3). However, prolonged exposure to the conditions that cause EEE can result in an increase in the generation of reactive oxygen species (ROS) such as singlet oxygen, superoxide anion (O2 ), and hydrogen peroxide (H2O2) (4, 8). If the accumulation of ROS under conditions of EEE exceeds the capacity of antioxidant systems to remove them, irreversible photooxidative damage to the chloroplast and the cell may occur. Thus, overproduction of ROS under EEE conditions can ultimately result in the permanent photodamage of leaf tissues (Fig. 1A).

Figure 1

Permanent photodamage and induction ofAPX2-LUC and APX2 in transgenicArabidopsis leaf tissue. Leaves of transgenic plant grown in LL (control) were exposed to EL (9). (A) Appearance of chlorosis on detached leaves after 2 hours in EL. (B) CCD camera image of relative luciferase activity (in RLUs) in detached leaves that were exposed to different times of EL (arrow indicates the chlorosis zone of the leaf). (C) Gel blot analysis of mRNA levels for APX2 andAPX3 in leaves that were exposed to different durations of EL (APX3 mRNA is shown as a loading control).

In our experimental system, EEE was generated by EL applied to LL-adapted Arabidopsis plants, resulting in the induction of antioxidant defense genes (as APX2) (8, 9). Leaves from transgenic Arabidopsis plants harboring anAPX2-LUC fusion (10) had no detectable luciferase activity when grown under LL conditions, but after challenging with EL, luciferase activity, which could be imaged, was induced (11) (Fig. 1B). The induction of the APX2-LUC transgene mirrored the induction of the native APX2 gene in the same plants, as determined by Northern (RNA) blotting (Fig. 1C) (8), and therefore could be used as a measure of activation of APX2expression. After 2 hours of exposure to EL, the leaves suffered photodamage and lost APX2-LUC and APX2 expression over most of the leaf area (Fig. 1, A through C).

Induction of APX2-LUC by EL could be diminished by infiltrating leaves with catalase but could not be diminished with superoxide dismutase (Fig. 2A). The effect of the catalase treatment occurred in a dose-dependent manner (12). The vacuum infiltration and incubation procedures did not affect either luciferase activity or the uptake of luciferin in control APX1-LUC transgenic lines that expressed this gene under LL conditions (8, 12, 13). Thus, H2O2 (but not O2 ) could be involved in the EL-induced expression of APX2. Unlike EL, H2O2 alone did not induce the expression of APX2-LUC sufficiently to be imaged (Fig. 2A), but the expression could be detected by the more sensitive in vitro assay (Table 1) (11).

Figure 2

Regulation of APX2-LUCexpression,F v/F m, and the protective role of H2O2 in transgenicArabidopsis leaf tissue. (A) CCD camera image of luciferase activity (in RLUs) in detached leaves treated (12) with H2O [control (C)], H2O2, superoxide dismutase (SOD), and catalase (CAT) for 2 hours in LL and then exposed to EL for 40 min. (B)F v/F m in detached leaves treated (12) with H2O (control) (diamonds) and H2O2 (crosses) for 120 min in LL and then exposed to EL for up to 150 min (parameters were measured in five different leaves that were obtained from three independent experiments, n = 15 ± SD, shown by error bars). (C) Protection against photodamage and permanent photodamage in leaves treated (11) with H2O2 and H2O for 2 hours in LL, then exposed for 2 hours (h) in EL, and reexposed for 2 hours in LL.

Table 1

Role of photosynthetic electron transport and H2O2 in the regulation of APX2-LUCexpression in transgenic Arabidopsis leaf tissue. Detached leaves were treated in LL with H2O (control), H2O2 (10 mM), DBMIB (12 μM ), DCMU (10 μM), or DCMU and H2O2 and exposed to EL for 1 hour (9–12). Luciferase activity was expressed as RLUs per gram of fresh weight. All parameters were measured in five different leaves obtained from three independent experiments (n = 15 ± SD). All treatments were statistically tested against the control. Levels of significance were calculated from the analysis of variance (ANOVA) data. ΦPSII, quantum yield of PS II electron transport; LL → EL, subsequent exposure to EL after LL treatments.

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Treatment of leaves with H2O2 and then with exposure to EL produced a lesser degree of photooxidative stress and caused a smaller induction of luciferase activity than in the H2O-treated controls (Fig. 2, B and C, and Table 1). Detached leaves that were pretreated with H2O2and exposed to EL showed a slower decline inF v/F m(F v, variable fluorescence of chlorophyll a;F m, maximal fluorescence of chlorophyll a) and a smaller decrease in photochemical quenching (q p) than H2O-treated control leaves showed (Fig. 2B and Table 1) (14), and the H2O2-treated leaves did not develop visible photodamage of leaf tissue (Fig. 2C). Similarly, treatment of maize seedlings and potato nodal explants with H2O2has been shown to protect against chilling in the dark and heat stress, respectively (15, 16). Our data show that H2O2 is involved in the acclimation to conditions evoked by EEE.

Treatment of detached leaves (12) with the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) before exposure to EL blocked the induction of luciferase activity (8).APX2-LUC was induced in LL by 2,5-dibromo- 6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) treatment (Table 1) (8). H2O2 did not relieve this effect of DCMU on the EL-mediated induction ofAPX2-LUC. Furthermore, the inductive effect of H2O2 observed in LL was also blocked by DCMU (Table 1). Thus, redox changes in electron transport through quinone B (QB) or plastoquinone (PQ) or both could be essential for the induction of APX2 (8, 17–19) (Table 1).

The above data (Fig. 2, A through C, and Table 1) suggested that H2O2 could act as a systemic messenger. To test this, we exposed leaves on approximately one-third of the rosette to EL for 30 min (hereafter called 1° leaves), and we measured leaves from the LL side of the rosette (hereafter called 2° leaves) (Fig. 3 and Table 2). APX2 expression in the 2° leaves was induced to ∼11% of the levels in EL-exposed 1° leaves, which was similar to that observed in H2O2-treated leaves (Table 1). This activation of APX2 expression in 2° leaves was associated with an increase in H2O2 content and changes inF v/F m (Table 2). Under these conditions, 1° leaves showed clear signs of photooxidative stress. Subsequently, a full exposure of partial EL-treated rosettes to further EL for 30 min exacerbated these stresses in 1° leaves, but 2° leaves showed acclimation to EEE and photooxidative stress. They displayed only a slight reduction inF v/F m, a slight decrease in q p, no further increase in H2O2 amounts, and less APX2induction than that in 1° leaves (Table 2). These data indicate that a systemic signal can control an acclimatory response to EEE.

Figure 3

Systemic induction of APX2-LUCexpression in transgenic Arabidopsis leaf tissue. Image of luciferase activity (in RLUs) (11). A part of the whole rosette (as shown) was exposed to EL for 40 min (the arrow indicates the apical region of the rosette). A typical primary (1°) EL-exposed leaf and a secondary (2°) LL-exposed leaf are shown.

Table 2

Systemic acquired acclimation to EL (9) ofAPX2-LUC transgenic Arabidopsis leaf tissue. Photosynthetic parameters, H2O2 content (in micromoles per gram of fresh weight), luciferase activity (in RLUs per gram of fresh weight), and mRNA levels [in relative units (RUs)] (APX2 mRNA level after 30 min in EL is 1; APX3mRNA level in LL is 1) after different light treatments. Treatment A, control rosette exposed to LL; treatment B, rosette partially exposed (∼30%) to EL for 30 min (1°, EL-exposed leaf; 2°, LL-exposed leaf); treatment C, rosette partially exposed to EL for 30 min, which was then completely exposed to a second EL treatment for another 30 min (1°, EL and EL–exposed leaf; 2°, LL and EL–exposed leaf). Parameters were measured in five different leaves that were obtained from three independent experiments (n = 15 ± SD). In RNA slot blot experiments, pooled samples of 15 leaves from three independent experiments were used. Treatments B1° and B2° were statistically tested against treatment A; treatments C1° and C2° were tested against B1° and B2°, respectively. Levels of significance were calculated from the ANOVA data.

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Our work allows a unified view of acclimatory responses to any fluctuating environmental condition that elicits EEE. When a leaf experiences a set of conditions such as EL, an induction of antioxidant defenses is one of the many cellular responses and is controlled at least in part by the redox status of the QB or PQ pool or both (8, 17–19) (Table 1). However, cells suffering these stresses also produce a systemic signal, a component of which is H2O2, which sets up an acclimatory response to EEE and, consequently, a photooxidative stress in unstressed regions of the plant (Table 2). Furthermore, because changes in the photosynthetic parameters have been observed in 2° leaves (Table 2), we suggest that a systemic signal can promote redox changes in the proximity of PSII in unstressed chloroplasts (Table 1), thus inducing protective mechanisms in remote chloroplasts and cells. We have termed this phenomenon “systemic acquired acclimation.”

  • * To whom correspondence should be addressed. E-mail: stanislaw.karpinski{at}


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