Identification of promoter sequences in the eukaryotic genome, by computer methods, is an important task of bioinformatics. However, this problem has not been solved since the best algorithms have a false positive probability of 10⁻³–10⁻⁴ per nucleotide. As a result of full genome analysis, there may be more false positives than annotated gene promoters. The probability of a false positive should be reduced to 10⁻⁶–10⁻⁸ to reduce the number of false positives and increase the reliability of the prediction. The method for multi alignment of the promoter sequences was developed. Then, mathematical methods were developed for calculation of the statistically important classes of the promoter sequences. Five promoter classes, from the rice genome, were created. We developed promoter classes to search for potential promoter sequences in the rice genome with a false positive number less than 10⁻⁸ per nucleotide. Five classes of promoter sequences contain 1740, 222, 199, 167 and 130 promoters, respectively. A total of 145,277 potential promoter sequences (PPSs) were identified. Of these, 18,563 are promoters of known genes, 87,233 PPSs intersect with transposable elements, and 37,390 PPSs were found in previously unannotated sequences. The number of false positives for a randomly mixed rice genome is less than 10⁻⁸ per nucleotide. The method developed for detecting PPSs was compared with some previously used approaches. The developed mathematical method can be used to search for genes, transposable elements, and transcript start sites in eukaryotic genomes.

Carotenoids are essential participants in photosynthesis and photo protection as well as growth, development, and stress response in plants. Phytoene desaturase (PDS; EC 1.3.5.5) is an enzyme that catalyzes the first stage of desaturation of 15-cis-phytoene (a precursor to all carotenoids). In this work, we examined for the first time the effect of PDS knockout in the genome of Nicotiana tabacum L. using the CRISPR-Cas9 system on activity of downstream genes involved in biosynthesis of carotenoids. Nine transgenic lines of tobacco were obtained with a mosaic editing of gene NtPDS; three versions of indels (350^351→ins^g#, 350^351→ins^t#, and t351→del(1nt)#) in the exon II resulting in the synthesis of a shortened non-functional version of the protein. The lines were characterized by spotted, green-white pigmentation of the leaves, altered flowering time and morphology as well as a reduced content of carotenoids and chlorophylls in the leaf tissue. A rise in the level of transcripts of phytoene synthase gene NtPSY2 was shown in late-flowering lines as compared to control material. Edited line L29 with the latest flowering showed a considerable increase in the level of transcripts of downstream structural genes of carotenogenesis. A reduction in the carotenoid content in the leaves with mosaic editing was accompanied by a decrease in the level of expression of MADS-box gene NtSEP1, the product of which presumably participates in regulation of transcription of the carotenoid biosynthesis genes. The obtained results may be used for further studies of regulation of biosynthesis of carotenoids and apocarotenoids in Solanaceae.

The plant response to stresses includes changes in starch metabolism regulated by a complex catalytic network, in which plastidic starch phosphorylase PHO1a is one of the key players. In this study, we used the CRISPR-Cas9 system to edit the PHO1a gene in four potato (Solanum tuberosum L.) cultivars, which resulted in the introduction of a radical mutation, G261V, into the PHO1a functional domain. The mutants had altered morphology and differed from wild-type plants in starch content in the roots and leaves. Exposure to cold stress revealed the differential response of parental and transgenic plants in terms of starch content and the expression of genes coding for β-amylases, amylase inhibitors, and stress-responsive MADS-domain transcription factors. These results suggest that the G261V mutation causes changes in the functional activity of PHO1a, which in turn affect the coordinated operation of starch catabolism enzymes both under normal and cold stress conditions, possibly through differential expression of MADS-domain transcription factors. Our results highlight a critical regulatory role of PHO1a in starch metabolism, root and shoot development, and stress response in potatoes.

Carotenoids are essential participants in photosynthesis and photo protection as well as growth, development, and stress response in plants. Phytoene desaturase (PDS; EC 1.3.5.5) is an enzyme that catalyzes the first stage of desaturation of 15-cis-phytoene (a precursor to all carotenoids). In this work, we examined for the first time the effect of PDS knockout in the genome of Nicotiana tabacum L. using the CRISPR-Cas9 system on activity of downstream genes involved in biosynthesis of carotenoids. Nine transgenic lines of tobacco were obtained with a mosaic editing of gene NtPDS; three versions of indels (350^351→ins^g#, 350^351→ins^t#, and t351→del(1nt)#) in the exon II resulting in the synthesis of a shortened non-functional version of the protein. The lines were characterized by spotted, green-white pigmentation of the leaves, altered flowering time and morphology as well as a reduced content of carotenoids and chlorophylls in the leaf tissue. A rise in the level of transcripts of phytoene synthase gene NtPSY2 was shown in late-flowering lines as compared to control material. Edited line L29 with the latest flowering showed a considerable increase in the level of transcripts of downstream structural genes of carotenogenesis. A reduction in the carotenoid content in the leaves with mosaic editing was accompanied by a decrease in the level of expression of MADS-box gene NtSEP1, the product of which presumably participates in regulation of transcription of the carotenoid biosynthesis genes. The obtained results may be used for further studies of regulation of biosynthesis of carotenoids and apocarotenoids in Solanaceae.