Salicylic acid and jasmonic acid biosynthetic pathways are simultaneously activated in transgenic Arabidopsis expressing the rolB/C gene from Ipomoea batatas.

The Agrobacterium rhizogenes root oncogenic locus (rol) genes interfere with hormone balance by altering their synthesis and/or recognition, giving rise to varied impacts on the physiological characteristics of plants and cell cultures. The homolog of the rolB and rolC genes from Ipomoea batatas, named Ib-rolB/C, similarly induces morphological and physiological alterations in transgenic Arabidopsis thaliana; however, its role in plant hormonal homeostasis has not been previously defined. In this study, we found that external application of salicylic acid (SA) and methyl jasmonate (MeJA) significantly upregulated Ib-rolB/C in detached I. batatas leaves. Furthermore, heterologous expression of Ib-rolB/C in A. thaliana markedly enhanced the accumulation of SA and MeJA, and to a lesser extent, elevated abscisic acid (ABA) levels, through the modulation of genes specific to hormone biosynthesis. Even though the RolB/RolC homolog protein has a notable structural resemblance to the RolB protein from A. rhizogenes, it exhibits a distinct localization pattern, predominantly residing in the cytoplasm and certain discrete subcellular structures, instead of the nucleus. Consequently, the functions of RolB/RolC in both naturally and artificially transgenic plants are linked to changes in the hormonal state of the cells, though the underlying signaling pathways remain to be elucidated.

Inhibition of the JAZ1 gene causes activation of camalexin biosynthesis in Arabidopsis callus cultures.

Indole alkaloid camalexin has potential medicinal properties such as suppressing the viability of leukemic but not normal cells. Camalexin is not produced in plants and an external factor is required to activate its biosynthesis. In this work, we stimulated camalexin biosynthesis in Arabidopsis calli by blocking one of repressors of the jasmonate pathway, the jasmonate ZIM-domain protein 1 (JAZ1) by using amiRNA targeting JAZ1 gene transcripts. Inhibition of the JAZ1 gene led to an increase in camalexin content from trace amounts in control culture to 9 µg/g DW in the jaz1 line without affecting growth. In addition, JAZ1 silencing enhanced tolerance to cold stress with simultaneous increasing camalexin content up to 30 µg/g DW. Real-time quantitative PCR determination of marker gene expression showed that effects caused by the JAZ1 silencing might be realized through crosslinking JA, ROS, and abscisic acid signaling pathways. Thus, targeting the distal components of signaling pathways can be suggested as a tool for bioengineering of secondary metabolism, along with standard techniques for targeting biosynthetic genes or genes encoding transcription factors.

Managing activity and Ca2+ dependence through mutation in the Junction of the AtCPK1 coordinates the salt tolerance in transgenic tobacco plants.

Calcium-dependent protein kinases (CDPKs) are Ca2+ 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 Ca2+ 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.

Inactivation of the auto-inhibitory domain in Arabidopsis AtCPK1 leads to increased salt, cold and heat tolerance in the AtCPK1-transformed Rubia cordifolia L cell cultures.

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.

Synthesis of bioactive silver nanoparticles using alginate, fucoidan and laminaran from brown algae as a reducing and stabilizing agent.

In this report, polysaccharides - alginate, fucoidan, laminaran - were isolated from marine algae Saccharina cichorioides and Fucus evanescens and their activity as a reducing and stabilizing agents in the biogenic synthesis of silver nanoparticles was evaluated. The cytotoxic and antibacterial properties of obtained nanoparticles were also assessed. It was found that all tested polysaccharides could be used as a reducing agent; however, their catalytic activities varied significantly in the following range alginate < fucoidan < laminaran. Nanoparticles demonstrated cytotoxicity against rat C6 glioma cells. It was considerably higher for alginate- and laminaran-obtained nanosilver samples compared to fucoidan. Additionally, silver nanoparticles possessed considerable antibacterial properties more pronounced in fucoidan-obtained samples. Our data demonstrate that different algal polysaccharides can be used for the synthesis of silver nanoparticles with varying bioactivities.

Activation of anthraquinone biosynthesis in long-cultured callus culture of Rubia cordifolia transformed with the rolA plant oncogene.

The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200 mg/l to 874 mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.

Increase in isoflavonoid content in Glycine max cells transformed by the constitutively active Ca2+ independent form of the AtCPK1 gene.

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 Ca2+ 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.

Silicon Crystals Formation Using Silicatein-Like Cathepsin of Marine Sponge Latrunculia oparinae.

The cDNA fragment encoding the catalytic domain of the new silicatein-like cathepsin enzyme LoCath was expressed in a strain Top10 of Escherichia coli, extracted and purified via nickel-affinity chromatography. Recombinant enzyme performed silica-polymerizing activity when mixed with water-soluble silica precursor-tetrakis-(2-hydroxyethyl)-orthosilicate. Scanning electron microscopy revealed hexagonal, octahedral and ß-tridimit crystals. Energy dispersion fluorescence X-ray spectrometry analysis showed that all these crystals consist of pure silicon oxide. It is the first report about the ability of marine sponge's cathepsin to polymerize silicon, as well as about the structure and composition of the silicon oxide crystal formed by recombinant cathepsin. Further study of the catalytic activity of silicatein and cathepsin will help to understand the biosilification processes in vivo, and will create basis for biotechnological use of recombinant proteins for silicon polymerization.

Expression profiles of calcium-dependent protein kinase genes (CDPK1-14) in Agrobacterium rhizogenes pRiA4-transformed calli of Rubia cordifolia under temperature- and salt-induced stresses.

Agrobacterium rhizogenes genetically transform plant cells naturally via horizontal gene transfer by the introduction of T-DNA from the Ri plasmid into genomic DNA to create favorable conditions for successful colonization. An intriguing feature of pRiA4-transformed cells is their recently discovered enhanced tolerance to abiotic stress stimuli and activation of antioxidant enzyme expression. The mechanism by which A. rhizogenes modulates the defense responses of transformed cells remains unclear. It has been established that calcium-dependent protein kinase (CDPK) genes mediate crosstalk of signaling pathways in plants, and these genes have been implicated in biotic and abiotic stress signaling. In this study, we identified fourteen CDPK genes from Rubia cordifolia and examined their expression in aerial plant organs as well as in non-transformed and A. rhizogenes A4-transformed calli. Expression of RcCDPK4, RcCDPK5, RcCDPK7, and RcCDPK10 was 1.2- to 3.9-fold higher in pRiA4-transformed cells than in non-transformed cells, whereas expression of RcCDPK1, RcCDPK9, RcCDPK11, and RcCDPK14 was 1.2- to 1.9-fold lower. Agrobacterium transformation substantially modified the transcriptional responses of specific RcCDPK isoforms in pRiA4-transformed cells under conditions of temperature- and salinity-induced stress. On the basis of the results, we suggest that A. rhizogenes T-DNA genes exert their diverse biological functions by altering the expression of various CDPK genes.

The production of class III plant peroxidases in transgenic callus cultures transformed with the rolB gene of Agrobacterium rhizogenes.

The production of plant peroxidases by plant cell cultures is of great interest because of the potential for industrial applications. We used plant cell cultures overexpressing the rolB gene to produce increased amounts of plant class III peroxidases. The rolB gene ensured the stable and permanent activation of peroxidase activity in the transformed callus cultures of different plants. In particular, the total peroxidase activity in transformed Rubia cordifolia cells was increased 23-86-fold, and the abundance of the major peroxidase gene transcripts was increased 17-125-fold (depending on the level of rolB expression) compared with non-transformed control calli. The peroxidase-activating effect of rolB was greater than that of other peroxidase inducers, such as external stresses and methyl jasmonate.

Molecular cloning and characterization of seven class III peroxidases induced by overexpression of the agrobacterial rolB gene in Rubia cordifolia transgenic callus cultures.

Here, seven new class III peroxidase genes of Rubia cordifolia L., RcPrx01-RcPrx07, were isolated and characterized. Expression of the Prx genes was studied in R. cordifolia aerial organs as well as in cells transformed with the rolB and rolC genes of Agrobacterium rhizogenes and cells transformed with the wild-type A. rhizogenes A4 strain. In rolC- and rolB-transformed cells, the rol genes were expressed under the control of the 35S promoter, whereas in A. rhizogenes A4-transformed cells the rol genes were expressed under the control of their native promoters. All studied peroxidase genes were greatly upregulated in rolB-overexpressing cells. In contrast, overexpression of the rolC gene and expression of the rol genes under the control of their native promoters had little effect on the abundance of peroxidase transcripts. In accordance with this observation, peroxidase activity was substantially increased in rolB cells and was slightly affected in other transformed cells. Our results indicate that rolB strictly affects the regulation of a set of seven R. cordifolia class III peroxidases.

Induction of anthraquinone biosynthesis in Rubia cordifolia cells by heterologous expression of a calcium-dependent protein kinase gene.

Calcium-dependent protein kinases (CDPKs) play an important role in plant cell responses to stress and pathogenic attack. In this study, we investigated the effect of heterologous expression of the Arabidopsis CDPK gene, AtCPK1, on anthraquinone production in transgenic Rubia cordifolia cells. AtCPK1 variants (a constitutively active, Ca(2+) -independent form and a non-active form used as a negative control) were transferred to callus cells by agrobacterial transformation. Overexpression of the constitutively active, Ca(2+) -independent form in R. cordifolia cells caused a 10-fold increase in anthraquinone content compared with non-transformed control cells, while the non-active form of AtCPK1 had no effect on anthraquinone production. Real-time PCR measurements showed that the activation of anthraquinone biosynthesis in transgenic calli correlated with the activation of isochorismate synthase gene expression. The activator effect of AtCPK1 was stable during prolonged periods of transgenic cell cultivation (more than 3 years) and the transgenic cultures exhibited high growth. Our results provide the first evidence that a CDPK gene can be used for the engineering of secondary metabolism in plant cells.

Decreased ROS level and activation of antioxidant gene expression in Agrobacterium rhizogenes pRiA4-transformed calli of Rubia cordifolia.

Microbe-plant interactions often lead to a decrease in the reactive oxygen species (ROS) level of plant cells, which allows pathogen survival through the suppression of plant immune responses. In the present investigation, we tested whether transformation of Rubia cordifolia cells by Agrobacterium rhizogenes had a similar effect. We isolated partial cDNA sequences of ascorbate peroxidase, catalase and Cu/Zn superoxide dismutase genes (RcApx1, RcApx2, RcApx3, RcCAT1, RcCAT2, RcCSD1, RcCSD2 and RcCSD3) from plant tissues, as well as pRiA4-transformed and normal calli of Rubia cordifolia, and studied their expression by real-time PCR. Transcription profiling revealed that ascorbate peroxidase (RcApx1) and Cu/Zn superoxide dismutase (RcCSD1) were the most abundant transcripts present in both plant tissues and non-transformed calli. Catalase genes were weakly expressed in these samples. The pRiA4-transformed calli showed enhanced expression of several genes encoding ROS-detoxifying enzymes. Confocal microscopy imaging revealed decreased ROS level in pRiA4-transformed calli compared to the control. These results demonstrate that A. rhizogenes, like other plant pathogens, uses a strategy aimed at decreasing ROS levels in host cells through the general upregulation of its antioxidant genes.

Effects of Ca(2+) channel blockers and protein kinase/phosphatase inhibitors on growth and anthraquinone production in Rubia cordifolia callus cultures transformed by the rolB and rolC genes.

The transformation of Rubia cordifolia L. cells by the 35S- rolB and 35S- rolC genes of Agrobacterium rhizogenes caused a growth inhibition of the resulting cultures and an induction of the biosynthesis of anthraquinone-type phytoalexins. Inhibitor studies revealed a striking difference between the rolC- and rolB-gene-transformed cultures in their sensitivity to verapamil, an L-type Ca(2+) channel blocker. The rolC culture possessed a 2-fold lowered resistance to the inhibitor than the normal culture, while the rolB culture was 4-fold more resistant to the treatment. Additionally, growth of the rolC culture was totally inhibited when the culture was grown in Ca(2+)-free medium, whereas growth of the rolB culture was reduced by less than half. We interpreted these results as evidence for a lack of calcium homeostasis in both transgenic cultures. Anthraquinone (AQ) production was not inhibited in the normal or transformed cultures by the Ca(2+) channel blockers verapamil and LaCl(3), or by diphenylene iodonium, an inhibitor of NADPH oxidase, or by the protein kinase inhibitor staurosporine. These results indicate that the induction of AQ production in non-transgenic and transgenic cultures does not proceed through the activation of the common Ca(2+)-dependent NADPH oxidase pathway that mediates signal transduction between an elicitor-receptor complex via transcriptional activation of defense genes. Okadaic acid and cantharidin, inhibitors of protein phosphatases 1 and 2A, caused an increase in AQ production in transgenic cultures. Okadaic acid stimulated AQ accumulation in the non-transformed culture, whereas cantharidin had no effect. These results show that different phosphatases are involved in AQ synthesis in normal and transgenic cultures of R. cordifolia.