Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves

In Arabidopsis leaves, high light stress induces rapid expression of a gene encoding a cytosolic ascorbate peroxidase (APX2), whose expression is restricted to bundle sheath cells of the vascular tissue. Imaging of chlorophyll fluorescence and the production of reactive oxygen species (ROS) indicated that APX2 expression followed a localised increase in hydrogen peroxide (H2O2) resulting from photosynthetic electron transport in the bundle sheath cells. Furthermore, leaf transpiration rate also increased prior to APX2 expression, suggesting that water status may also be involved in the signalling pathway. Abscisic acid stimulated APX2 expression. Exposure of ABA-insensitive mutants (abi1-1, abi2-1) to excess light resulted in reduced levels of APX2 expression and confirmed a role for ABA in the signalling pathway. ABA appears to augment the role of H2O2 in initiating APX2 expression. This regulation of APX2 may reflect a functional organisation of the leaf to resolve two conflicting physiological requirements of protecting the sites of primary photosynthesis from ROS and, at the same time, stimulating ROS accumulation to signal responses to changes in the light environment.     

鲜切加工加速荸荠组织衰老与H2O2累积的关系

以荸荠为材料,研究了鲜切加工加速组织衰老与活性氧代谢的关系.结果表明:鲜切加工提高了荸荠切片抗氧化酶(超氧化物歧化酶、抗坏血酸-过氧化物酶和过氧化氢酶)的活性;但同时明显刺激了O2-产生,促进了H2O2累积,加速了抗坏血酸在贮藏后期的损失,加强了膜脂过氧化作用和增加了电解质渗出率.统计分析表明H2O2含量、丙二醛含量、电解质渗出率三者之间存在正相关性.H2O2组织定位结果也证实鲜切加速组织衰老与H2O2累积密切相关.完整荸荠组织O2-产生比较平稳,抗氧化酶活性维持稳定,H2O2未有明显累积.     

Induction of ASCORBATE PEROXIDASE 2 expression in wounded Arabidopsis leaves does not involve known wound-signalling pathways but is associated with changes in photosynthesis

ASCORBATE PEROXIDASE 2 (APX2) encodes a key enzyme of the antioxidant network. In excess light-stressed Arabidopsis leaves, photosynthetic electron transport (PET), hydrogen peroxide (H(2)O(2)) and abscisic acid (ABA) regulate APX2 expression. Wounded leaves showed low induction of APX2 expression, and when exposed to excess light, APX2 expression was increased synergistically. Signalling pathways dependent upon jasmonic acid (JA), chitosan and ABA were not involved in the wound-induced expression of APX2, but were shown to require PET and were preceded by a depressed rate of CO(2) fixation. This led to an accumulation of H(2)O(2) in veinal tissue. Diphenyl iodonium (DPI), which has been shown previously to be a potent inhibitor of H(2)O(2) accumulation in the veins of wounded leaves, prevented induction of APX2 expression probably by inhibition of PET. Thus, the weak induction of APX2 expression in wounded leaves may require H(2)O(2) and PET only. As in other environmental stresses, wounding of leaves resulted in decreased photosynthesis leading to increased reactive oxygen species (ROS) production. This may signal the induction of many 'wound-responsive' genes not regulated by JA-dependent or other known JA-independent pathways.     

Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress.

The Arabidopsis gene APX3 that encodes a putative peroxisomal membrane-bound ascorbate peroxidase was expressed in transgenic tobacco plants. APX3-expressing lines had substantial levels of APX3 mRNA and protein. The H2O2 can be converted to more reactive toxic molecules, e.g. .OH, if it is not quickly removed from plant cells. The expression of APX3 in tobacco could protect leaves from oxidative stress damage caused by aminotriazole which inhibits catalase activity that is found mainly in glyoxysomes and peroxisomes and leads to accumulation of H2O2 in those organelles. However, these plants did not show increased protection from oxidative damage caused by paraquat which leads to the production of reactive oxygen species in chloroplasts. Therefore, protection provided by the expression of APX3 seems to be specific against oxidative stress originated from peroxisomes, not from chloroplasts, which is consistent with the hypothesis that APX3 is a peroxisomal membrane-bound antioxidant enzyme.     

Ascorbate peroxidase 1 plays a key role in the response of Arabidopsis thaliana to stress combination

Within their natural habitat plants are subjected to a combination of different abiotic stresses, each with the potential to exacerbate the damage caused by the others. One of the most devastating stress combinations for crop productivity, which frequently occurs in the field, is drought and heat stress. In this study we conducted proteomic and metabolic analysis of Arabidopsis thaliana plants subjected to a combination of drought and heat stress. We identified 45 different proteins that specifically accumulated in Arabidopsis in response to the stress combination. These included enzymes involved in reactive oxygen detoxification, malate metabolism, and the Calvin cycle. The accumulation of malic enzyme during the combined stress corresponded with enhanced malic enzyme activity, a decrease in malic acid, and lower amounts of oxaloacetate, suggesting that malate metabolism plays an important role in the response of Arabidopsis to the stress combination. Cytosolic ascorbate peroxidase 1 (APX1) protein and mRNA accumulated during the stress combination. When exposed to heat stress combined with drought, an APX1-deficient mutant (apx1) accumulated more hydrogen peroxide and was significantly more sensitive to the stress combination than wild type. In contrast, mutants deficient in thylakoid or stromal/mitochondrial APXs were not more sensitive to the stress combination than apx1 or wild type. Our findings suggest that cytosolic APX1 plays a key role in the acclimation of plants to a combination of drought and heat stress.     

Expression of the <Emphasis Type="Italic">Apx</Emphasis> gene family during leaf senescence of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H₂O₂ scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.     

Singlet oxygen imaging in Arabidopsis thaliana leaves under photoinhibition by excess photosynthetically active radiation

Arabidopsis thaliana leaves were infiltrated with DanePy (3-( N -diethylaminoethyl)- N -dansyl)aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1 H -pyrrole), a double, fluorescent and spin sensor of singlet oxygen. DanePy fluorescence was imaged by laser scanning microscopy. We found that DanePy penetrated into chloroplasts but did not alter the functioning of the photosynthetic electron transport as assessed by chlorophyll fluorescence induction. In imaging, DanePy fluorescence was well distinct from chlorophyll fluorescence. Photoinhibition by excess photosynthetically active radiation caused quenching of DanePy fluorescence in the chloroplasts but not in other cell compartments. When leaves were infiltrated with dansyl, the fluorescent group in DanePy, there was no fluorescence quenching during photoinhibition. This shows that the fluorescence quenching of DanePy is caused by the conversion of its pyrrol group into nitroxide, i.e. it was caused by the reaction of singlet oxygen with the double sensor and not by artifacts. These data provide direct experimental evidence for the localization of singlet oxygen production to chloroplasts in vivo.