Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.
Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein-protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione.
Department of Stress Biology and Plant Pathology, CEBAS-CSIC, Campus Universitario de Espinardo, E-30100 Murcia, Spain. Electronic address: acalderon@cebas.csic.es.
Aingeru
A
Calderón
Department of Stress Biology and Plant Pathology, CEBAS-CSIC, Campus Universitario de Espinardo, E-30100 Murcia, Spain. Electronic address: amortiz@cebas.csic.es.
Ana
A
Ortiz-Espín
Centre for Plant Biotechnology and Genomics (CBGP; UPM-INIA), Campus de Montegancedo, Universidad Politécnica de Madrid, Pozuelo de Alarcón, E-28223 Madrid, Spain. Electronic address: raquel.iglesias@upm.es.
Raquel
R
Iglesias-Fernández
Centre for Plant Biotechnology and Genomics (CBGP; UPM-INIA), Campus de Montegancedo, Universidad Politécnica de Madrid, Pozuelo de Alarcón, E-28223 Madrid, Spain. Electronic address: p.carbonero@upm.es.
Pilar
P
Carbonero
Department of Physiology, Faculty of Medicine, University of Valencia, Av. Blasco Ibañez 15, E-46010 Valencia, Spain. Electronic address: Federico.V.Pallardo@uv.es.
Federico Vicente
FV
Pallardó
Department of Stress Biology and Plant Pathology, CEBAS-CSIC, Campus Universitario de Espinardo, E-30100 Murcia, Spain. Electronic address: fsevilla@cebas.csic.es.
Francisca
F
Sevilla
Department of Stress Biology and Plant Pathology, CEBAS-CSIC, Campus Universitario de Espinardo, E-30100 Murcia, Spain. Electronic address: ajimenez@cebas.csic.es.
Ana
A
Jiménez
D018929:Cell Culture Techniques / Q000379:methods; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D005978:Glutathione / Q000096:biosynthesis / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000166:cytology / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism
D018809:Proliferating Cell Nuclear Antigen; D013879:Thioredoxins; D005978:Glutathione
Cell cycle; Glutathione; Nucleus; Overexpression; Proliferating cell nuclear antigen (PCNA); Thioredoxin o1; Tobacco BY-2 cells