Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR

We have previously shown that stromal and thylakoid-bound ascorbate peroxidase (APX) isoenzymes of spinach chloroplasts arise from a common pre-mRNA by alternative splicing in the C-terminus of the isoenzymes [Ishikawa, Yoshimura, Tamoi, Takeda and Shigeoka (1997) Biochem. J. 328, 795–800]. To explore the production of mature, functional mRNA encoding chloroplast APX isoenzymes, reverse transcriptase-mediated PCR and S1 nuclease protection analysis were performed with poly(A)+ RNA or polysomal RNA from spinach leaves. As a result, four mRNA variants, one form of thylakoid-bound APX (tAPX-I) and three forms of stromal APX (sAPX-I, sAPX-II and sAPX-III), were identified. The sAPX-I and sAPX-III mRNA species were generated through the excision of intron 11; they encoded the previously identified sAPX protein. Interestingly, the sAPX-II mRNA was generated by the insertion of intron 11 between exons 11 and 12. The use of this insertional sequence was in frame with the coding sequence and would lead to the production of a novel isoenzyme containing a C-terminus in which a seven-residue sequence replaced the last residue of the previously identified sAPX. The recombinant novel enzyme expressed in Escherichia coli showed the same enzymic properties (except for molecular mass) as the recombinant sAPX from the previously identified sAPX-I mRNA, suggesting that the protein translated from the sAPX-II mRNA is functional as a soluble APX in vivo. The S1 nuclease protection analysis showed that the expression levels of mRNA variants for sAPX and tAPX isoenzymes are in nearly equal quantities throughout the spinach leaves grown under normal conditions. The present results demonstrate that the expression of chloroplast APX isoenzymes is regulated by a differential splicing efficiency that is dependent on the 3´-terminal processing of ApxII, the gene encoding the chloroplast APX isoenzymes.

Download Full-text

RNA metabolism is the primary target of formamide in vivo

Scientific Reports ◽

10.1038/s41598-017-16291-8 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 4

Author(s):

Rafael Hoyos-Manchado ◽

Félix Reyes-Martín ◽

Charalampos Rallis ◽

Enrique Gamero-Estévez ◽

Pablo Rodríguez-Gómez ◽

...

Keyword(s):

Rna Metabolism ◽

Primary Target

Download Full-text

Antisense reduction of thylakoidal ascorbate peroxidase in Arabidopsis enhances Paraquat-induced photooxidative stress and Nitric Oxide-induced cell death

Planta ◽

10.1007/s00425-005-1485-9 ◽

2005 ◽

Vol 221 (6) ◽

pp. 757-765 ◽

Cited By ~ 43

Author(s):

Delia Tarantino ◽

Candida Vannini ◽

Marcella Bracale ◽

Manuela Campa ◽

Carlo Soave ◽

...

Keyword(s):

Nitric Oxide ◽

Cell Death ◽

Ascorbate Peroxidase ◽

Photooxidative Stress

Download Full-text

Comparison of defence responses to Botrytis cinerea infection in tomato plants propagated in vitro and grown in vivo

Acta Agrobotanica ◽

10.5586/aa.1996.012 ◽

2013 ◽

Vol 49 (1-2) ◽

pp. 115-121 ◽

Cited By ~ 1

Author(s):

Jacek Patykowski ◽

Elżbieta Kuźniak ◽

Henryk Urbaniak

Keyword(s):

Superoxide Dismutase ◽

Botrytis Cinerea ◽

Ascorbate Peroxidase ◽

Guaiacol Peroxidase ◽

Activity Increase ◽

Tomato Plants ◽

Defence Responses ◽

Defence Reactions

Defence reactions: O2 - generation, superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase activities after B. cinerea infection in tomato plants propagated in vitro and grown in vivo have been compared. Infection resulted in rapid O2 - generation. Superoxide dismutase activity increase was slower than O2 - response. In plants propagated in vitro catalase and guaiacol peroxidase activities after infection were induced less strongly than in plants grown in vivo. K2HPO4 pretreatment of plants grown in vitro enhanced significantly the activities of catalase and guaiacol peroxidase after infection. Slight restriction of B. cinerea infection development in in vitro propagated plants pretreated with K2HP04 was observed.

Download Full-text

Biosynthesis of Nitrogenous Phospholipids in Spinach Leaves

Canadian Journal of Biochemistry ◽

10.1139/o74-071 ◽

1974 ◽

Vol 52 (6) ◽

pp. 469-482 ◽

Cited By ~ 38

Author(s):

M. O. Marshall ◽

M. Kates

Keyword(s):

Microsomal Fraction ◽

Enzyme System ◽

Supernatant Fraction ◽

Exchange Reactions ◽

Methylation Pathway ◽

Ethanolamine Kinase ◽

Spinach Leaves

Pathways for biosynthesis of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), in spinach leaves have been studied both in vivo (whole leaves and leaf slices) and in vitro (cell-free leaf fractions). Biosynthesis of PS was shown to occur by the action of a particle-bound CDP-diglyceride: serine phosphatidyltransferase, and PE by the action of a PS-decarboxylase localized in the 100 000 × g supernatant fraction. PE was also formed by the operation of the CDP-ethanolamine:diglyceride phosphorylethanolamine transferase, localized in the microsomal fraction. The presence of ethanolamine kinase required for formation of phosphorylethanolamine was demonstrated in vitro, but not the presence of CTP:phosphorylethanolamine cytidyltransferase; however, the latter is presumed present on the basis of in vivo results. Operation of the methylation pathway for biosynthesis of PC was established in vivo, and direct methylation of phosphatidyl-N-methylethanolamine to phosphatidyl-N,N-dimethylethanolamine (PE-diMe) and of PE-diME to PC by S-adenosylmethionine was demonstrated with a particulate enzyme system localized in the microsomal fraction; direct methylation of PE itself could not be shown in this system. PC was also synthesized by the CDP-choline:diglyceride phosphorylcholine transferase system localized in the microsomal fraction. Synthesis of PE and PC by Ca2+-stimulated exchange reactions with ethanolamine and choline, respectively, could be demonstrated, but at low rates. However, no synthesis of PS by exchange reactions with serine could be detected.

Download Full-text

Chloroplast division in spinach leaves examined by scanning electron microscopy and freeze-etching

Journal of Cell Science ◽

10.1242/jcs.46.1.87 ◽

1980 ◽

Vol 46 (1) ◽

pp. 87-96

Author(s):

N. Chaly ◽

J.V. Possingham ◽

W.W. Thomson

Keyword(s):

Electron Microscopy ◽

Green Light ◽

Cell Area ◽

Low Intensity ◽

Spinach Leaf ◽

Freeze Etching ◽

Leaf Disks ◽

Scanning Electron ◽

Spinach Leaves

Spinach leaf disks were cultured for 5 days in low-intensity green light and then were transferred to high-intensity white light. Harvests over the next 16 h established that cell area increased by about 80% and chloroplast number per cell increased by about 65%, while the percentage of dumbbell-shaped chloroplasts per cell decreased by 65%. Freeze-etch replicas of fixed and unfixed leaf disks, as well as scanning electron-microscope preparations of fixed material, contained dumbbell-shaped chloroplasts constricted to various degrees. Freeze-etch replicas of unfixed cells from young leaf bases, in which the number of chloroplasts per cell is known to be rapidly increasing, also contained many constricted chloroplasts. It is concluded that dumbbell-shaped chloroplasts occur in vivo and represent a stage in the division of chloroplasts.

Download Full-text

Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.11.2505 ◽

1996 ◽

Vol 40 (11) ◽

pp. 2505-2510 ◽

Cited By ~ 122

Author(s):

J Tankovic ◽

B Perichon ◽

J Duval ◽

P Courvalin

Keyword(s):

Staphylococcus Aureus ◽

Streptococcus Pneumoniae ◽

Gene Amplification ◽

Low Frequency ◽

Quinolone Resistance ◽

Fluoroquinolone Resistance ◽

Primary Target ◽

Total Dna

We have analyzed by gene amplification and sequencing mutations in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes of fluoroquinolone-resistant Streptococcus pneumoniae mutants obtained during therapy or in vitro. Mutations leading to substitutions in ParC were detected in the two mutants obtained in vivo, BM4203-R (substitution of a histidine for an aspartate at position 84 [Asp-84-->His]; Staphylococcus aureus coordinates) and BM4204-R (Ser-80-->Phe), and in two mutants obtained in vitro (Ser-80-->Tyr). An additional mutant obtained in vitro, BM4205-R3, displayed a higher level of fluoroquinolone resistance and had a mutation in gyrA leading to a Ser-84-->Phe change. We could not detect any mutation in the three remaining mutants obtained in vitro. Total DNA from BM4203-R, BM4204-R, and BM4205-R3 was used to transform S. pneumoniae CP1000 by selection on fluoroquinolones. For the parC mutants, transformants with phenotypes indistinguishable from those of the donors were obtained at frequencies (5 x 10(-3) to 8 x 10(-3)) compatible with monogenic transformation. By contrast, transformants were obtained at a low frequency (4 x 10(-5)), compatible with the transformation of two independent genes, for the gyrA mutant. Resistant transformants of CP1000 were also obtained with an amplified fragment of parC from BM4203-R and BM4204-R but not with a gyrA fragment from BM4205-R3. All transformants had mutations identical to those in the donors. These data strongly suggest that ParC is the primary target for fluoroquinolones in S. pneumoniae and that BM4205-R3 is resistant to higher levels of the drugs following the acquisition of two mutations, including one in gyrA.

Download Full-text

Melatonin synthesis enzymes interact with ascorbate peroxidase to protect against oxidative stress in cassava

Journal of Experimental Botany ◽

10.1093/jxb/eraa267 ◽

2020 ◽

Vol 71 (18) ◽

pp. 5645-5655 ◽

Cited By ~ 1

Author(s):

Yujing Bai ◽

Jingru Guo ◽

Russel J Reiter ◽

Yunxie Wei ◽

Haitao Shi

Keyword(s):

Oxidative Stress ◽

Antioxidant Capacity ◽

Stress Tolerance ◽

Ascorbate Peroxidase ◽

Stress Responses ◽

Direct Interaction ◽

Ros Scavenging ◽

Melatonin Synthesis

Abstract Melatonin is an important indole amine hormone in animals and plants. The enzymes that catalyse melatonin synthesis positively regulate plant stress responses through modulation of the accumulation of reactive oxygen species (ROS). However, the relationship between melatonin biosynthetic enzymes and ROS-scavenging enzymes has not been characterized. In this study, we demonstrate that two enzymes of the melatonin synthesis pathway in Manihot esculenta (MeTDC2 and MeASMT2) directly interact with ascorbate peroxidase (MeAPX2) in both in vitro and in vivo experiments. Notably, in the presence of MeTDC2 and MeASMT2, MeAPX2 showed significantly higher activity and antioxidant capacity than the purified MeAPX2 protein alone. These findings indicate that MeTDC2–MeAPX2 and MeASMT2–MeAPX2 interactions both activate APX activity and increase antioxidant capacity. In addition, the combination of MeTDC2, MeASMT2, and MeAPX2 conferred improved resistance to hydrogen peroxide in Escherichia coli. Moreover, this combination also positively regulates oxidative stress tolerance in cassava. Taken together, these findings not only reveal a direct interaction between MeTDC2, MeASMT2, and MeAPX2, but also highlight the importance of this interaction in regulating redox homoeostasis and stress tolerance in cassava.

Download Full-text