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

Comparative analysis of the complete plastomes of garlic Allium sativum and bulb onion Allium cepa

Sequencing and comparative characterization of plant plastid genomes, or plastomes, is an important tool for modern phylogenetic and taxonomic studies, as well as for understanding the plastome evolution. The genus Allium L. (family Amaryllidaceae) incorporates more than 900 species, includes economically signifi­cant vegetable crops such as garlic A. sativum, onion A. cepa, leek A. porrum, etc. In this work, the plastome of garlic A. sativum has been completely sequenced. The A. sativum plastome is 153172 bp in size. It consists of a large unique (LSC, 82035 bp) and small unique (SSC, 18015 bp) copies, separated by inverted repeats (IRa and IRb) of 26561 bp each. In the garlic plastome, 134 genes have been annotated: 82 protein-coding genes, 38 tRNA genes, 8 rRNA genes, and 6 pseudogenes. Comparative analysis of A. sativum and A. cepa plastomes reveals differences in the sizes of structural elements and spacers at the inverted repeat bound­aries. The total numbers of genes in A. sativum and A. cepa are the same, but the gene composition is dif­ferent: the rpl22 gene is functional in A. sativum, being a pseudogene in A. cepa; conversely, the rps16 gene is a pseudogene in A. sativum and a protein-coding gene in A. cepa. In the A. sativum and A. cepa plastomes, 32 SSR sequences have been identified. More than half of them are dinucleotides, and the remaining are tetra-, penta-, and hexanucleotides at the same time, trinucleotides were absent. The compared plastomes differ in the numbers of certain SSRs, and some are present in only one of the species

5′-UTR allelic variants and expression of the lycopene-ɛ-cyclase <i>LCYE</i> gene in maize (<i>Zea mays</i> L.) inbred lines of Russian selection

In breeding, biofortification is aimed at enriching the edible parts of the plant with micronutrients. Within the framework of this strategy, molecular screening of collections of various crops makes it possible to determine allelic variants of genes, new alleles, and the linkage of allelic variants with morphophysiological traits. The maize (Zea mays L.) is an important cereal and silage crop, as well as a source of the main precursor of vitamin A – β-carotene, a derivative of the β,β-branch of the carotenoid biosynthesis pathway. The parallel β,ε-branch is triggered by lycopene-ε-cyclase LCYE, a low expression of which leads to an increase in provitamin A content and is associated with the variability    of the 5’-UTR gene regulatory sequence. In this study, we screened a collection of 165 maize inbred lines of Russian selection for 5’-UTR LCYE allelic variants, as well as searched for the dependence of LCYE expression levels on the 5’-UTR allelic variant in the leaves of 14 collection lines. 165 lines analyzed were divided into three groups carrying alleles A2 (64 lines), A5 (31) and A6 (70), respectively. Compared to A2, allele A5 contained two deletions (at positions -267–260 and -296–290 from the ATG codon) and a G251→T substitution, while allele A6 contained one deletion (-290–296) and two SNPs (G251→T, G265→T). Analysis of LCYE expression in the leaf tissue of seedlings from accessions of 14 lines differing in allelic variants showed no associations of the 5’-UTR LCYE allele type with the level of gene expression. Four lines carrying alleles A2 (6178-1, 6709-2, 2289-3) and A5 (5677) had a significantly higher level of LCYE gene expression (~0.018–0.037) than the other 10 analyzed lines (~0.0001–0.004), among which all three allelic variants were present

Dependence of the content of starch and reducing sugars on the level of expression of the genes of β-amylases StBAM1 and StBAM9 and the amylase inhibitor StAI during long-term low-temperature storage of potato tubers

Solanum tuberosum L. is the most important non-grain starch crop with a potential yield of 38–48 t/ha and a starch content of 13.2–18.7 %. Potato tubers are stored at a low temperature (2–4 °C) in a state of physiological dormancy. A disadvantage of this type of storage is the degradation of starch and the accumulation of reducing sugars (cold-induced sweetening), including due to an increase in the activity of β-amylases that hydrolyze starch to maltose. In this study, a comparative analysis of the β-amylase (StBAM1, StBAM9) and amylase inhibitor (StAI ) gene expression, as well as starch and reducing sugar content in tubers during long-term low-temperature storage (September, February, April) was performed using potato cultivars Nadezhda, Barin, Krasavchik, Severnoe siyanie and Utro. The β-amylase genes, StBAM9 and one of the two StBAM1 homologs (with the highest degree of homology with AtBAM1), were selected based on phylogenetic analysis data. Evaluation of the expression of these genes and the amylase inhibitor gene showed a tendency to decrease in transcription for all analyzed cultivars. The starch content also significantly decreased during tuber storage. The amount of reducing sugars increased in the September–April period, while in February–April, their content did not change (Krasavchik), decreased (Barin, Severnoe siyanie) or continued to grow (Utro, Nadezhda). It can be assumed that the gene activity of StBAM1 and StBAM9 correlates with the amount of starch (positively) and monosaccharides (negatively). The level of StAI expression, in turn, may be directly dependent on the level of StBAM1 expression. At the same time, there is no relationship between the degree of cultivar predisposition to cold-induced sweetening and the expression profile of the StBAM1, StBAM9, and StAI genes

Structural and functional features of phytoene synthase isoforms PSY1 and PSY2 in pepper Capsicum annuum L. cultivars

The fruits of various pepper cultivars are characterized by a different colour, which is determined by the pigment ratio; carotenoids dominate in ripe fruits, while chlorophylls, in immature fruits. A key regulator of carotenoid biosynthesis is the phytoene synthase encoded by the PSY gene. The Capsicum annuum genome contains two isoforms of this enzyme, localized in leaf (PSY2) and fruit (PSY1) plastids. In this work, the complete PSY1 and PSY2 genes were identified in nine C. annuum cultivars, which differ in ripe fruit colour. PSY1 and PSY2 sequence variability was 2.43 % (69 SNPs) and 1.21 % (36 SNPs). The most variable were PSY1 proteins of the cultivars ‘Maria’ (red-fruited) and ‘Sladkij shokolad’ (red-brown-fruited). All identified PSY1 and PSY2 homologs contained the phytoene synthase domain HH-IPPS and the transit peptide. In the PSY1 and PSY2 HH-IPPS domains, functionally significant sites were determined. For all accessions studied, the active sites (YAKTF and RAYV), aspartate-rich substrate-Mg2+-binding sites (DELVD and DVGED), and other functional residues were shown to be conserved. Transit peptides were more variable, and their similarity in the PSY1 and PSY2 proteins did not exceed 78.68 %. According to the biochemical data obtained, the largest amounts of chlorophylls and carotenoids across the cultivars studied were detected in immature and ripe fruits of the cv. ‘Sladkij shokolad’ and ‘Shokoladnyj’. Also, ripe fruits of the cv. ‘Nesozrevayuschij’ (green-fruited) were marked by significant chlorophyll content, but a minimum of carotenoids. The PSY1 and PSY2 expression patterns were determined in the fruit pericarp at three ripening stages in ‘Zheltyj buket’, ‘Sladkij shokolad’, ‘Karmin’ and ‘Nesozrevayuschij’, which have different ripe fruit colours: yellow, red-brown, dark red and green, respectively. In the leaves of the cultivars studied, PSY1 expression levels varied significantly. All cultivars were characterized by increased PSY1 transcription as the fruit ripened; the maximum transcription level was found in the ripe fruit of ‘Sladkij shokolad’, and the lowest, in ‘Nesozrevayuschij’. PSY2 transcripts were detected not only in the leaves and immature fruits, but also in ripe fruits. Assessment of a possible correlation of PSY1 and PSY2 transcription with carotenoid and chlorophyll content revealed a direct relationship between PSY1 expression level and carotenoid pigmentation during fruit ripening. It has been suggested that the absence of a typical pericarp pigmentation pattern in ‘Nesozrevayuschij’ may be associated with impaired chromoplast formation

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

Homeobox genes encoding WOX transcription factors in the flowering parasitic plant Monotropa hypopitys

The formation and maintenance of plant stem cell populations are controlled by the WOX family of homeobox-containing transcription factors. The evolution of WOX genes is considered to be one of the main reasons for flower morphology and plant architecture diversity. The stem cell regulation mechanism is considered to be conserved among flowering plants and most thoroughly studied in Arabidopsis thaliana as a model. The angiosperms morphological diversity implies that there are species-specific features inherent to this mechanism, while the basic signaling is maintained. The unique flowering achlorophyllous mycoheterotrophic plant Monotropa hypopitys obtains nutrients from the tree roots through the mycorrhizal symbiosis. In inductive conditions, the reproductive stem with bracts and an inflorescence at the top is developed from an adventitious root bud. Like other plants, M. hypopitys forms the inflorescence, flower and root meristems, presumably using conserved mechanisms regulating stem cell niche. The study of M. hypopitys homeobox genes should contribute to the knowledge about the function of WOX transcription factors and further understanding of the stem cells control mechanisms in mycoheterotrophic species. The aim of the present study was to analyze M. hypopitys root, bracts and flower transcriptomes obtained from two individual flowering plants. In total, five WOX genes have been identified and characterized by their structure, phylogeny, expression pattern, and possible functions. The assumption is that the MhyWUS1 and MhyWUS2 genes maintain the stem cell population in the inflorescence and flower meristems, MhyWOX13 has a role in the control of root stem cell niche, seed pod formation, flowering initiation, and basic cellular processes, MhyWOX4 functions in the control of cambium stem cells, and MhyWOX2 participates in the differentiation of egg cells and zygotes

Transcription factors MhyFIL1 and MhyFIL3 <i>(Monotropa hypopitys)</i> determine the asymmetric development of above-ground lateral organs in plants

It is believed that the complete mycoheterotroph pinesap Monotropa hypopitys adaptively evolved from a photosynthetic mycorrhizal ancestor, which had lost its photosynthetic apparatus and vegetative organs (stem and leaves). The aerial part of the plant is a reproductive axis with sterile bracts and inflorescence with a flower type canonical for higher plants. The origin of leaves and leaf-like lateral organs is associated, among other factors, with the evolution of the YABBY genes, which are divided into“vegetative” and evolutionarily recent“reproductive” genes, with regard to their expression profiles. The study of the vegetative YABBY genes in pinesap will determine whether their functions (identification of cell identity on the abaxial surface of the lateral organs) are preserved in the leafless plant. In this study, the structural and phylogenetic analysis of the pinesap vegetative genes MhyFIL1 and MhyFIL3 is performed, the main conserved domains and motifs of the encoded proteins are characterized, and it is confirmed that the genes belong to the vegetative clade YABBY3/FIL. The effect of heterologous ectopic expression of the MhyFIL1 and MhyFIL3 genes on the phenotype of transgenic tobacco Nicotiana tabacum is evaluated. The leaves formed by both types of plants, 35S::MhyFIL1 and 35S::MhyFIL3, were narrower than in control plants and were twisted due to the changed identity of adaxial surface cells. Also, changes in the architecture of the aerial part and the root system of transgenic plants, including aberrant phyllotaxis and arrest of the shoot and root apical meristem development, were noted. Some of the 35S::MhyFIL1 and 35S::MhyFIL3 plants died as early as the stage of the formation of the first leaves, others did not bloom, and still others had a greatly prolonged vegetation period and formed fewer flowers than normal ones. The flowers had no visible differences from the control except for fragile pedicles. Thus, the absence of structural changes from the M. hypopitys flower in comparison to autotrophic species and the effect of MhyFIL1/3 heterologous expression on the development of tobacco plants indicate the preservation of the functions of the vegetative YABBY genes by the MhyFIL1/3 genes in pinesap. Moreover, the activity of YABBY transcription factors of the FIL clade in M. hypopitys is not directly related to the loss of the ability of pinesap to form leaves during the evolutionary transition from autotrophic nutrition to heterotrophy

Identification and characterization of mRNAs of receptor-like kinases MhyGSO1 and MhyGSO2 in flowering parasitic plant Monotropa hypopitys

Plant organ formation is based on the balance of the programmed cell division and positional differentiation maintained by intercellular communication mediated by signaling molecules and receptors. In Arabidopsis thaliana, two paralogous leucine-rich repeat receptor-like kinases, GASSHO1 and GASSHO2, redundantly participate in the regulation of various root cells identity and functioning and the proper epidermis patterning. The GASSHO genes are characterized mainly in A. thaliana. Their significance in combination with the conservation of basic developmental processes justifies the study of GASSHO kinases in other plant species with different nutrition and developmental features. The aim of this work was to identify the GASSHO genes in an angiosperm plant, pinesap Monotropa hypopitys, which is a non-photosynthetic achlorophyllous mycoheterotroph. In different tissues (roots with buds, bracts, and flowers) of two individual plants at the late flowering stage, the transcriptomic data search identified 3’-partial mRNAs of two paralogous genes, MhyGASSHO1 (MhyGSO1) and MhyGSO2. Structural analysis of the encoded amino acid sequences revealed conserved domains, specific for leucine-rich repeat receptor-like kinases, in MhyGSO1, and the N-terminal leucine-rich domain in MhyGSO2. Phylogenetic analysis of MhyGASSHOs confirmed their homology with GSO1 and GSO2 kinases of the Rosids and Asterids representatives. The closest homologues of MhyGSO1 and MhyGSO2 were GSO1 and GSO2, respectively, of the Solanales members (Asterids). Quantification of the MhyGSO1 and MhyGSO2 transcripts revealed expression of both genes in flowers and bracts, and MhyGSO1 – also in roots with buds. In combination with known data about GSO1 and GSO2, it allowed us to assume the redundant activity of MhyGASSHO paralogues in signaling pathways, in particular, in response to exogenous sucrose and in development of reproductive organs and embryonic inflorescences

ИЗУЧЕНИЕ ЭФФЕКТ ГЕТЕРОЗИСА ПЕРЦА СЛАДКОГО ПРИ ПОДБОРЕ РОДИТЕЛЬСКИХ ПАР С ИСПОЛЬЗОВАНИЕМ ДАННЫХ МОЛЕКУЛЯРНОГО АНАЛИЗА

Based on the results of molecular-genetic analysis of pepper accessions of VNIISSOK's breeding, the parental lines were selected. The diallel crossing among them has been conducted. The F1 hybrids were developed. The hybrids vigor (heterosis) for main agronomic traits was estimated. The best pepper hybrids were selected. The potential correlation between genetic divergence determined by molecular markers and heterosis effect is speculated.По результатам молекулярно-генетического анализа сортообразцов перца селекции ВНИИССОК подобраны родительские пары и проведены диаллельные скрещивания между ними. В результате получены гибриды F1, рассчитан эффект гетерозиса по основным хозяйственно ценным признакам. Выявлены лучшие гетерозисные гибридные комбинации. Проведены исследования по выявлению взаимосвязи между уровнем генетической дивергентности, определенной с помощью молекулярного маркирования, и эффектом гетерозиса