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.

5'-UTR allelic variants and expression of the lycopene-ɛ-cyclase LCYE gene in maize (Zea mays 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 - ß-carotene, a derivative of the ß,ß-branch of the carotenoid biosynthesis pathway. The parallel ß,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.

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.

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 amplif ication 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 ac cessions 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-specif ic fragments identif ied in this work can become the basis for the development of AFLP markers for traits of resistance to stress factors.

Dependence of the content of starch and reducing sugars on the level of expression of the genes of ß-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 ß-amylases that hydrolyze starch to maltose. In this study, a comparative analysis of the ß-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 ß-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.

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.

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

At all stages of f lowering, 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 f lower 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 f loral 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 conf irmed 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 f lowers, 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 f lowered 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 f lowers and capsules than non-transgenic plants. The f lower 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 f lower meristem development and the pathway of anthocyanin biosynthesis in petals.

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.

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.

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 color, 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 color. 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 colors: 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 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.

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.

Diversification of the Homeotic AP3 Clade MADS-Box Genes in Asteraceae Species Chrysanthemum morifolium L. and Helianthus annuus L.

The structure and phylogeny of MADS-box genes HAM91 of sunflower (Helianthus annuus) and CDM115 of chrysanthemum (Chrysanthemum morifolium) were characterized. It is shown that these genes encode MADS-domain transcription factors, which are orthologs of TM6 (Solanum lycopersicum) and APETALA3 (Arabidopsis thaliana), respectively. We obtained two types of transgenic tobacco plants (Nicotiana tabacum) with constitutive expression of HAM91 and CDM115 genes. Both types of plants flowered later than the control plants and formed more flowers and seed pods. The weight of seeds of 35S::CDM115 plants was significantly lower than in the control and 35S::HAM91 plants, which may indicate to a change in the identity of ovules in 35S::CDM115.

Ectopic Expression of the Homeotic MADS-Box Gene HAM31 (Helianthus annuus L.) in Transgenic Plants Nicotiana tabacum L. Affects the Gynoecium Identity.

The structure of the MADS-box gene HAM31 of the sunflower (Helianthus annuus) was characterized. It is shown that the product of this gene is an ortholog of the B-class MADS transcription factor PISTILLATA (Arabidopsis thaliana). Two types of transgenic tobacco plants (Nicotiana tabacum) with the constitutive expression of the HAM31 gene in the sense and antisense orientation were obtained. The 35S::HAM31s plants formed flowers with an altered gynoecium identity, whereas 35S::HAM31as plants did not differ from the control.

[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.

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 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.

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.

YABBY3-Orthologous Genes in Wild Tomato Species: Structure, Variability, and Expression.

Evolution of the genes encoding YABBY transcription factors is believed to be one of the key reasons for flat leaf emergence from the radially symmetrical stem and gynoecium diversity. YABBY genes determine the identity of the abaxial surface of all aboveground lateral organs in seed plants. In the present study, complete sequences of YABBY3-orthologous genes were identified and characterized in 13 accessions of cultivated and wild tomato species with diverse morphophysiology of leaves, flowers, and fruits. The obtained gene sequences showed high homology (95-99%) and an identical exon-intron structure with the known S. lycopersicum YABBY3 gene, and they contained sequences that encode the conserved HMG-like YABBY and Cys2Cys2-zinc-finger domains. In total, in the analyzed YABBY3 genes, 317 variable sites were found, wherein 8 of 24 exon-specific SNPs were nonsynonymous. In the vegetative and reproductive organs of red-fruited and green-fruited tomato species, YABBY3 gene expression was similar to that in S. pimpinellifolium described earlier, but it demonstrated interspecies differences at the leaf-, bud- and flower-specific expression levels.

Ectopic expression of the HAM59 gene causes homeotic transformations of reproductive organs in sunflower (Helianthus annuus L.).

The function of the HAM59 MADS-box gene in sunflower (Helianthus annuus L.) was studied to clarify homeotic C activity in the Asteraceae plant family. For the first time, transgenic sunflower plants with a modified pattern of HAM59 expression were obtained. It was shown that the HAM59 MADS-box transcription factor did mediate C activity in sunflower. In particular, it participated in termination of the floral meristem, repression of the cadastral function of A-activity, and together with other C-type sunflower protein HAM45-in the specification of the identity of stamens and pistils.