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Calcium-dependent protein kinases (CDPKs) are very effective calcium signal decoders due to their unique structure, which mediates substrate-specific [Ca 2+ ] cyt signalling through phosphorylation. However, Ca 2+ -dependence makes it challenging to study CDPKs. This work focused on the effects of the overexpression of native and modified forms of the AtCPK1 gene on the tolerance of tobacco plants to heat and cold. We studied the interaction between the calcium and signalling systems of abscisic acid (ABA) at various temperatures. The hormonal state, stress-induced senescence, and expression of important corresponding genes were investigated. We showed that inactivation of the autoinhibitory domain of the modified constitutively active form of AtCPK1 has a positive effect on resistance not only to long-term cold but also to heat. We showed that the constitutively active form of AtCPK1 under nonstressed conditions activated biosynthesis of ABA, but a decrease in ABA content was detected upon heat exposure. On the basis of our results, we can assume that this effect is achieved through the CPK-dependent activation of salicylic acid (SA) signalling. The obtained data shed light on heat-associated molecular processes and support the possibility of using intradomain modifications of CDPK both for comprehensive study of its functional features and as a bioengineering tool., 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 © 2024 Elsevier GmbH. All rights reserved.)

Main Conclusion: Long-term cultured calli may experience a biosynthetic shift due to the IAA-dependent expression of the rolA gene, which also affects ROS metabolism. The "hairy root" syndrome is caused by the root-inducing Ri-plasmid of Rhizobium rhizogenes, also known as Agrobacterium rhizogenes. The Ri-plasmid contains genes known as rol genes or root oncogenic loci, which promote root development. The important implications of the rolA gene from the T-DNA include reduced plant size, resistance to infections, and the activation of specialised metabolism. Nevertheless, rolA does not belong to the plast gene group because its function is still uncertain. Recent investigations have shown two important effects of the rolA gene. First, the production of secondary metabolites has changed in long-term cultivated rolA-transgenic calli of Rubia cordifolia L. Second, the expression of both the rolA and rolB genes has a strong auxin-dependent antagonistic effect on reactive oxygen species (ROS) homeostasis. In this work, we attempted to elucidate two rolA gene phenomena: what caused the secondary metabolism of long-term cultured calli to change? How does the individual expression of the rolA gene affect ROS homeostasis? We analysed SNPs in the 5' untranslated region and coding region of the rolA gene. These mutations do not affect the known essential amino acids of the RolA proteins. Notably, in the promoter of the rolA gene, an ACTTTA motif for auxin-mediated transcription factors was identified. Using two separate cell cultures, we demonstrated the strong auxin dependence of rolA gene expression. The expression of genes involved in ROS metabolism decreased in response to an auxin-mediated increase in rolA gene expression. Two assumptions can be made. The long-term cultivation of calli may cause changes in the hormonal state of the culture over time, which may modulate the action of the RolA protein. Moreover, auxin-dependent expression of the rolA gene led to a decrease in ROS metabolism. It can be assumed that the antagonistic interaction between rolA and rolB prevents strong rolB-induced auxin sensitivity and oxidative bursts to balance the cell state., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no competing interests. Ethics approval: Not applicable. Consent to participate: Not applicable. Consent for publication: All the authors whose names appeared on the submission approved the version to be published and agreed to be accountable for all aspects of the work in ensuring that the questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 one of the main Ca 2+ decoders in plants. Among them, Arabidopsis thaliana AtCPK1 is one of the most studied CDPK genes as a positive regulator of plant responses to biotic and abiotic stress. The mutated form of AtCPK1 , in which the autoinhibitory domain is inactivated ( AtCPK1-Ca ), provides constitutive kinase activity by mimicking a stress-induced increase in the Ca 2+ flux. In the present study, we performed a proteomic analysis of Vitis amurensis calli overexpressing the AtCPK1-Ca form using untransformed calli as a control. In our previous studies, we have shown that the overexpression of this mutant form leads to the activation of secondary metabolism in plant cell cultures, including an increase in resveratrol biosynthesis in V. amurensis cell cultures. We analyzed upregulated and downregulated proteins in control and transgenic callus cultures using two-dimensional gel electrophoresis, and Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF). In calli transformed with AtCPK1-Ca , an increased amounts of pathogenesis-related proteins were found. A quantitative real-time PCR analysis confirmed this result.

Calcium-dependent protein kinases (CDPKs) regulate plant development and stress responses. However, the interaction of these protein kinases with the abscisic acid (ABA) stress hormone signalling system has not been studied in detail. In Arabidopsis , AtCPK1 plays an important role in the acclimation of plants to environmental stresses. Phylogenetic and molecular analyses showed that, among 50 isoforms of Glycine max (L.) Merrill CDPKs, the GmCDPK27/GmCDPK48, GmCDPK5/GmCDPK24, and GmCDPK10/GmCDPK46 paralogous pairs were the isoforms most related to AtCDPK1. We investigated the expression of the corresponding six GmCDPKs genes during treatment with cold, heat, and salt stress. Wild soybean was the most resistant to stresses, and among the three cultivars studied (Sfera, Hodgson, and Hefeng25), Sfera was close to the wild type in terms of resistance. GmCDPK5 and GmCDPK10 were the most responsive to stress treatments, especially in wild soybean, compared with cultivars. Among the studied GmCDPK isoforms, only GmCDPK5 expression increased after treatment with abscisic acid (ABA) in a dose- and time-dependent manner. Targeted LC-MS/MS analysis of endogenous ABA levels showed that wild soybean and Sfera had nearly twice the ABA content of Hodgson and Hefeng25. An analysis of the expression of marker genes involved in ABA biosynthesis showed that GmNCED1 -gene-encoding 9-cis-epoxycarotenoid dioxygenase 1 is induced to the greatest extent in wild soybean and Sfera under salt, cold, and heat exposure. Our data established a correlation between the induction of GmCDPK5 and ABA biosynthesis genes. GmCDPK5 is an interesting target for genetic and bioengineering purposes and can be used for genetic editing, overexpression, or as a marker gene in soybean varieties growing under unfavourable conditions.

Main Conclusion: Increased flavonol accumulation and enhanced drought tolerance in A4-rolB-overexpressing plants can be explained by the cooperative action of the SA and ROS signalling pathways. Clarification of function of the A4-rolB plast gene from pRiA4 of Rhizobium rhizogenes will allow a better understanding of the biological principles of the natural transformation process and its use as a tool for plant bioengineering. In the present study, we investigated whether the overexpression of A4-rolB gene could regulate two important processes, flavonoid biosynthesis and drought tolerance. In addition, we investigated some aspects of the possible machinery of the A4-rolB-induced changes in plant physiology, such as crosstalk of the major signalling systems. Based on the data obtained in this work, it can be presumed that constitutive overexpression of A4-rolB leads to the activation of the salicylic acid signalling system. An increase in flavonol accumulation and enhanced drought tolerance can be explained by the cooperative action of SA and ROS pathways., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

The cultivation of soybean plants is one of the most important crop production sectors in the world. Isoflavones are an important defence against pathogens in soybeans. The aim of the present study was to analyse isoflavone biosynthesis in wild and cultivated soybeans grown in the field conditions in an unfavourable climate. We analysed by LCMS-IT-TOF the composition and content of isoflavonoids, productivity and fungal disease resistance of wild and cultivated. The Hefeng25 and Sfera varieties have the highest isoflavonoid content and fungal tolerance. We have shown a 3-fold increase of total isoflavonoids in Sfera, comparing with wild type, and 4- and 7-fold increases of total isoflavone aglycones in Hefeng25 and Sfera, respectively. Accordingly, the expression of genes encoding enzymes of the isoflavonoid biosynthetic pathway was also maximal in these cultivars. Thus, biosynthetic status is an important indicator of soybean productivity and resistance to pathogens in adverse climates., (Copyright © 2020 Elsevier Ltd. 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.)

Calcium-dependent protein kinases (CDPKs) represent a class within a multigene family that plays an important role in biotic and abiotic plant stress responses and is involved in the regulation of secondary metabolite biosynthesis. Our previous study showed that overexpression of the mutant constitutively active Ca 2+ independent form of the AtCPK1 gene (AtCPK1-Ca) significantly increased the biosynthesis of anthraquinones and stilbenes in Rubia cordifolia L. and Vitis amurensis Rupr. transgenic cell cultures, respectively. Here, we have established transgenic calli of soybean plants Glycine max (L.) Merr. that express the AtCPK1-Ca gene. Heterologous expression of the AtCPK1-Ca gene provoked a 5.2-fold increase in total isoflavone production up to 208.09 mg/L, along with an increase in isoflavone aglycones production up to 6.60 mg/L, which is 3-fold greater than that of the control culture. The production of prenylated isoflavones significantly increased, reaching 3.78 mg/L, 13-fold higher than in the control culture. The expression levels of 4-coumarate:CoA ligases, isoflavone synthases, 2-hydroxyisoflavanone dehydratase, isoflavone dimethylallyltransferase, and coumestrol 4-dimethylallyltransferase genes in transgenic cell cultures significantly increased. Thus, heterologous expression of the AtCPK1-Ca gene can be used to bioengineer plant cell cultures that produce isoflavonoids., (Copyright © 2018 Elsevier Ltd. All rights reserved.)