O. A. Shulga

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.

Expression of PVX coat protein gene under the control of extensin-gene promoter confers virus resistance on transgenic potato plants

Tuber discs of potato (Solanum tuberosum L.) cultivars Desirée and Gracia were infected by Agrobacterium tumefaciens carrying a binary vector with the coat protein gene of potato virus X controlled by the carrot extensin gene long-transcript promoter. Several transgenic potato plants have been obtained by direct regeneration of shoots on culture medium with kanamycin used for selection. The presence of the coat protein gene was proved by Southern hybridization in several transformants. Its low but detectable expression level was shown by Northern and Western analysis. Ethephon treatment resulted in a five-fold increase in the amounts of the coat protein mRNA. The majority of transformants exhibited reduced accumulation of virus RNA in inoculated leaves. Potentials in the use of an ethylene-inducible promoter in the production of virus-resistant transgenic plants will be discussed.

MADS-box genes controlling inflorescence morphogenesis in sunflower

MADS-box genes play an important role in plant ontogeny, particularly, in the regulation of floral organ induction and development. Eight full-length cDNAs of HAM genes (HelianthusannuusMADS) have been isolated from sunflower. They encode MADS-box transcription factors expressed in inflorescence tissues. In the frames of the ABCDE model, the HAM proteins were classified according to their structural homology to known MADS-box transcription factors. The HAM45 and HAM59 genes encode the homeotic C function and are involved in the control of the identity of pistil and stamens, while the HAM75 and HAM92 genes determine the A function and identity of floral and inflorescence meristems and petal identity. The HAM31, HAM2, HAM63, and HAM91 genes encode the B function and are involved in the formation of petals and stamens; and the HAM137 gene encodes the E function. Analysis of the expression of HAM genes in sunflower has demonstrated that the structural and functional differences between the ray and tubular flowers in the inflorescence could be a consequence of the lack of HAM59 expression during ray flower initiation

Genetic regulation of inflorescence development in chrysanthemum.

Flowering is a conserved process in higher plants and is controlled by a network of hierarchically interacting genes [1]. The study of this process in the model plants Arabidopsis thaliana, Petunia hybrida , and Antirrhinum majus gave rise to the development of the ABCDE model [2–4]. In this model, A-activity determines sepal and petal identity; B-activity determines petal and stamen identity; C-activity, the identity of the stamen and carpel; D-activity, the identity of the ovule; and E-activity is involved in the petal, stamen, and carpel identity determination. Each activity is determined by a group of genes, most of which encode the MADSbox transcription factors [5]. It was suggested that the MADS-box proteins formed quaternary complexes that regulated the transcription of target genes [4, 6]. In A. thaliana , the petal identity is determined by the complex AP1/SEP3/PI/AP3 (which corresponds to A/E/B/B activities); the stamen identity is determined by AG/SEP3/PI/AP3 (C/E/B/B); and the carpel identity, by SEP3/SEP3/AG/AG complex (E/E/C/C).

Transcriptome-Wide Characterization of the MADS-Box Family in Pinesap Monotropa hypopitys Reveals Flowering Conservation in Non-photosynthetic Myco-Heterotrophs

Pinesap Monotropa hypopitys is a myco-heterotrophic non-photosynthetic higher angiosperm perennial plant devoid of vegetative organs and with standard flower arrangement. The MADS-domain transcription factor family has multiple regulatory functions in plant life cycle and is implicated in flower evolution and diversity. This study is the first to describe the MADS-box genes in a myco-heterotrophic eudicot. By performing the M. hypopitys transcriptome-wide analysis, we identified 30 MADS-box genes belonging to the major clades in the MIKCc and MIKC* lineages. Among them, MhyMADS18 is suggested as an ancestral gene of the cluster comprising AP1/FUL, FLC, SEP, and AGL6 clades. The RNA-seq profiling of the MhyMADS expression in the flowering plant revealed mRNAs specific to bracts, flowers, and roots with adventitious buds. Our results represent systematic and expression analysis of the pinesap MADS-box genes that may be involved in seed dormancy regulation, flowering time control, and flower organ identity specification. The obtained data should further our understanding of the roles played by the MADS-box genes in developmental regulation of myco-heterotrophic plants.

Influence of ectopic expression of Asteraceae MADS box genes on plant ontogeny in tobacco

Plant MADS box transcription factors play key roles in many developmental processes, including the transition to reproductive phase and determination of floral meristem and organs identity. Here we describe the obtaining and characterization of transgenic Nicotiana tabacum L. plants with constitutive expression of Asteraceae MADS box genes CDM111, CDM41, CDM8,CDM77, CDM44 (Chrysanthemum morifolium L.) and HAM92, HAM75 (Helianthus annuus L.). Phylogenetic analysis confirmed that CDM111, HAM75 and HAM92 belong to APETALA1 (AP1), CDM41 and CDM8—FRUITFULL (FUL), CDM44—SEPALLATA3 (SEP3), and CDM77—ASTERACEAE.SEP3 (AST.SEP3) clades. Overexpression of Chrysanthemum and HelianthusAP1/FUL-like genes in tobacco plants resulted in early flowering, shortened stem and decreased number of leaves, which confirmed the functional similarity of Asteraceae AP1/FUL-like factors to AP1 and FUL. This observation testified the conservatism of processes taking place in different plants including Asteraceae. The yeast GAL4 two- and three-hybrid analysis of interactions between CDM77 and other CDM proteins revealed that CDM77 shares similar interaction map with Gerbera SEP-proteins GRCD1 and GRCD2. Overexpression of CDM44 in tobacco caused early flowering without any alterations in vegetative tissues, while overexpression of CDM77 did not reveal any visible developmental changes, which verified the functional similarity between CDM44 and SEP3, and assumed the unique role of CDM77 as whorl- and flower-type specific C-function partner.

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

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

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.

Profiling of microRNAs in wild type and early flowering transgenic Chrysanthemum morifolium by deep sequencing

Here, we performed comparative miRNA profiling in wild type and early flowering transgenic Chrysanthemum morifolium with constitutive expression of APETALA1 (AP1)-like gene, HAM92 (Helianthus annuus). Six sRNA libraries constructed from leaves and shoot apexes after the short day photoperiod initiation, as well as from opened inflorescence after anthesis were sequenced and analyzed. A total of 324 members (163 families) of putative conserved miRNAs and 30 candidate novel miRNAs specific for C. morifolium (cmo-miRNAs) were identified. Bioinformatic analysis revealed 427 and 138 potential mRNA targets for conserved and novel cmo-miRNAs, respectively. These genes were described in Gene Ontology terms and found to be implicated in a broad range of signaling pathways. Plant- and tissue-specific expression of 9 highly conserved cmo-miRNAs was compared between wild type and transgenic chrysanthemum lines with ectopic expression of AP1-like genes HAM92 and CDM111 (C. morifolium), using RT-qPCR and cmo-miR162a as a reference miRNA. The results of our study provide a framework for further investigation of miRNA evolution and functions in higher plants, as well as their roles in flowering control.

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.