INTRASPECIFIC VARIABILITY OF THE Sus1 SUCROSE SYNTHASE GENE IN Pisum sativum ACCESSIONS

The pea Pisum sativum is widely cultivated in Russia as well as over the world. Pea productivity depends on the ability of the pea plant to get into a symbiosis with nodule bacteria. It was previously shown that the strength of the symbiotic activity depends on the activity of plant sucrose cleavage enzymes. Sucrose synthase Sus1 is one of the most important enzymes involved in carbohydrate metabolism. Sucrose synthase cleaves sucrose into UDP-glucose and fructose. This paper is devoted to characterization of Sus1 gene intraspecific variability in 14 Pisum sativum accessions. The length of the identified Sus1 gene varied from 3514 bp to 3532 bp. All identified genes had a similar structure and contained 13 exons and 12 introns. According to their structure, they were assigned to the SUS1-group of dicotyledonous plants. In nucleotide sequences, 125 SNPs were identified. In addition to SNPs, intron sequences contained six indels, thus their length varied from 1093 bp to 1111 bp. The most variable was the intron III. In coding sequences, 47 SNPs were found, wherein the most variable was the exon II. 16 exon SNPs led to amino acid substitutions. Six of them were deleterious and may potentially influence protein folding and stability. All the conservative motifs and active sites were detected in the translated amino acid sequences. It was shown that their sequences were invariable in all the tested accessions. Computational analysis of the amino acid sequences has predicted Sus1 tertiary structure. The protein is a tetramer and each subunit in its turn consists of three domains. The phylogenetic analysis using identified Pisum Sus1 sequences and homologous sucrose synthase genes revealed that the Sus1 and Sus3 genes are closer to each other than to Sus2. It was also proposed that the sucrose synthase family genes had diverged before legumes split into species

Vitamin C in fleshy fruits: biosynthesis, recycling, genes, and enzymes

L-ascorbic acid (vitamin C) is a plant secondary metabolite that has a variety of functions both in plant tissues and in the human body. Plants are the main source of vitamin C in human nutrition, especially citrus, rose hip, tomato, strawberry, pepper, papaya, kiwi, and currant fruits. However, in spite of the biological significance of L-ascorbic acid, the pathways of its biosynthesis in plants were fully understood only in 2007 by the example of a model plant Arabidopsis thaliana. In the present review, the main biosynthetic pathways of vitamin C are described: the L-galactose pathway, L-gulose pathway, galacturonic and myo-inositol pathway. To date, the best studied is the L-galactose pathway (Smyrnoff–Wheeler pathway). Only for this pathway all the enzymes and the entire cascade of reactions have been described. For other pathways, only hypothetical metabolites are proposed and not all the catalyzing enzymes have been identified. The key genes participating in ascorbic acid biosynthesis and accumulation in fleshy fruits are highlighted. Among them are L-galactose pathway proteins (GDP-mannose phosphorylase (GMP, VTC1), GDP-D-mannose epimerase (GME), GDP-L-galactose phosphorylase (GGP, VTC2/VTC5), L-galactose-1-phosphate phosphatase (GPP/VTC4), L-galactose-1-dehydrogenase (GalDH), and L-galactono1,4-lactone dehydrogenase (GalLDH)); D-galacturonic pathway enzymes (NADPH-dependent D-galacturonate reductase (GalUR)); and proteins, controlling the recycling of ascorbic acid (dehydroascorbate reductase (DHAR1) and monodehydroascorbate reductase (MDHAR)). Until now, there is no clear and unequivocal evidence for the existence of one predominant pathway of vitamin C biosynthesis in fleshy fruits. For example, the L-galactose pathway is predominant in peach and kiwi fruits, whereas the D-galacturonic pathway seems to be the most essential in grape and strawberry fruits. However, in some plants, such as citrus and tomato fruits, there is a switch between different pathways during ripening. It is noted that the final ascorbic acid content in fruits depends not only on biosynthesis but also on the rate of its oxidation and recirculation

Analysis of the structure and function of the tomato <i>Solanum lycopersicum</i> L. MADS-box gene <i>SlMADS5</i>

At all stages of flowering, a decisive role is played by the family of MADS-domain transcription factors, the combinatorial action of which is described by the ABCDE-model of flower development. The current volume of data suggests a high conservatism of ABCDE genes in angiosperms. The E-proteins SEPALLATA are the central hub of the MADS-complexes, which determine the identity of the floral organs. The only representative of the SEPALLATA3 clade in tomato Solanum lycopersicum L., SlMADS5, is involved in determining the identity of petals, stamens, and carpels; however, data on the functions of the gene are limited. The study was focused on the SlMADS5 functional characterization. Structural and phylogenetic analyses of SlMADS5 confirmed its belonging to the SEP3 clade. An in silico expression analysis revealed the absence of gene transcripts in roots, leaves, and shoot apical meristem, and their presence in flowers, fruits, and seeds at different stages of development. Two-hybrid analysis showed the ability of SlMADS5 to activate transcription of the target gene and interact with TAGL1. Transgenic plants Nicotiana tabacum L. with constitutive overexpression of SlMADS5 cDNA flowered 2.2 times later than the control; plants formed thickened leaves, 2.5–3.0 times thicker stems, 1.5–2.7 times shortened internodes, and 1.9 times fewer flowers and capsules than non-transgenic plants. The flower structure did not differ from the control; however, the corolla petals changed color from light pink to magenta. Analysis of the expression of SlMADS5 and the tobacco genes NtLFY, NtAP1, NtWUS, NtAG, NtPLE, NtSEP1, NtSEP2, and NtSEP3 in leaves and apexes of transgenic and control plants showed that SlMADS5 mRNA is present only in tissues of transgenic lines. The other genes analyzed were highly expressed in the reproductive meristem of control plants. Gene transcripts were absent or were imperceptibly present in the leaves and vegetative apex of the control, as well as in the leaves and apexes of transgenic lines. The results obtained indicate the possible involvement of SlMADS5 in the regulation of flower meristem development and the pathway of anthocyanin biosynthesis in petals

ПОЛИМОРФИЗМ САХАРОЗОСИНТАЗНОГО ДОМЕНА ГЕНОВ SUS4 СОРТОВ КАРТОФЕЛЯ РОССИЙСКОЙ, БЕЛОРУССКОЙ И КАЗАХСТАНСКОЙ СЕЛЕКЦИИ

The potato is one of the main strategic crops in the Russian Federation, Belarus and Kazakhstan. Currently, we have achieved significant advances in the understanding of metabolic mechanism of carbohydrate and interconversion «sucrose – starch» in potato tubers. Sucrose synthase (Sus) is a key enzyme in the breakdown of sucrose. Sucrose synthase (Sus) is catalyzing a reversible reaction of conversion sucrose and UDP into fructose and UDP-glucose. The identification and subsequent characterization of the genes encoding plant sucrose synthase is the first step towards understanding their physiological roles and metabolic mechanism involved in carbohydrate accumulation in potato tubers. In the present work the nucleotide and amino acid polymorphism of the Sus4 gene fragments containing sequences of the sucrose synthase domain were analyzed. Sus4 gene fragments (intron III – exon VI) in 9 potato cultivars of Russian, Kazakh and Belarusian breeding were analyzed. The polymorphism of the Sus4 sucrose synthase domain sequences was first examined. The length of analyzed fragment varied from 977 b.p. (cultivars Favorit, Karasaiskii, Miras) to 1013 b.p. (cultivars Zorochka, Manifest, Elisaveta, Bashkirskii). It was demonstrated that the examined sequences contained point mutations, as well as insertions and deletions. The common polymorphism level was 5.82%. It was shown that the examined sequences contained 58 SNPs and 4 indels. The most variable were introns IV (12.4%) and V (9.18%). The most variable was exon IV. 7 allelic variants were detected. 6 different amino acid sequences specific to different varieties were also identified.Картофель является одной из основных стратегических сельскохозяйственных культур в Российской Федерации, Республике Беларусь и Республике Казахстан. В настоящее время достигнуты значительные успехи в понимании процессов углеводного метаболизма и взаимного превращения «сахароза - крахмал» в клубнях картофеля. Показано, что одними из ключевых генов, определяющих качество картофельной продукции, являются гены метаболизма сахарозы. Сахарозосинтаза (Sus) один из основных ферментов распада сахарозы. Сахарозосинтаза (Sus) осуществляет обратимый гидролиз сахарозы до УДФ-глюкозы и фруктозы. Идентификация новых генов, кодирующих растительные сахарозосинтазы, и анализ их нуклеотидных последовательностей – важный шаг к пониманию физиологической роли и метаболических механизмов, связанных с обменом углеводов в клубнях картофеля. В данной работе приведен анализ нуклеотидного и аминокислотного полиморфизма сахарозосинтазного домена гена Sus4. Изучена последовательность фрагмента гена Sus4 с интрона III по экзон VI у 9 сортов картофеля россий- ской, казахской и белорусской селекции. Длина полученных фрагментов варьировала от 977 п.н. (у сортов Фаворит, Карасайский, Мирас) до 1013 п.н. (у сортов Зорочка, Манифест, Елизавета, Башкирский). Полученные последовательности содержали как единичные мутации, так и инсер- ции и делеции. Общий уровень полиморфизма изученных последовательностей составил 5.82%. Всего в исследованном фрагменте найдено 58 SNPs и 4 индели. Наиболее вариабельные интроны - IV (12,4%) и V (9,18%). Наиболее вариабельный экзон - IV. Показано наличие 7 аллельных вариантов и 6 вариантов аминокислотных последовательностей

VARIABILITY OF A FRAGMENT OF THE ACID VACUOLAR INVERTASE PAIN-1 GENE IN POTATO CULTIVARS

Acid vacuolar invertase Pain-1 participates in the regulation of starch and sucrose contents in cells. This enzyme is also involved in plant response to abiotic stress. For the first time Pain-1 gene fragment (exon V – exon VII) polymorphism was determined in 19 potato varieties. A total of 25 SNPs were detected. A new SNP, С1895, was found in exon VII. Five of eight SNPs located in exons led to amino acid substitutions. Three of them were radical. It was shown that the conservative C-terminal domain contained three variable amino acids

THE POLYMORPHISM OF THE SUS4 SUCROSE SYNTHASE DOMAIN SEQUENCES IN RUSSIAN, BELORUSSIAN AND KAZAKH POTATO CULTIVARS

The potato is one of the main strategic crops in the Russian Federation, Belarus and Kazakhstan. Currently, we have achieved significant advances in the understanding of metabolic mechanism of carbohydrate and interconversion «sucrose – starch» in potato tubers. Sucrose synthase (Sus) is a key enzyme in the breakdown of sucrose. Sucrose synthase (Sus) is catalyzing a reversible reaction of conversion sucrose and UDP into fructose and UDP-glucose. The identification and subsequent characterization of the genes encoding plant sucrose synthase is the first step towards understanding their physiological roles and metabolic mechanism involved in carbohydrate accumulation in potato tubers. In the present work the nucleotide and amino acid polymorphism of the Sus4 gene fragments containing sequences of the sucrose synthase domain were analyzed. Sus4 gene fragments (intron III – exon VI) in 9 potato cultivars of Russian, Kazakh and Belarusian breeding were analyzed. The polymorphism of the Sus4 sucrose synthase domain sequences was first examined. The length of analyzed fragment varied from 977 b.p. (cultivars Favorit, Karasaiskii, Miras) to 1013 b.p. (cultivars Zorochka, Manifest, Elisaveta, Bashkirskii). It was demonstrated that the examined sequences contained point mutations, as well as insertions and deletions. The common polymorphism level was 5.82%. It was shown that the examined sequences contained 58 SNPs and 4 indels. The most variable were introns IV (12.4%) and V (9.18%). The most variable was exon IV. 7 allelic variants were detected. 6 different amino acid sequences specific to different varieties were also identified

Effect of a Radical Mutation in Plastidic Starch Phosphorylase PHO1a on Potato Growth and Cold Stress Response

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