Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture

Abstract 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 PsTrx o 1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrx o 1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrx o 1 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, PsTrx o 1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrx o 1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrx o 1 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 PsTrx o 1 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 Trx o 1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione.