Circadian Regulation of Expression of Carotenoid Metabolism Genes (PSY2, LCYE, CrtRB1, and NCED1) in Leaves of Tomato Solanum lycopersicum L.

The circadian dynamics of the expression of key genes of carotenoid metabolism (PSY2, LCYE, CrtRB1, and NCED1) in the photosynthetic tissue of tomato Solanum lycopersicum L. (cultivar Korneevsky) plants was characterized. An in silico analysis of the gene expression pattern was carried out and a high level of their transcripts was detected in the leaf tissue. qRT-PCR analysis of gene expression was performed at six time points during the day and showed the highest levels of PSY2, LCYE, and NCED1 transcripts in the second half of the light phase and CrtRB1 at the end of the dark phase. The content and composition of carotenoids in leaf tissue in the middle of the day was determined; it was shown that the leaf accumulates 1.5 times more compounds of the ɛ/ß-branch of carotenoid biosynthesis pathway than compounds of the ß/ß-branch.

Potato Solanum tuberosum L. Phytoene Synthase Genes (StPSY1, StPSY2, and StPSY3) Are Involved in the Plant Response to Cold Stress.

The structure and phylogeny of the Solanum tuberosum L. phytoene synthase genes StPSY1, StPSY2, and StPSY3 were characterized. Their expression was studied in potato seedlings exposed to cold stress in the dark phase of the diurnal cycle to simulate night cooling. All of the three genes were activated as the temperature decreased, and the greatest response was observed for StPSY1. StPSY3 was for the first time shown to respond to cold stress and photoperiod. A search for cis-regulatory elements was carried out in the promoter regions and 5'-UTRs of the StPSY genes, and the regulation of all three genes proved associated with the response to light. A high level of cold-induced activation of StPSY1 was tentatively attributed to the presence of cis elements associated with sensitivity to cold and ABA.

Expression Levels of Flowering Time Genes (CONZ1, GIGZ1A, GIGZ1B, FKF1A, and FKF1B) in Seedlings under Long-Day Conditions Differentiates Early and Late Zea mays L. Lines.

Phenophase durations, including the timing of flowering and ripeness, were characterized in 20 inbred lines of the maize Zea mays L. Expression of key flowering initiation genes (CONZ1, GIGZ1a, GIGZ1b, ZmFKF1a, and ZmFKF1b) under long-photoperiod conditions was studied in seedlings of six maize lines that differed in ripeness time. Significantly lower transcription levels of all of the five genes were found in early-ripening lines compared with late-ripening lines. Similar expression patterns were observed for the GIGZ1a and GIGZ1b paralogous genes, while ZmFKF1a significantly predominated in expression over its paralog ZmFKF1b.

Functional Diversification of the Carotenoid-cis-trans-Isomerases CrtISO, CrtISO-L1, and CrtISO-L2 in Tomato Species (Solanum, Section Lycopersicon).

The expression of the genes of carotenoid-cis-trans-isomerases CrtISO, CrtISO-L1, and CrtISO-L2 was studied in comparison with the content of carotenoids in tomato species with different ripe fruit colors: green (Solanum habrochaites), yellow (S. cheesmaniae), and red (S. pimpinellifolium and S. lycopersicum). More ancient origin of CrtISO-L2 in relation to CrtISO and CrtISO-L1 was shown. A similar content of total carotenoids (leaves) and ß-carotene (ripe fruits) between the samples was found. Unlike the fruits of S. habrochaites and S. cheesmaniae, the red fruits accumulated lycopene and 20-30 times greater total carotenoids. The highest level of transcripts both in leaves and in ripe fruits was detected for CrtISO. The CrtISO-L1 and CrtISO-L2 genes were transcribed at high levels in leaves and at low levels in fruits, except for the high expression of CrtISO-L2 in S. lycopersicum fruits. No correlation between the content of carotenoids and the level of gene expression in the fruit was observed. In the leaves, a positive correlation between the amount of carotenoids and the levels of CrtISO-L1 and CrtISO-L2 transcripts was found.

Structural and Functional Diversification of SEPALLATA Genes TM5 and RIN in Tomato Species (Section Lycopersicon).

New TOMATO MADS 5 (TM5) homologous genes were identified in evolutionarily recent, red-fruited and more ancient, wild green-fruited tomato species. It was shown that the identified TM5 homologs belong to the SEPALLATA3 clade; thus, the SEP subfamily diversification was characterized. For the first time, the TM5 and RIN co-expression pattern was determined in flowers, immature green fruits, and ripe fruits of Solanum lycopersicum and in five wild tomato species. It was shown that, regardless of the species, the level of TM5 transcription in flowers was higher than that of RIN, whereas in fruits it was lower than the level of RIN transcription. The data obtained suggest that TM5, together with other transcription factors RIN and SlCMB1, is involved in the regulation of fruit development and ripening.

Variability and Phylogeny of the Pepper Phytoene Synthase Paralogs PSY1 and PSY2 in Species of Various Capsicum Complexes.

Genes homologous to PSY1 and PSY2 that encode phytoene synthase isoforms in Capsicum species C. baccatum, C. chinense, C. frutescens, C. tovarii, C. eximium, and C. chacoense were identified. High conservatism of functionally significant sites of phytoene synthases of the analyzed accessions was revealed. It was found that only PSY1-based clustering of pepper species corresponds to the traditional Capsicum phylogeny; C. eximium was a part of the Purple corolla complex, and C. chacoense was equidistant from Annuum and Baccatum clades. The absence of significant differences between PSY1 and PSY2 of yellow-fruited C. chinense and red-fruited pepper accessions was shown. The yellow color of C. chinense fruit may be the result of both decreased PSY1 expression and increased PSY2 transcription. Thus, it was demonstrated that the acquired fruit pigmentation retains strict phylogenetic limitations, which, however, can be overcome using artificial selection for the activity of phytoene synthase PSY1.

Identification and Expression Analysis of Chitinase Genes in Pitchers of Nepenthes sp. during Development.

Fifteen chitinases of classes I-V were identified in the transcriptomes of pitchers and adult leaves of the carnivorous plant Nepenthes sp. Ten of these chitinases were identified for the first time, including the chitinases of classes II and V. The expression levels of all found chitinase genes in leaves and at three stages of pitcher development were determined. The maximum level of transcriptional activity in an open pitcher was observed for the genes encoding chitinase NChi4 (class II) and its isoforms. The expression levels of these genes significantly increased as the pitcher developed. In addition, for the first time, transcription of the genes encoding chitinases of all five classes was detected in the leaves of this plant.

Identification and Analysis of VTC2 Homologs Encoding the Key Enzyme of L-Ascorbic Acid Biosynthesis in Tomato Species (Solanum Section of Lycopersicon).

The complete nucleotide sequences of 11 wild and cultivated tomato GDP-L-galactose-phosphorylase- 1 homologs (VTC2) were identified and characterized. The overall level of nucleotide variability was 9.19%. Totally 25 out of 54 cDNA SNPs were non-synonymous and resulted in amino acid substitutions. In the group of green-fruited tomato species, more SNPs were detected than in the group of red-fruited species, but the percentage of non-synonymous substitutions in red-fruited tomatoes was significantly higher (66.7% vs. 37.5%). In the translated amino acid sequences, the GDF-L-galactose-hexose-1-phosphate-guanyltransferase domain and the highly conserved HIT-motif were identified and a conserved motif specific for VTC2 of Solanoideae species was detected.

Coexpression of YABBY1 and YABBY3 Genes in Lateral Organs of Tomato Species (Solanum, Section Lycopersicon).

The interspecific polymorphism and the expression patterns of the genes encoding the YABBY1 and YABBY3 transcription factors of cultivated tomato Solanum lycopersicum and wild species S. chmielewskii, S. peruvianum, and S. habrochaites are characterized. The possibility of an inverse relationship between the level of YABBY1 and YABBY3 genes coexpression and the size of leaves and flowers of the tomato species studied is shown. The phylogeny of the genes suggests an earlier emergence of YABBY1 compared to YABBY3 as well as the origin of these paralogs from a common ancestor before the divergence of dicotyledonous plants into Rosids and Asterids.

TAI vacuolar invertase orthologs: the interspecific variability in tomato plants (Solanum section Lycopersicon).

Understanding the genetic mechanisms underlying carbohydrate metabolism can promote the development of biotechnological advances in fruit plants. The flesh tomato fruit represents an ideal system for examining the role of sucrose cleavage enzymes in fruit development, and wild tomato species differing in storage sugars serve as an excellent research material for this purpose. Plant vacuolar invertase is a key enzyme of sucrose metabolism in the sink organs. In the present study, we identified complete gene sequences encoding the TAI vacuolar invertase in 11 wild and one cultivated tomato accessions of the Solanum section Lycopersicon. The average level of interspecific polymorphism in TAI genes was 8.58%; however, in the green-fruited tomatoes, the TAI genes contained 100 times more SNPs than those in the red-fruited accessions. The TAI proteins demonstrated 8% variability, whereas the red-fruited species had none. A TAI-based phylogenetic tree revealed two main clusters containing self-compatible and self-incompatible species, which concurs with the previous crossability-based division and demonstrates that the TAI genes reflect the evolutionary relationships between the red- and green-fruited tomatoes. Furthermore, we detected differential expression patterns of the TAI genes in the fruits of wild and cultivated tomatoes, which corresponded to sugar composition. The polymorphism analysis of the TAI acid invertases of Solanum section Lycopersicon species will contribute to the understanding of the genetic potential of TAI genes to impact tomato breeding through genetic engineering of the carbohydrate composition in the fruit.

[Identification and expression analysis of receptor-like kinase gene ERECTA in mycoheterotrophic plant Monotropa hypopitys].

The precise spatial-temporal coordination of cell division and differentiation is necessary for the correct formation of tissues, organs, and the organism as a whole. This coordination has been implemented by the intercellular communication mediated by signaling molecules and receptors that selectively recognize them. Membrane receptor kinases of ERECTA family regulate inflorescence and flower structure, the formation of root epidermis and adaptation responses. The characterization of the ERECTA genes of flowering plant pinesap Monotropa hypopitys with unique development features can enrich the knowledge about the kinase ERECTA functions and conserved development processes with their participation. Transcriptomic and genomic search with the subsequent structural-phylogenetic analysis identified the mRNA of a gene of serine-threonine kinase receptor with leucine-rich repeats of MhyERL1, which is the only ortholog of the ERECTA family kinases of pinesap. A quantitative analysis of the MhyERL1 gene transcripts has revealed its expression in all analyzed pinesap tissues with maximum levels in the flowers. MhyERL1 is probably involved in defining the inflorescence and flower architecture, and the formation of the pinesap root epidermis. The cascades involving ERL1 are apparently conserved. The exception are pathways associated with the development of above-ground vegetative structures, and the immune response to fungal pathogens probably lost in the process of the pinesap adaptation to unfavorable environmental conditions.

[Analysis of microsatellite loci variability in rare and endemic species Allium regelianum A.K. Becker ex Iljin].

SSR analysis of rare and endemic species Allium regelianum, which grows in the south of Russia, was performed for the first time. Variability analysis of 88 accessions of A. regelianum was carried out using four highly polymorphic microsatellite loci (PIC value ranged from 0.55 to 0.72). SSR-analysis made it possible to revealed polymorphism within and among the populations of A. regelianum from Volgograd region. Analysis of Wright's F-statistics and the analysis of molecular variance showed that more than 90% of total genetic variation of the species was attributed to the differences within the populations and less than 10% of the differences were found among the populations. Cluster analysis of 46 accessions of A. regelianum from three populations of Volgograd region and principal coordinate analysis of all accessions did not reveal differentiation among the populations.

Homeotic MADS-box genes encoding LeMADS-MC orthologues in wild tomato species (genus Solanum).

New full-length genes encoding the LeMADS-MC transcription factor orthologues were identified and functionally characterized in wild tomato species S. cheesmaniae and S. habrochaites. A comparative analysis of the encoded proteins and LeMADS-MC of the cultivated tomato species S. lycopersicum revealed two major amino acid residues substitutions: V155E in the K-domain of ShaMADS-MC (S. habrochaites) and S80L in the I-region of SchMADS-MC (S. cheesmaniae). Structural differences of the C-terminal regions of MC and the canonical euAP1 proteins indicate possible chromosomal rearrangements in the Solanoideae subfamily, which, however, did not change the main known conserved euAP1 functions.

Identification and expression analysis of chitinase genes in parasitic plant Monotropa hypopitys.

Genes encoding six chitinases, five of which belong to classes I (MhCHI3 and MhCHI4), IV (MhCHI1), V (MhCHI5), and VII (MhCHI2), were identified in the transcriptome of the parasitic mixoheterotrophic plant Monotropa hypopitys. The transcription level of MhCHI5 and MhCHI1 was low; however, in the leaves (bracts) and roots it was higher than in flowers. MhCHI4 transcripts were detected primarily in the flowers and were almost absent in the roots, whereas the expression level of MhCHI3 was relatively high in all organs but maximum in the leaves (bracts).

Identification and characterization of the flower meristem identity gene MhyLFY in mycoheterotrophic plant Monotropa hypopitys.

The gene encoding the transcription factor LEAFY was identified in the genome of the mycoheterotrophic plant, pinesap Monotropa hypopitys. In the transcriptomes of roots, bracts, and flowers of flowering pinesaps, the MhyLFY gene expression was absent. These data suggest the conservativeness of the LFY-dependent mechanism of flower meristem identity and flower formation in heterotrophic species with some differences associated to the specificity of development and the structure of such plants. The pinesap flowering under the control of the transcription factor MhyLFY may be initiated either in an embryonic inflorescence during spring dormancy release of adventitious root buds or in an inflorescence of a growing reproductive stem after photoperiodic induction.

Diversification of the Homoeologous Lr34 Sequences in Polyploid Wheat Species and Their Diploid Progenitors.

Allopolyploidization induces a multiple processes of genomic reorganization, including the structurally functional diversification of the homoeologous genes. An example of such diversification is the appearance of the Lr34 gene on chromosome 7D of bread wheat T. aestivum (BAD), the gene conferring durable, race non-specific protection against three fungal pathogens. In this study, we focused on the variability of a functionally critical region between exons 10-12 of Lr34 among diploid progenitors of wheat genomes and their respective polyploids. In the diploid A-genome species, two basic forms of the studied region have been revealed: (1) non-functional forms containing stop codons, or/and frameshifts (T. monococcum/T. urartu) and (2) forms with no such a mutations (T. boeoticum). The Lr34 sequence of T. urartu containing a TGA stop codon was inherited by the first tetraploid T. dicoccoides (BA), and then reorganized in some accessions of this species due to the insertion of an LTR retroelement in exon 10. Besides T. boeoticum, the second form of the Lr34 sequence is also characteristic of A. speltoides, which presumably donated this form to all polyploid descendants bearing B-genome. No differences were found between the D-genome-specific Lr34 sequences studied here and downloaded from databases, implying the highest level of conservation of the Lr34 predecessor throughout evolution. The sequence data were later used to construct phylograms, and apparent peculiarities in the evolution of the studied region of Lr34 genes discussed.

[Analysis of the ITS1/ITS2 nuclear spacers and the secondary structure of 5.8S rRNA gene in endemic species Bellevalia sarmatica (Pall. ex Georgi) Woronow and related species of the subfamily scilloideae].

Sequence variability of the ITS spacers and 5.8S rRNA gene was examined in 11 accessions of the subfamily Scilloideae, including seven accessions of rare and endangered species Bellevalia sarmatica from Volgograd region. The intraspecific polymorphism level of the examined ITS1­5.8S­ITS2 sequence of B. sarmatica accessions constituted 1.3%. The phylogenetic position of B. sarmatica within the genus Bellevalia was determined. It was demonstrated that B. sarmatica belonged to the section Nutantes, and the most closely related species were B. webbiana and B. dubia. Nucleotide substitutions in the 5.8S rRNA gene sequence of the analyzed Scilloideae accessions were identified and studied. The predicted secondary structure of 5.8S rRNA gene was constructed. It was demonstrated that in the examined accessions, mutations in the 5.8S rRNA gene were mainly localized in the third hairpin region and had no effect on the secondary structure of the 5.8S rRNA molecule.

[Sorbitol-6-Phosphate Dehydrogenase Gene Polymorhism in Malus Mill. (Rosaceae)].

The sorbitol-6-phosphate dehydrogenase gene (S6PDH) sequences of six representatives of the genus Malus, which belong to five different taxonomic sections, were examined for the first time. The exon-intron structure and polymorphism of the nucleotide and amino acid sequences of these genes was characterized. The intraspecific polymorphism of the S6PDH gene was assessed for the first time in 40 Russian and foreign apple (Malus domestica) cultivars. It was demonstrated that the interspecific polymorphism level of the S6PDH coding sequences in the studied. representatives of the genus Malus was 4%, and the intraspecific polymorphism level of M. domestica cultivars was very low, constituting 0.96%.

[Intraspecific Variability of ITS Sequences in the Parasitic Plant Monotropa hypopitys L. from the European Russian Populations].

Intraspecific sequence polymorphism in the ITS region of the nuclear ribosomal operon was examined in ten populations of the parasitic plant Monotropa hypopitys from Belgorod oblast, Vologda oblast, and Kaluga oblast of Russia. Based on an analysis of the identified nucleotide substitutions and indels in the ITS regions, the M. hypopitys populations were divided into two groups (A and B). Four new ITS haplotypes of M. hypopitys were identified.

5.8S rRNA sequence and secondary structure in Monotropa hypopitys and related Ericaceae species.

This is the first study to investigate the secondary structure of 5.8S rRNA in M. hypopitys and related Ericaceae species. The identified nucleotide substitutions are localized mostly at the 3' and 5' ends of the gene, in the region of the third hairpin, and do not significantly affect the topology of the secondary structure of the 5.8S rRNA molecule.