Assessment of energy partitioning in PSII complexes using chlorophyll fluorescence: reviewing the different approaches toward the definition of a unified method

Based on the examination and quantitative comparison of the approaches used to assess the energy partitioning in photosystem II, the unified method was proposed to calculate the contribution of the components of nonphotochemical quenching.     

The role of antioxidant enzymes in photoprotection

The enzymatic component of the antioxidant system is discussed as one of the defensive mechanisms providing protection against excessive light absorption in plants. We present an analysis of attempts to improve stress tolerance by means of the creation of transgenic plants with elevated antioxidant enzyme activities and conclude that the effect of such transgenic manipulation strongly depends on the manner in which the stress is imposed. The following factors may diminish the differences in photosynthetic performance between transgenic plants and wild type under field conditions: effective functioning of the thermal dissipation mechanisms providing a primary line of defense against excessive light, long-term adjustments of the antioxidant system and other photoprotective mechanisms, the relatively low level of control over electron transport exerted by the Water–Water cycle, especially under warm conditions, and a decrease in the content of the transgenic product during leaf aging.     

The Arabidopsis ascorbate peroxidase 3 is a peroxisomal membrane-bound antioxidant enzyme and is dispensable for Arabidopsis growth and development

Ascorbate peroxidase (APX) exists as several isoforms that are found in various compartments in plant cells. The cytosolic and chloroplast APXs appear to play important roles in antioxidation metabolism in plant cells, yet the function of peroxisomal APX is not well studied. In this study, the localization of a putative peroxisomal membrane-bound ascorbate peroxidase, APX3 from Arabidopsis, was confirmed by studying the green fluorescent protein (GFP)-APX3 fusion protein in transgenic plants. GFP-APX3 was found to co-localize with a reporter protein that was targeted to peroxisomes by the peroxisomal targeting signal 1. The function of APX3 in Arabidopsis was investigated by analysing an APX3 knockout mutant under normal and several stress conditions. It was found that loss of function in APX3 does not affect Arabidopsis growth and development, suggesting that APX3 may not be an important antioxidant enzyme in Arabidopsis, at least under the conditions that were tested, or the function of APX3 could be compensated by other antioxidant enzymes in plant cells.     

Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.     

Enhanced photochemical light utilization and decreased chilling-induced photoinhibition of photosystem II in cotton overexpressing genes encoding chloroplast-targeted antioxidant enzymes

The aim of this study was to determine whether increases in stromal superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) via transformation could reduce photosystem (PS) II photoinhibition at low temperature for cotton (Gossypium hirsutum L.) plants and to determine by what mechanism this protection may be realized. During 3-h exposures of lincomycin-treated leaf discs to 10 degrees C and a photon flux density of 500 &mgr;mol m-2 s-1, all transgenic plants exhibited significantly greater PSII activity and O2 evolution than did wild-type plants. Also, the rate constant of PSII photoinactivation was significantly lower for all transgenic plants than for wild-type plants. No significant differences existed between genotypes in non-photochemical quenching of chlorophyll a fluorescence and the regulated component of the thermal dissipation of excitation energy. The relationship between changes in variable to maximum chlorophyll fluorescence (Fv/Fm) and the time-dependent averaged excessive light exposure was similar for all genotypes. This observation excluded the possibility that differences in PSII photodamage were due to improvements in the direct protection of PSII from active oxygen by antioxidant enzyme overproduction. Similar decreases in Fv/Fm during the stress treatment for all genotypes when leaves were pre-treated with 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) suggested that the effect of overproduction involved events downstream of PSII in the electron transfer pathway. Since all transgenic plants exhibited a significantly higher photochemical quenching of chlorophyll fluorescence during the chilling treatment, we concluded that, under the conditions used in this study, the enhancement of the protection of PSII from photodamage by increasing the stromal antioxidant enzyme activity in cotton leaves was due to the maintenance of a higher rate of electron transport and, consequently, a lower reduction state of QA.     

Minimization of the Photon Energy Absorbed by ‘Closed’ Reaction Centers of Photosystem 2 as a Photoprotective Strategy in Higher Plants

Photoinactivation of photosystem 2 (PS2) results from absorption of so-called excessive photon energy. Chlorophyll a fluorescence can be applied to quantitatively estimate the portion of excessive photons by means of the parameter E = (F – F₀)/F_m, which reflects the share of the absorbed photon energy that reaches the reaction centers (RCs) of PS2 complexes with Q_A in the reduced state (closed RCs). Data obtained for cotton (Gossypium hirsutum), bean (Phaseolus vulgaris), and arabidopsis (Arabidopsis thaliana) suggest a linear relationship between the total amount of the photon energy absorbed in excess (excessive irradiation) and the decline in PS2 activity, though the slope may differ depending on the species. This relationship was sensitive not only to the leaf temperature but also to treatment with methyl viologen. Such observations imply that the intensity of the oxidative stress as well as the plant's ability to detoxify active oxygen species may interact to determine the damaging potential of the excessive photons absorbed by PS2 antennae. Energy partitioning in PS2 complexes was adjusted during adaptation to irradiation and in response to a decrease in leaf temperature to minimize the excitation energy that is trapped by closed PS2 RCs. The same amount of the excessive photons absorbed by PS2 antennae led to a greater decrease in PS2 activity at warmer temperatures, however, the delay in the development of non-photochemical and photochemical energy quenching under lower temperature resulted in faster accumulation of excessive photons during induction. Irradiance response curves of EF suggest that, at high irradiance (above 700 mol m^–2 s^–1), steady-state levels of this parameter tend to be similar regardless of the leaf temperature.     

Protecting cotton photosynthesis during moderate chilling at high light intensity by increasing chloroplastic antioxidant enzyme activity.

This study examined the effect of increasing chloroplastic superoxide dismutase (SOD), ascorbate peroxidase (APX), or glutathione reductase (GR) activity via plant transformation of cotton on the initial recovery of photosynthesis following exposures to 10 degrees C and high photon flux density (PFD). Growing wild-type or non-expressing segregate plants (controls) and transformants at two PFDs (600 micromol m(-2) s(-1) and full sun) resulted in a range of total antioxidant enzyme activities. Total SOD activities above that for control leaves grown in full sun did not substantially improve the recoveries of CO(2)-saturated photosynthesis, especially for stress treatments lasting more than 1 h, while elevated APX or GR activity did improve recoveries after 1-3 h of the chilling treatment. No synergistic effects were noted when the activities of more than one antioxidant enzyme were elevated in transgenic hybrids. Although these results suggest that the protection of photosynthesis can be realized by reducing either superoxide or H(2)O(2) levels, thereby reducing the possibility of hydroxyl radical formation, the situation is complicated, since elevated APX or GR activity can improve recoveries even when additional SOD activity has no effect. In conclusion, to enhance the protection of photosynthesis using stroma-targeted antioxidant enzymes, enhancing metabolism associated with H(2)O(2) is more effective than enhancing the capacity for superoxide scavenging. Although small, the improvement in the protection of photosynthetic capacity may be sufficient to improve cotton yield in temperate regions with large diurnal temperature fluctuations.     

Transgenic overproduction of glutathione reductase does not protect cotton, Gossypium hirsutum (Malvaceae), from photoinhibition during growth under chilling conditions

In some studies, tissues from plants that have been genetically transformed to overproduce antioxidant enzymes sustain less damage when abruptly exposed to short-term chilling in the laboratory. However, few studies have examined the performance of transgenic plants during longer-term growth under chilling conditions. We compared growth of transgenic cotton that overproduces glutathione reductase (GR+; ～40-fold overproduction) to growth of the wild type in a controlled environment chamber as leaf temperature was lowered from 28° to 14°C over 9 d and for a subsequent 9-d period at 14°C. In wild-type and GR+ cotton, chilling temperatures resulted in decreased dark-adapted F(v)/F(m) (the ratio of variable to maximal fluorescence; a measure of maximum photosystem II quantum yield) and mid-light period photosystem II quantum yield, coupled with increased 1 - q(P) (a nonlinear estimate of the reduction state of the primary quinone acceptor of photosystem II). The capacity for photosynthetic oxygen evolution decreased during the first portion of the chilling exposure, but recovered slightly during the second half. At no point during the chilling exposure did the performance of GR+ plants differ significantly from that of wild-type plants in any of the above parameters. The absence of an effect of GR overproduction under longer-term chilling may be explained, in part, by the fact that wild-type cotton acclimated to chilling by upregulating native GR activity.     

A Chlorophyll Fluorescence Analysis of the Allocation of Radiant Energy Absorbed in Photosystem 2 Antennae of Cotton Leaves During Exposure to Chilling

When dark-acclimated cotton (Gossypium hirsutum L. cv. Coker 312) leaves, pre-treated with lincomycin to inhibit chloroplast protein repair processes, were exposed to 10 °C and a PPFD of 500 mol m^-2 s^-1, the proportion of excitation energy entering photochemistry (P) increased, but only to 5 % of the total energy absorbed at steady state levels of P, which were reached at 40 min of irradiation. Thermal dissipation (D) of absorbed energy increased throughout the 360 min irradiation period and accounted for the greatest portion of absorbed energy at 10 °C. When D was partitioned into constitutive (D_CON), regulated (D_REG), and photoinhibitory (D_PI) components, it was primarily composed of D_REG, the readily reversible portion of D. However, the induction of D was slow at 10 °C. Sixty minutes were required for D to reach 70 % of the energy absorbed. Considerable absorption of energy in excess of that utilized in photochemistry or dissipated thermally (designated as E) occurred, especially during induction of P and D. Over the irradiation period, the time-dependent averaged E exhibited an inverse, linear relationship with the ratio of variable (F_v) to maximum (F_m) fluorescence (PS2 efficiency) and a linear relationship with D_PI. We propose that time-dependent averaged E may be useful for estimating the potential for damage to PS2 under stressful environmental conditions.