In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana

Background: MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). Results: We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. Conclusion: This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during development, points to mechanisms other than transcriptional control. Together this information is essential to understand the role of these proteins in the regulatory processes that drive floral development and leads to new hypotheses

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

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

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

Effectiveness of the barley cultivar ‘Bios 1’ as source material for breeding

Background. The efficiency of breeding depends largely on the breeding method and the choice of the source material. Hybridization and mutagenesis, combined with selection, are the basic techniques in the development of promising breeding materials and adaptable cultivars of spring barley.Object and methods. The research was implemented in 2002–2019. The material for the research were 948 breeding lines (Federal Agricultural Science Center of the North-East, Kirov) developed through hybridization with cv. ‘Bios 1’, and 190 mutant samples (Vyatka State Agricultural Academy, Kirov) obtained as a result of treating barley seeds with sodium carbonate and irradiation with laser and far-red light in various combinations. The study was conducted in accordance with approved standard techniques.Results and conclusions. Various collection accessions and breeding lines were involved in crosses with cv. ‘Bios 1’. The lines were studied according to the full-scale scheme of the breeding process. As a result, only one breeding line, 52-15, having ‘Bios 1’ in its pedigree, was approved for testing in the competitive variety trial in 2019. These results attested to a low combination capacity of cv. ‘Bios 1’ and to the inefficiency of its further use in hybridization as a parent form. In Vyatka State Agricultural Academy, 190 mutant forms of barley were produced using ‘Bios 1’ as the initial form. According to the results of laboratory experiments and competitive variety trials, 5 mutant forms of barley were identified as promising. Spring barley samples, combining high yield with a set of traits valuable for breeding, were selected for further breeding work: breeding line 52-15, and mutant forms M 4-16-3, M 9-5-3 and M 11- 13 Kha. The new barley accession M 8-3-013, maturing 8 days earlier than the original cultivar, having a long (8.9 cm), wellgrained (24.3 grains) and productive (1.31 g) ear, is submitted for the State Variety Trials

Genome variability of domestic tomato varieties: data from AFLP analysis

Tomato Solanum lycopersicum L. is one of the main vegetable crops, accessions and cultivars of which are characterized by a low level of genomic polymorphism. Introgressive tomato breeding uses related wild Solanum species to improve cultivars for stress tolerance and fruit quality traits. The aim of this work was to evaluate the genome variability of 59 cultivars and perspective breeding lines of S. lycopersicum and 11 wild tomato species using the AFLP method. According to the AFLP analysis, four combinations of primers E32/M59, E32/M57, E38/M57, and E41/M59, which had the highest PIC (polymorphism information content) values, were selected. In the process of genotyping a collection of 59 cultivars/lines of S. lycopersicum and 11 wild tomato accessions, the selected primers revealed 391 fragments ranging in size from 80 to 450 bp, of which 114 fragments turned out to be polymorphic and 25 were unique. Analysis of the amplification spectra placed wild tomato accessions into separate clades. Sister clades included cultivars of FSCV breeding resistant to drought and/or cold and, in part, to late blight, Alternaria, Septoria, tobacco mosaic virus and blossom end rot, as well as tomato accessions not characterized according to these traits, which suggests that they have resistance to stress factors. In accessions of distant clades, there was clustering on the basis of resistance to Verticillium, cladosporiosis, Fusarium, tobacco mosaic virus, gray rot, and blossom end rot. The combination of accessions according to their origin from the originating organization was shown. The primer combinations E32/M59, E32/M57, E38/M57 and E41/M59 were shown to be perspective for genotyping tomato cultivars to select donors of resistance to various stress factors. The clade-specific fragments identified in this work can become the basis for the development of AFLP markers for traits of resistance to stress factors

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

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

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

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