DREB1 and DREB2 Genes in Garlic (Allium sativum L.): Genome-Wide Identification, Characterization, and Stress Response.

Dehydration-responsive element-binding (DREB) transcription factors (TFs) of the A1 and A2 subfamilies involved in plant stress responses have not yet been reported in Allium species. In this study, we used bioinformatics and comparative transcriptomics to identify and characterize DREB A1 and A2 genes redundant in garlic (Allium sativum L.) and analyze their expression in A. sativum cultivars differing in the sensitivity to cold and Fusarium infection. Eight A1 (AsaDREB1.1-1.8) and eight A2 (AsaDREB2.1-2.8) genes were identified. AsaDREB1.1-1.8 genes located in tandem on chromosome 1 had similar expression patterns, suggesting functional redundancy. AsaDREB2.1-2.8 were scattered on different chromosomes and had organ- and genotype-specific expressions. AsaDREB1 and AsaDREB2 promoters contained 7 and 9 hormone- and stress-responsive cis-regulatory elements, respectively, and 13 sites associated with TF binding and plant development. In both Fusarium-resistant and -sensitive cultivars, fungal infection upregulated the AsaDREB1.1-1.5, 1.8, 2.2, 2.6, and 2.8 genes and downregulated AsaDREB2.5, but the magnitude of response depended on the infection susceptibility of the cultivar. Cold exposure strongly upregulated the AsaDREB1 genes, but downregulated most AsaDREB2 genes. Our results provide the foundation for further functional analysis of the DREB TFs in Allium crops and could contribute to the breeding of stress-tolerant varieties.

Genome-Wide Identification, Expression, and Response to Fusarium Infection of the SWEET Gene Family in Garlic (Allium sativum L.).

Proteins of the SWEET (Sugar Will Eventually be Exported Transporters) family play an important role in plant development, adaptation, and stress response by functioning as transmembrane uniporters of soluble sugars. However, the information on the SWEET family in the plants of the Allium genus, which includes many crop species, is lacking. In this study, we performed a genome-wide analysis of garlic (Allium sativum L.) and identified 27 genes putatively encoding clade I-IV SWEET proteins. The promoters of the A. sativum (As) SWEET genes contained hormone- and stress-sensitive elements associated with plant response to phytopathogens. AsSWEET genes had distinct expression patterns in garlic organs. The expression levels and dynamics of clade III AsSWEET3, AsSWEET9, and AsSWEET11 genes significantly differed between Fusarium-resistant and -susceptible garlic cultivars subjected to F. proliferatum infection, suggesting the role of these genes in the garlic defense against the pathogen. Our results provide insights into the role of SWEET sugar uniporters in A. sativum and may be useful for breeding Fusarium-resistant Allium cultivars.

ZmDREB2.9 Gene in Maize (Zea mays L.): Genome-Wide Identification, Characterization, Expression, and Stress Response.

Dehydration-responsive element-binding (DREB) transcription factors of the A2 subfamily play key roles in plant stress responses. In this study, we identified and characterized a new A2-type DREB gene, ZmDREB2.9, in the Zea mays cv. B73 genome and compared its expression profile with those of the known A2-type maize genes ZmDREB2.1-2.8. ZmDREB2.9 was mapped to chromosome 8, contained 18 predicted hormone- and stress-responsive cis-elements in the promoter, and had two splice isoforms: short ZmDREB2.9-S preferentially expressed in the leaves, embryos, and endosperm and long ZmDREB2.9-L expressed mostly in the male flowers, stamens, and ovaries. Phylogenetically, ZmDREB2.9 was closer to A. thaliana DREB2A than the other ZmDREB2 factors. ZmDREB2.9-S, ZmDREB2.2, and ZmDREB2.1/2A were upregulated in response to cold, drought, and abscisic acid and may play redundant roles in maize stress resistance. ZmDREB2.3, ZmDREB2.4, and ZmDREB2.6 were not expressed in seedlings and could be pseudogenes. ZmDREB2.7 and ZmDREB2.8 showed similar transcript accumulation in response to cold and abscisic acid and could be functionally redundant. Our results provide new data on Z. mays DREB2 factors, which can be used for further functional studies as well as in breeding programs to improve maize stress tolerance.

Thaumatin-like Protein (TLP) Genes in Garlic (Allium sativum L.): Genome-Wide Identification, Characterization, and Expression in Response to Fusarium proliferatum Infection.

Plant antifungal proteins include the pathogenesis-related (PR)-5 family of fungi- and other stress-responsive thaumatin-like proteins (TLPs). However, the information on the TLPs of garlic (Allium sativum L.), which is often infected with soil Fusarium fungi, is very limited. In the present study, we identified 32 TLP homologs in the A. sativum cv. Ershuizao genome, which may function in the defense against Fusarium attack. The promoters of A. sativumTLP (AsTLP) genes contained cis-acting elements associated with hormone signaling and response to various types of stress, including those caused by fungal pathogens and their elicitors. The expression of AsTLP genes in Fusarium-resistant and -susceptible garlic cultivars was differently regulated by F. proliferatum infection. Thus, in the roots the mRNA levels of AsTLP7-9 and 21 genes were increased in resistant and decreased in susceptible A. sativum cultivars, suggesting the involvement of these genes in the garlic response to F. proliferatum attack. Our results provide insights into the role of TLPs in garlic and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.

Characterization of 15-cis-ζ-Carotene Isomerase Z-ISO in Cultivated and Wild Tomato Species Differing in Ripe Fruit Pigmentation.

Isomerization of 9,15,9'-tri-cis-ζ-carotene mediated by 15-cis-ζ-carotene isomerase Z-ISO is a critical step in the biosynthesis of carotenoids, which define fruit color. The tomato clade (Solanum section Lycopersicon) comprises the cultivated tomato (Solanum lycopersicum) and 12 related wild species differing in fruit color and, thus, represents a good model for studying carotenogenesis in fleshy fruit. In this study, we identified homologous Z-ISO genes, including 5'-UTRs and promoter regions, in 12 S. lycopersicum cultivars and 5 wild tomato species (red-fruited Solanum pimpinellifolium, yellow-fruited Solanum cheesmaniae, and green-fruited Solanum chilense, Solanum habrochaites, and Solanum pennellii). Z-ISO homologs had a highly conserved structure, suggesting that Z-ISO performs a similar function in tomato species despite the difference in their fruit color. Z-ISO transcription levels positively correlated with the carotenoid content in ripe fruit of the tomatoes. An analysis of the Z-ISO promoter and 5'-UTR sequences revealed over 130 cis-regulatory elements involved in response to light, stresses, and hormones, and in the binding of transcription factors. Green- and red/yellow-fruited Solanum species differed in the number and position of cis-elements, indicating changes in the transcriptional regulation of Z-ISO expression during tomato evolution, which likely contribute to the difference in fruit color.

Characterization of RIN Isoforms and Their Expression in Tomato Fruit Ripening.

Ripening of tomato fleshy fruit is coordinated by transcription factor RIN, which triggers ethylene and carotenoid biosynthesis, sugar accumulation, and cell wall modifications. In this study, we identified and characterized complete sequences of the RIN chromosomal locus in two tomato Solanum lycopersicum cultivars, its rin/RIN genotype, and three wild green-fruited species differing in fruit color and composition. The results reveal that S. lycopersicum cultivars and some wild species (S. pennellii, S. habrochaites, and S. huaylasense) had a 3'-splicing site enabling the transcription of RIN1i and RIN2i isoforms. The other wild species (S. arcanum, S. chmielewskii, S. neorickii, and S. peruvianum) had a 3'-splicing site only for RIN2i, which was consistent with RIN1i and RIN2i expression patterns. The genotype rin/RIN, which had an extended 3'-terminal deletion in the rin allele, mainly expressed the chimeric RIN-MC transcript, which was also found in cultivars (RIN/RIN). The RIN1, but not RIN2, protein is able to induce the transcription of the reporter gene in the Y2H system, which positively correlated with the transcription profile of RIN1i and RIN target genes. We suggest that during fruit ripening, RIN1 activates ripening-related genes, whereas RIN2 and RIN-MC act as modulators by competing for RIN-binding sites in gene promoters, which should be confirmed by further studies on the association between RIN-splicing mechanisms and tomato fruit ripening.

Pathogenesis-Related Genes of PR1, PR2, PR4, and PR5 Families Are Involved in the Response to Fusarium Infection in Garlic (Allium sativum L.).

Plants of the genus Allium developed a diversity of defense mechanisms against pathogenic fungi of the genus Fusarium, including transcriptional activation of pathogenesis-related (PR) genes. However, the information on the regulation of PR factors in garlic (Allium sativum L.) is limited. In the present study, we identified AsPR genes putatively encoding PR1, PR2, PR4, and PR5 proteins in A. sativum cv. Ershuizao, which may be involved in the defense against Fusarium infection. The promoters of the AsPR1-5 genes contained jasmonic acid-, salicylic acid-, gibberellin-, abscisic acid-, auxin-, ethylene-, and stress-responsive elements associated with the response to plant parasites. The expression of AsPR1c, d, g, k, AsPR2b, AsPR5a, c (in roots), and AsPR4a(c), b, and AsPR2c (in stems and cloves) significantly differed between garlic cultivars resistant and susceptible to Fusarium rot, suggesting that it could define the PR protein-mediated protection against Fusarium infection in garlic. Our results provide insights into the role of PR factors in A. sativum and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.

Nepenthes × ventrata Transcriptome Profiling Reveals a Similarity Between the Evolutionary Origins of Carnivorous Traps and Floral Organs.

The emergence of the carnivory syndrome and traps in plants is one of the most intriguing questions in evolutionary biology. In the present study, we addressed it by comparative transcriptomics analysis of leaves and leaf-derived pitcher traps from a predatory plant Nepenthes ventricosa × Nepenthes alata. Pitchers were collected at three stages of development and a total of 12 transcriptomes were sequenced and assembled de novo. In comparison with leaves, pitchers at all developmental stages were found to be highly enriched with upregulated genes involved in stress response, specification of shoot apical meristem, biosynthesis of sucrose, wax/cutin, anthocyanins, and alkaloids, genes encoding digestive enzymes (proteases and oligosaccharide hydrolases), and flowering-related MADS-box genes. At the same time, photosynthesis-related genes in pitchers were transcriptionally downregulated. As the MADS-box genes are thought to be associated with the origin of flower organs from leaves, we suggest that Nepenthes species could have employed a similar pathway involving highly conserved MADS-domain transcription factors to develop a novel structure, pitcher-like trap, for capture and digestion of animal prey during the evolutionary transition to carnivory. The data obtained should clarify the molecular mechanisms of trap initiation and development and may contribute to solving the problem of its emergence in plants.

Genome-Wide Identification and Expression of Chitinase Class I Genes in Garlic (Allium sativum L.) Cultivars Resistant and Susceptible to Fusarium proliferatum.

Vegetables of the Allium genus are prone to infection by Fusarium fungi. Chitinases of the GH19 family are pathogenesis-related proteins inhibiting fungal growth through the hydrolysis of cell wall chitin; however, the information on garlic (Allium sativum L.) chitinases is limited. In the present study, we identified seven class I chitinase genes, AsCHI1-7, in the A. sativum cv. Ershuizao genome, which may have a conserved function in the garlic defense against Fusarium attack. The AsCHI1-7 promoters contained jasmonic acid-, salicylic acid-, gibberellins-, abscisic acid-, auxin-, ethylene-, and stress-responsive elements associated with defense against pathogens. The expression of AsCHI2, AsCHI3, and AsCHI7 genes was constitutive in Fusarium-resistant and -susceptible garlic cultivars and was mostly induced at the early stage of F. proliferatum infection. In roots, AsCHI2 and AsCHI3 mRNA levels were increased in the susceptible and decreased in the resistant cultivar, whereas in cloves, AsCHI7 and AsCHI5 expression was decreased in the susceptible but increased in the resistant plants, suggesting that these genes are involved in the garlic response to Fusarium proliferatum attack. Our results provide insights into the role of chitinases in garlic and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.

Differential Regulation of Phytoene Synthase PSY1 During Fruit Carotenogenesis in Cultivated and Wild Tomato Species (Solanum section Lycopersicon).

In plants, carotenoids define fruit pigmentation and are involved in the processes of photo-oxidative stress defense and phytohormone production; a key enzyme responsible for carotene synthesis in fruit is phytoene synthase 1 (PSY1). Tomatoes (Solanum section Lycopersicon) comprise cultivated (Solanum lycopersicum) as well as wild species with different fruit color and are a good model to study carotenogenesis in fleshy fruit. In this study, we identified homologous PSY1 genes in five Solanum section Lycopersicon species, including domesticated red-fruited S. lycopersicum and wild yellow-fruited S. cheesmaniae and green-fruited S. chilense, S. habrochaites and S. pennellii. PSY1 homologs had a highly conserved structure, including key motifs in the active and catalytic sites, suggesting that PSY1 enzymatic function is similar in green-fruited wild tomato species and preserved in red-fruited S. lycopersicum. PSY1 mRNA expression directly correlated with carotenoid content in ripe fruit of the analyzed tomato species, indicating differential transcriptional regulation. Analysis of the PSY1 promoter and 5'-UTR sequence revealed over 30 regulatory elements involved in response to light, abiotic stresses, plant hormones, and parasites, suggesting that the regulation of PSY1 expression may affect the processes of fruit senescence, seed maturation and dormancy, and pathogen resistance. The revealed differences between green-fruited and red-fruited Solanum species in the structure of the PSY1 promoter/5'-UTR, such as the acquisition of ethylene-responsive element by S. lycopersicum, could reflect the effects of domestication on the transcriptional mechanisms regulating PSY1 expression, including induction of carotenogenesis during fruit ripening, which would contribute to red coloration in mature fruit.

The expression pattern of the Pho1a genes encoding plastidic starch phosphorylase correlates with the degradation of starch during fruit ripening in green-fruited and red-fruited tomato species.

Genes encoding plastidic starch phosphorylase Pho1a were identified in 10 tomato species (Solanum section Lycopersicon). Pho1a genes showed higher variability in green-fruited than in red-fruited tomato species, but had an extremely low polymorphism level compared with other carbohydrate metabolism genes and an unusually low ratio of intron to exon single nucleotide polymorphisms (SNPs). In red-fruited species, Pho1a was expressed in all analysed tissues, including fruit at different developmental stages, with the highest level in mature green fruit, which is strong sink organ importing sucrose and accumulating starch. In green-fruited species Solanum peruvianum and Solanum arcanum, the Pho1a expression level was similar in mature green and ripe fruit, whereas in Solanum chmielewskii, it was higher in ripe fruit, and in Solanum habrochaites, the dynamics of fruit-specific Pho1a expression was similar to that in red-fruited tomatoes. During fruit development, in red-fruited Solanum lycopersicum, sucrose level was low, the monosaccharide content increased; in green-fruited S. peruvianum, the sucrose concentration increased and those of monosaccharides decreased. In both species, the starch content and Pho1a expression were downregulated. The evolutionary topology based on Pho1a sequences was consistent with the current division of tomatoes into red-fruited and green-fruited species, except for S. habrochaites.

The YABBY Genes of Leaf and Leaf-Like Organ Polarity in Leafless Plant Monotropa hypopitys.

Monotropa hypopitys is a mycoheterotrophic, nonphotosynthetic plant acquiring nutrients from the roots of autotrophic trees through mycorrhizal symbiosis, and, similar to other extant plants, forming asymmetrical lateral organs during development. The members of the YABBY family of transcription factors are important players in the establishment of leaf and leaf-like organ polarity in plants. This is the first report on the identification of YABBY genes in a mycoheterotrophic plant devoid of aboveground vegetative organs. Seven M. hypopitys YABBY members were identified and classified into four clades. By structural analysis of putative encoded proteins, we confirmed the presence of YABBY-defining conserved domains and identified novel clade-specific motifs. Transcriptomic and qRT-PCR analyses of different tissues revealed MhyYABBY transcriptional patterns, which were similar to those of orthologous YABBY genes from other angiosperms. These data should contribute to the understanding of the role of the YABBY genes in the regulation of developmental and physiological processes in achlorophyllous leafless plants.

Deep-sequence profiling of miRNAs and their target prediction in Monotropa hypopitys.

Myco-heterotroph Monotropa hypopitys is a widely spread perennial herb used to study symbiotic interactions and physiological mechanisms underlying the development of non-photosynthetic plant. Here, we performed, for the first time, transcriptome-wide characterization of M. hypopitys miRNA profile using high throughput Illumina sequencing. As a result of small RNA library sequencing and bioinformatic analysis, we identified 55 members belonging to 40 families of known miRNAs and 17 putative novel miRNAs unique for M. hypopitys. Computational screening revealed 206 potential mRNA targets for known miRNAs and 31 potential mRNA targets for novel miRNAs. The predicted target genes were described in Gene Ontology terms and were found to be involved in a broad range of metabolic and regulatory pathways. The identification of novel M. hypopitys-specific miRNAs, some with few target genes and low abundances, suggests their recent evolutionary origin and participation in highly specialized regulatory mechanisms fundamental for non-photosynthetic biology of M. hypopitys. This global analysis of miRNAs and their potential targets in M. hypopitys provides a framework for further investigation of miRNA role in the evolution and establishment of non-photosynthetic myco-heterotrophs.

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.

A Bsister MADS-box gene involved in ovule and seed development in petunia and Arabidopsis.

MADS-domain transcription factors are essential for proper flower and seed development in angiosperms and their role in determination of floral organ identity can be described by the 'ABC model' of flower development. Recently, close relatives of the B-type genes were identified by phylogenetic studies, which are referred to as B(sister) (B(s)) genes. Here, we report the isolation and characterization of a MADS-box B(s) member from petunia, designated FBP24. An fbp24 knock-down line appeared to closely resemble the Arabidopsis B(s) mutant abs and a detailed and comparative analysis led to the conclusion that both FBP24 and ABS are necessary to determine the identity of the endothelial layer within the ovule. Protein interaction studies revealed the formation of higher-order complexes between B(s)-C-E and B(s)-D-E type MADS-box proteins, suggesting involvement of these specific complexes in determination of endothelium identity. However, although there are many similarities between the two genes and their products and functions, interestingly FBP24 cannot replace ABS in Arabidopsis. The results presented here demonstrate the importance of the comparative analysis of key regulatory genes in various model systems to fully understand all aspects of plant development.

Control of floral meristem determinacy in petunia by MADS-box transcription factors.

The shoot apical meristem (SAM), a small group of undifferentiated dividing cells, is responsible for the continuous growth of plants. Several genes have been identified that control the development and maintenance of the SAM. Among these, WUSCHEL (WUS) from Arabidopsis (Arabidopsis thaliana) is thought to be required for maintenance of a stem cell pool in the SAM. The MADS-box gene AGAMOUS, in combination with an unknown factor, has been proposed as a possible negative regulator of WUS, leading to the termination of meristematic activity within the floral meristem. Transgenic petunia (Petunia hybrida) plants were produced in which the E-type and D-type MADS-box genes FLORAL BINDING PROTEIN2 (FBP2) and FBP11, respectively, are simultaneously overexpressed. These plants show an early arrest in development at the cotyledon stage. Molecular analysis of these transgenic plants revealed a possible combined action of FBP2 and FBP11 in repressing the petunia WUS homolog, TERMINATOR. Furthermore, the ectopic up-regulation of the C-type and D-type homeotic genes FBP6 and FBP7, respectively, suggests that they may also participate in a complex, which causes the determinacy in transgenic plants. These data support the model that a transcription factor complex consisting of C-, D-, and E-type MADS-box proteins controls the stem cell population in the floral meristem.

Identification and characterization of four chrysanthemum MADS-box genes, belonging to the APETALA1/FRUITFULL and SEPALLATA3 subfamilies.

Four full-length MADS-box cDNAs from chrysanthemum, designated Chrysanthemum Dendrathema grandiflorum MADS (CDM) 8, CDM41, CDM111, and CDM44, have been isolated and further functionally characterized. Protein sequence alignment and expression patterns of the corresponding genes suggest that CDM8 and CDM41 belong to the FRUITFULL (FUL) clade, CDM111 is a member of the APETALA1 (AP1) subfamily, and CDM44 is a member of the SEPALLATA3 (SEP3) subfamily of MADS-box transcription factors. Overexpression of CDM111 in Arabidopsis plants resulted in an aberrant phenotype that is reminiscent of the phenotype obtained by ectopic expression of the AP1 gene. In addition, CDM111 was able to partially complement the ap1-1 mutant from Arabidopsis, illustrating that CDM111 is the functional equivalent to AP1. Yeast two- and three-hybrid studies were performed to investigate the potential protein interactions and complexes in which these chrysanthemum MADS-box proteins are involved. Based on these studies, we conclude that CDM44 is most likely the SEP3 functional equivalent, because the CDM44 protein interacts with CDM proteins of the AP1/FUL and AG subfamilies, and as a higher order complex with the heterodimer between the presumed B-type CDM proteins.