Anastasiya N. Bubnova

BACKGROUND: Breakthroughs in genome editing technology have made it possible to precisely alter target nucleotides in plant DNA/RNA. Such changes can be identical or comparable to natural mutations, or achieved through conventional mutagenesis, providing the scientific basis for recognizing gene-edited (GE) plants of this category as analogous to plants created through traditional breeding. Risk assessment of off-target editing effects, as well as the methodology and criteria for distinguishing GE plants from transgenic ones, remain unresolved issues for the scientifically based regulation of plant genetic engineering in Russia. AIM: This study aimed to develop methodological approaches and a decision-making tree for determining the status of plants obtained using genome editing technology, allowing for the registration of the GE plant and its subsequent cultivation. METHODS: The objects of this analytical study are GE organisms of plant origin. RESULTS: The result of this work is a comprehensive strategy for minimizing the risks of off-target editing effects and assessing the biosafety of GE plant, namely: a) “safe design” at the concept stage; b) molecular genetic analysis of the obtained GE plants using instrumental methods; c) analysis of the data required for the biosafety assessment of SDN-1 and SDN-2 types of GE plants; d) criteria and a decision-making tree for determining the status of plants obtained using genome editing technology. For instrumental confirmation a modified plant’s status, we proposed to analyze by PCR/RT-PCR of the regions around target editing sites with a length of 1000 base pairs, centered relative to the break site, with subsequent sequencing, and for a more in-depth analysis, the k-mer method. CONCLUSION: The developed methodological approaches, criteria, requirements, and decision tree will enable the classification of plants obtained using genome editing technology. Thus, the ability to determine whether a plant is transgenic (SDN-3) or gene-edited (SDN-1, SDN-2) will enable the establishment of appropriate regulatory and control measures.

In this study, promoter sequences predicted by the MAHDS method in the Oryza sativa genome were analyzed using three genome annotations: RefSeq NCBI, Rice Genome Annotation Project, and Ensembl. Part of the predicted promoters was found to be located near annotated genes, which indicates their potential functional role. The remaining sequences, considered as potentially novel regulatory elements, were examined using YAPP for the presence of core promoter motifs and their functional combinations. All analyzed predicted promoters contain either an Inr or a TATA motif—the key elements involved in transcription initiation. The identified motif combinations suggest a high likelihood of transcriptional activity in these sequences, and the consistency of the results with annotated data and CAGE-seq signals supports the reliability and applicability of the MAHDS method.

The latest research has shown that chitosan acts as a growth stimulator and elicitor in plants, including resistance to biotic and abiotic factors. However, increasing concentrations could possibly make chitosan a source of stress for plants. In this study, we investigated the effect of low-molecular-weight chitosan hydrolysate on the root development of tomato (Solanum lycoperscum) cultivars Red Cherry, Lel, and Tytan. The growth rate change, together with total phenolic content, phenylalanine ammonia-lyase (PAL) activity, and gene expression, were studied in relation to tomatoes. High concentrations of chitosan negatively affect the growth of tomato seedlings and contribute to changes in the tropism of the seedling roots. After the addition of chitosan hydrolysate, the PAL activity and the total phenolic content decreased 24 h later. PAL is a key enzyme in the biosynthesis of many plant stress factors. An analysis of the tomato PAL gene family was carried out. The SlPAL gene expression in the seedlings of cv. Cherry increased 1.5 times after 48 h, while in cv. Lel, the expression stably decreased in the presence of chitosan. The obtained results are supposed to aid our understanding of the mechanisms underlying the effects of chitosan on plant development and further its successful application in agriculture as well as in research on plant stress.

The exact identification of promoter sequences remains a serious problem in computational biology, as the promoter prediction algorithms under development continue to produce false-positive results. Therefore, to fully assess the validity of predicted sequences, it is necessary to perform a comprehensive test of their properties, such as the presence of downstream transcribed DNA regions behind them, or chromatin accessibility for transcription factor binding. In this paper, we examined the promoter sequences of chromosome 1 of the rice Oryza sativa genome from the Database of Potential Promoter Sequences predicted using a mathematical algorithm based on the derivation and calculation of statistically significant promoter classes. In this paper TATA motifs and cis-regulatory elements were identified in the predicted promoter sequences. We also verified the presence of potential transcription start sites near the predicted promoters by analyzing CAGE-seq data. We searched for unannotated transcripts behind the predicted sequences by de novo assembling transcripts from RNA-seq data. We also examined chromatin accessibility in the region of the predicted promoters by analyzing ATAC-seq data. As a result of this work, we identified the predicted sequences that are most likely to be promoters for further experimental validation in an in vivo or in vitro system.