Determination of Protein Carbonylation and Proteasome Activity in Seeds

Xia, Qiong; El-Maarouf-Bouteau, Hayat; Bailly, Christophe; Meimoun, Patrice

doi:10.1007/978-1-4939-3759-2_16

Cited by 8 publications

(3 citation statements)

References 25 publications

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“…Protein carbonyl content was assessed using a modified method described by Levine [99] and Xia [100]. A 100 µg amount of protein was resuspended in 100 µL 6% SDS, 40 mM sodium acetate pH 5, and insoluble protein was separated by centrifugation at 15,000× g for 5 min.…”

Section: Quantitative Analysis Of Protein Carbonylationmentioning

confidence: 99%

Enhanced Carbonylation of Photosynthetic and Glycolytic Proteins in Antibiotic Timentin-Treated Tobacco In Vitro Shoot Culture

Andriūnaitė

Rugienius

Tamošiūnė

et al. 2022

Plants

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Antibiotics are used in plant in vitro tissue culture to eliminate microbial contamination or for selection in genetic transformation. Antibiotic timentin has a relatively low cytotoxic effect on plant tissue culture; however, it could induce an enduring growth-inhibiting effect in tobacco in vitro shoot culture that persists after tissue transfer to a medium without antibiotic. The effect is associated with an increase in oxidative stress injury in plant tissues. In this study, we assessed changes of reactive oxygen species accumulation, protein expression, and oxidative protein modification response associated with enduring timentin treatment-induced growth suppression in tobacco (Nicotiana tabacum L.) in vitro shoot culture. The study revealed a gradual 1.7 and 1.9-fold increase in superoxide (O2•−) content at the later phase of the propagation cycle for treatment control (TC) and post-antibiotic treatment (PA) shoots; however, the O2•− accumulation pattern was different. For PA shoots, the increase in O2•− concentration occurred several days earlier, resulting in 1.2 to 1.4-fold higher O2•− concentration compared to TC during the period following the first week of cultivation. Although no protein expression differences were detectable between the TC and PA shoots by two-dimensional electrophoresis, the increase in O2•− concentration in PA shoots was associated with a 1.5-fold increase in protein carbonyl modification content after one week of cultivation, and protein carbonylation analysis revealed differential modification of 26 proteoforms involved in the biological processes of photosynthesis and glycolysis. The results imply that the timentin treatment-induced oxidative stress might be implicated in nontranslational cellular redox balance regulation, accelerates the development of senescence of the shoot culture, and contributes to the shoot growth-suppressing effect of antibiotic treatment.

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Section: Quantitative Analysis Of Protein Carbonylationmentioning

confidence: 99%

Enhanced Carbonylation of Photosynthetic and Glycolytic Proteins in Antibiotic Timentin-Treated Tobacco In Vitro Shoot Culture

Andriūnaitė

Rugienius

Tamošiūnė

et al. 2022

Plants

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“…The content of carbonylated proteins was determined by derivatizing carbonyls from proteins using 2.4-dinitrophenylhydrazine (DNPH), as performed by Xia et al (2016). The carbonyl content was calculated using the absorption coefficient of 22 000 M −1 cm -1 at 370 nm.…”

Section: Quantification Of Superoxide Anion Hydrogen Peroxide Mda And...mentioning

confidence: 99%

“…The carbonylated protein content was quantified through 0.3 g of seeds macerated in buffer containing Hepes-NaOH (10 mM, pH 7.5), protease inhibitor cocktail (0.1%), and β-mercaptoethanol (0.07%). The determination of carbonylated proteins was performed by derivatizing protein carbonyls using 2.4-dinitrophenylhydrazine (DNPH) (Xia et al, 2016).…”

Section: Quantification Of Ros and Oxidative Damagementioning

confidence: 99%

Effects of high temperature on morphophysiological, ultrastructural, and biochemical parameters during the germination of two forest species

Reis¹

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Climate change resulting from anthropogenic actions has increased global temperature. These temperature increases will likely influence the survival of part of the species. Among the environmental factors, the temperature is one of the most critical for affecting the physiological, biochemical, and molecular factors of the seed. Temperature increases is considered one of the environmental factors that help in the overproduction of reactive oxygen species (ROS). The urgency of understanding seed responses to climate change is particularly important. A detailed overview of seed responses to heat stress can help formulate appropriate strategies for species conservation and preservation. Knowledge about the capacity of forest seeds to deal with temperature changes is still scarce. In this sense, the objectives of this work were: 1. To investigate the morphophysiological, biochemical, and ultrastructural changes during germination of Melanoxylon brauna seeds under heat stress; 2. Evaluate the effects of temperature on morphology, generation of ROS, antioxidant system, and mobilization of reserves in Ormosia coarctata seeds; 3. Characterize the effects of diphenyleneiodonium (DPI) on M. brauna germination, internal anatomy, hydrogen peroxide (H 2 O 2 ) content, and activity of reserve enzymes and antioxidant enzymes under optimal conditions (25 °C) and stress by heat (40°C). To evaluate the internal morphology the seeds were radiographed. Ultrastructural and anatomical parameters were evaluated using transmission electron microscopy and light microscopy. ROS production, malondialdehyde (MDA) and glucose content, carbonyl proteins, and enzyme activity (superoxide dismutase - SOD, ascorbate peroxidase - APX, catalase - CAT, peroxidase - POX, glucose-6-phosphate dehydrogenase - G6PDH, lipase, α- and β-amylase and protease) were measured by spectrophotometric analysis. The high temperature causes a reduction in the percentage and speed of germination and affects the internal morphology of M. brauna and O. coarctcta seeds. In M. brauna seeds, heat stress decreases respiratory rates and compromises the structure of mitochondria. ROS content and protein carbonylation in seeds submitted to 40 °C increased in relation to 25 °C and 35°C. The activities of the enzymes SOD, APX, CAT, and POX were significantly reduced in seeds subjected to heat stress. Glucose content, G6PDH (M. brauna), and lipase activity also decreased in seeds exposed to high- temperature stress. Seed exposure to DPI decreased germination percentage at 25 °C and does not affect germination at 40 °C. DPI caused less reduction in the loosening of the outer layers of the integument and reduced the H 2 O 2 content and activity of all tested enzymes. In summary, heat stress negatively affects germination, promotes oxidative stress, and induces damage to mitochondrial ultrastructure, seed morphology, and anatomy. The data obtained in this study contribute to a better understanding of the effects of high temperatures on the germination of native seeds, in addition to showing the contribution of ROS to germination. Keywords: Climate change. Heat stress. Melanoxylon brauna. Ormosia coarctata.

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A Simplified Method to Assay Protein Carbonylation by Spectrophotometry

Moreau

Issakidis‐Bourguet

2022

Methods in Molecular Biology

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Determination of Protein Carbonylation and Proteasome Activity in Seeds

Cited by 8 publications

References 25 publications

Enhanced Carbonylation of Photosynthetic and Glycolytic Proteins in Antibiotic Timentin-Treated Tobacco In Vitro Shoot Culture

Enhanced Carbonylation of Photosynthetic and Glycolytic Proteins in Antibiotic Timentin-Treated Tobacco In Vitro Shoot Culture

Effects of high temperature on morphophysiological, ultrastructural, and biochemical parameters during the germination of two forest species

A Simplified Method to Assay Protein Carbonylation by Spectrophotometry

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