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Gene transfer from Agrobacterium to plants is the best studied example of horizontal gene transfer (HGT) between prokaryotes and eukaryotes. The rol genes of A. rhizogenes (Rhizobium rhizogenes) provide uncontrolled root growth, or "hairy root" syndrome, the main diagnostic feature. In the present study, we investigated the stable pRiA4-transformed callus culture of Rubia cordifolia L. While untransformed callus cultures need PGRs (plant growth regulators) as an obligatory supplement, pRiA4 calli is able to achieve long-term PGR-free cultivation. For the first time, we described the pRiA4-transformed callus cultures' PGR-dependent ROS status, growth, and specialized metabolism. As we have shown, expression of the rolA and rolB but not the rolC genes is contradictory in a PGR-dependent manner. Moreover, a PGR-free pRiA4 transformed cell line is characterised as more anthraquinone (AQ) productive than an untransformed cell culture. These findings pertain to actual plant biotechnology: it could be the solution to troubles in choosing the best PGR combination for the cultivation of some rare, medicinal, and woody plants; wild-type Ri-plants and tissue cultures may become freed from legal controls on genetically modified organisms in the future. We propose possible PGR-dependent relationships between rolA and rolB as well as ROS signalling targets. The present study highlighted the high importance of the rolA gene in the regulation of combined rol gene effects and the large knowledge gap in rolA action., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Calcium-dependent protein kinases (CDPKs) are Ca 2+ decoders in plants. AtCPK1 is a positive regulator in the plant response to biotic and abiotic stress. Inactivation of the autoinhibitory domain of AtCPK1 in the mutated form KJM23 provides constitutive activity of the kinase. In the present study, we investigated the effect of overexpressed native and mutant KJM23 forms on salinity tolerance in Nicotiana tabacum. Overexpression of native AtCPK1 provided tobacco resistance to 120 mM NaCl during germination and 180 mM NaCl during long-term growth, while the resistance of plants increased to 240 mM NaCl during both phases of plant development when transformed with KJM23. Mutation in the junction KJM4, which disrupted Ca 2+ induced activation, completely nullified the acquired salt tolerance up to levels of normal plants. Analysis by confocal microscopy showed that under high salinity conditions, overexpression of AtCPK1 and KJM23 inhibited reactive oxygen species (ROS) accumulation to levels observed in untreated plants. Quantitative real-time PCR analysis showed that overexpression of AtCPK1 and KJM23 was associated with changes in expression of genes encoding heat shock factors. In all cases, the KJM23 mutation enhanced the effect of AtCPK1, while the KJM4 mutation reduced it to the control level. We suggest that the autoinhibitory domains in CDPKs could be promising targets for manipulation in engineering salt-tolerant plants., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Calcium-dependent protein kinases (CDPKs) are essential regulators of plant growth and development, biotic and abiotic stress responses. Inactivation of the auto-inhibitory domain (AID) of CDPKs provides the constitutive activity. This study investigated the effect of overexpressed native and constitutive active (AtCPK1-Ca) forms of the AtCPK1 gene on abiotic stress tolerance and the ROS/redox system in Rubia cordifolia transgenic callus lines. Overexpression of the native AtCPK1 increased tolerance to salinity and cold almost in two times, when AtCPK1-Ca - in three times compare to control culture. A more interesting effect of overexpression of the AtCPK1 and AtCPK1-Ca was observed for heat resistance. The native form of AtCPK1 increased resistance to heating by 45%, while the AtCPK1-Ca increased by 80%. At the same time, another type of mutation of the AID (AtCPK1-Na, not active) did not affect the tolerance of the cell culture to stresses. We suppose, in this process, the ROS/redox system might be involved. Levels of intracellular ROS, ROS-generating enzymes expression and activities (Rbohs, Prx) and ROS-detoxifying enzymes (SOD, Cat, Apx and Prx) changed in a coordinated manner and in strict interconnection, depending of the callus growth phase and correlated with improved stress tolerance caused by AtCPK1. Because overexpression of both the AtCPK1 and AtCPK1-Ca did not significantly change callus growth, we propose that inactivation of AID of the AtCPK1 or its ortholog, might be an interesting instrument for improvement of plant cells resistance to abiotic stress., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)