CRISPR/Cas9-Mediated Knockout of HOS1 Reveals Its Role in the Regulation of Secondary Metabolism in Arabidopsis thaliana.

In Arabidopsis, the RING finger-containing E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) functions as a main regulator of the cold signaling. In this study, CRISPR/Cas9-mediated targeted mutagenesis of the HOS1 gene in the first exon was performed. DNA sequencing showed that frameshift indels introduced by genome editing of HOS1 resulted in the appearance of premature stop codons, disrupting the open reading frame. Obtained hos1 Cas9 mutant plants were compared with the SALK T-DNA insertion mutant, line hos1-3, in terms of their tolerance to abiotic stresses, accumulation of secondary metabolites and expression levels of genes participating in these processes. Upon exposure to cold stress, enhanced tolerance and expression of cold-responsive genes were observed in both hos1-3 and hos1 Cas9 plants. The hos1 mutation caused changes in the synthesis of phytoalexins in transformed cells. The content of glucosinolates (GSLs) was down-regulated by 1.5-times, while flavonol glycosides were up-regulated by 1.2 to 4.2 times in transgenic plants. The transcript abundance of the corresponding MYB and bHLH transcription factors, which are responsible for the regulation of secondary metabolism in Arabidopsis, were also altered. Our data suggest a relationship between HOS1-regulated downstream signaling and phytoalexin biosynthesis.

Linking Brassinosteroid and ABA Signaling in the Context of Stress Acclimation.

The important regulatory role of brassinosteroids (BRs) in the mechanisms of tolerance to multiple stresses is well known. Growing data indicate that the phenomenon of BR-mediated drought stress tolerance can be explained by the generation of stress memory (the process known as 'priming' or 'acclimation'). In this review, we summarize the data on BR and abscisic acid (ABA) signaling to show the interconnection between the pathways in the stress memory acquisition. Starting from brassinosteroid receptors brassinosteroid insensitive 1 (BRI1) and receptor-like protein kinase BRI1-like 3 (BRL3) and propagating through BR-signaling kinases 1 and 3 (BSK1/3) → BRI1 suppressor 1 (BSU1) -‖ brassinosteroid insensitive 2 (BIN2) pathway, BR and ABA signaling are linked through BIN2 kinase. Bioinformatics data suggest possible modules by which BRs can affect the memory to drought or cold stresses. These are the BIN2 → SNF1-related protein kinases (SnRK2s) → abscisic acid responsive elements-binding factor 2 (ABF2) module; BRI1-EMS-supressor 1 (BES1) or brassinazole-resistant 1 protein (BZR1)-TOPLESS (TPL)-histone deacetylase 19 (HDA19) repressor complexes, and the BZR1/BES1 → flowering locus C (FLC)/flowering time control protein FCA (FCA) pathway. Acclimation processes can be also regulated by BR signaling associated with stress reactions caused by an accumulation of misfolded proteins in the endoplasmic reticulum.

Green synthesis of silver nanoparticles using transgenic Nicotiana tabacum callus culture expressing silicatein gene from marine sponge Latrunculia oparinae.

In the present investigation, transgenic tobacco callus cultures and plants overexpressing the silicatein gene LoSilA1 from marine sponge Latrunculia oparinae were obtained and their bioreduction behaviour for the synthesis of silver nanoparticles (AgNPs) was studied. Synthesized nanoparticles were characterized using UV-visible spectroscopy, Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic flame electron microscopy (AFM) and nanoparticle tracking analysis (NTA). Our measurements showed that the reduction of silver nitrate produced spherical AgNPs with diameters in the range of 12-80 nm. The results of XRD analysis proved the crystal nature of the obtained AgNPs. FTIR analysis indicated that particles are reduced and stabilized in solution by the capping agent, which is likely to be proteins present in the callus extract. Interestingly, the reduction potential of LoSiLA1-transgenic callus line was increased three-fold compared with the empty vector-transformed calli. The synthesized AgNPs were found to exhibit strong antibacterial activity against Escherichia coli and Agrobacterium rhizogenes. The present study reports the first evidence for using genetic engineering for activation of the reduction potential of plant cells for synthesis of biocidal AgNPs.

Critical analysis of protein signaling networks involved in the regulation of plant secondary metabolism: focus on anthocyanins.

Anthocyanin biosynthesis in Arabidopsis is a convenient and relatively simple model for investigating the basic principles of secondary metabolism regulation. In recent years, many publications have described links between anthocyanin biosynthesis and general defense reactions in plants as well as photomorphogenesis and hormonal signaling. These relationships are complex, and they cannot be understood intuitively. Upon observing the lacuna in the Arabidopsis interactome (an interaction map of the factors involved in the regulation of Arabidopsis secondary metabolism is not available), we attempted to connect various cellular processes that affect anthocyanin biosynthesis. In this review, we revealed the main signaling protein modules that regulate anthocyanin biosynthesis. To our knowledge, this is the first reconstruction of a network of proteins involved in plant secondary metabolism.

New opportunities for the regulation of secondary metabolism in plants: focus on microRNAs.

Plant cell cultures are of particular interest in industrial applications as a source of biologically active substances. It is difficult, however, to achieve stable production of secondary metabolites for many plant cell cultures using classical techniques. Novel approaches should be developed for removal of the inhibitor blocks that prevent pathway activation and shift the regulatory balance to the activation of entire biosynthetic pathways. MicroRNAs (miRNAs) are small RNAs that play important regulatory roles in various biological processes. Only recently miRNAs have been demonstrated as active in secondary metabolism regulation. In this work, we summarize recent data on the emerging approaches based on regulation of secondary metabolism by miRNAs.

CDPK-driven changes in the intracellular ROS level and plant secondary metabolism.

Heterologous expression of a constitutively active calcium-dependent protein kinase (CDPK) gene was previously shown to increase secondary metabolite production in cultured cells of Rubia cordifolia, but the critical question of how CDPK activates secondary metabolism remains to be answered. In this article, we report that the expression of the Arabidopsis CDPK gene, AtCPK1, in R. cordifolia cells caused moderate and stable elevation of intracellular reactive oxygen species (ROS) levels. In contrast, the non-active, mutated AtCPK1 gene did not cause such an effect. The active AtCPK1 also increased cell size, likely by restricting cell division. These results are consistent with the model in which constitutive expression of AtCPK1 mimics the effects of elicitors, acting on secondary metabolism via the activation of ROS production.