Identification and Characterization of MIKCc-Type MADS-Box Genes in the Flower Organs of Adonis amurensis.

Adonis amurensis is a perennial herbaceous flower that blooms in early spring in northeast China, where the night temperature can drop to -15 °C. To understand flowering time regulation and floral organogenesis of A. amurensis, the MIKCc-type MADS (Mcm1/Agamous/ Deficiens/Srf)-box genes were identified and characterized from the transcriptomes of the flower organs. In this study, 43 non-redundant MADS-box genes (38 MIKCc, 3 MIKC*, and 2 Mα) were identified. Phylogenetic and conserved motif analysis divided the 38 MIKCc-type genes into three major classes: ABCDE model (including AP1/FUL, AP3/PI, AG, STK, and SEPs/AGL6), suppressor of overexpression of constans1 (SOC1), and short vegetative phase (SVP). qPCR analysis showed that the ABCDE model genes were highly expressed mainly in flowers and differentially expressed in the different tissues of flower organs, suggesting that they may be involved in the flower organ identity of A. amurensis. Subcellular localization revealed that 17 full-length MADSs were mainly localized in the nucleus: in Arabidopsis, the heterologous expression of three full-length SOC1-type genes caused early flowering and altered the expression of endogenous flowering time genes. Our analyses provide an overall insight into MIKCc genes in A. amurensis and their potential roles in floral organogenesis and flowering time regulation.

SiMADS34, an E-class MADS-box transcription factor, regulates inflorescence architecture and grain yield in Setaria italica.

KEY MESSAGE: A novel MADS-box member SiMADS34 is essential for regulating inflorescence architecture and grain yield in Setaria italica. MADS-box transcription factors participate in regulating various developmental processes in plants. Inflorescence architecture is one of the most important agronomic traits and is closely associated with grain yield in most staple crops. Here, we isolated a panicle development mutant simads34 from a foxtail millet (Setaria italica (L.) P. Beauv.) EMS mutant library. The mutant showed significantly altered inflorescence architecture and decreased grain yield. Investigation of agronomic traits revealed increased panicle width by 16.8%, primary branch length by 10%, and number of primary branches by 30.9%, but reduced panicle length by 25.2%, and grain weight by 25.5% in simads34 compared with wild-type plants. Genetic analysis of a simads34 × SSR41 F2 population indicated that the simads34 phenotype was controlled by a recessive gene. Map-based cloning and bulked-segregant analysis sequencing demonstrated that a single G-to-A transition in the fifth intron of SiMADS34 in the mutant led to an alternative splicing event and caused an early termination codon in this causal gene. SiMADS34 mRNA was expressed in all of the tissues tested, with high expression levels at the heading and panicle development stages. Subcellular localization analysis showed that simads34 predominantly accumulated in the nucleus. Transcriptome sequencing identified 241 differentially expressed genes related to inflorescence development, cell expansion, cell division, meristem growth and peroxide stress in simads34. Notably, an SPL14-MADS34-RCN pathway was validated through both RNA-seq and qPCR tests, indicating the putative molecular mechanisms regulating inflorescence development by SiMADS34. Our study identified a novel MADS-box member in foxtail millet and provided a useful genetic resource for inflorescence architecture and grain yield research.

Genome-wide analysis of Jatropha curcas MADS-box gene family and functional characterization of the JcMADS40 gene in transgenic rice.

BACKGROUND: Physic nut (Jatropha curcas), an inedible oilseed plant, is among the most promising alternative energy sources because of its high oil content, rapid growth and extensive adaptability. Proteins encoded by MADS-box family genes are important transcription factors participated in regulating plant growth, seed development and responses to abiotic stress. However, there has been no in-depth research on the MADS-box genes and their roles in physic nut. RESULTS: In our study, 63 MADS-box genes (JcMADSs) were identified in the physic nut genome, and classed into five groups (MIKCC, Mα, Mß, Mγ, MIKC*) according to phylogenetic comparison with Arabidopsis homologs. Expression profile analysis based on RNA-seq suggested that many JcMADS genes had the strongest expression in seeds, and seven of them responded in leaves to at least one abiotic stressor (drought and/or salinity) at one or more time points. Transient expression analysis and a transactivation assay indicated that JcMADS40 is a nucleus-localized transcriptional activator. Plants overexpressing JcMADS40 did not show altered plant growth, but the overexpressing plants did exhibit reductions in grain size, grain length, grain width, 1000-seed weight and yield per plant. Further data on the reduced grain size in JcMADS40-overexpressing plants supported the putative role of JcMADS genes in seed development. CONCLUSIONS: This study will be useful in order to further understand the process of MADS-box genes involved in regulating growth and development in addition to their functions in abiotic stress resistance, and will eventually provide a theoretical basis for the functional investigation and the exploitation of candidate genes for the molecular improvement of physic nut.

Comparative transcriptomic analysis of the flower induction and development of the Lei bamboo (Phyllostachys violascens).

BACKGROUND: Bamboo is a very important forest resource. However, the prolonged vegetative stages and uncertainty of flowering brings difficulties in bamboo flowers sampling. Until now, the flowering mechanism of bamboo is still unclear. RESULTS: In this study, three successive stages of flowering buds and the corresponding vegetative buds (non-flowering stage) from Lei bamboo (Phyllostachys violascens) were collected for transcriptome analysis using Illumina RNA-Seq method. We generated about 442 million clean reads from the above samples, and 132,678 unigenes were acquired with N50 of 1080 bp. A total of 7266 differentially expressed genes (DEGs) were determined. According to expression profile and gene function analysis, some environmental stress responsive and plant hormone-related DEGs were highly expressed in the inflorescence meristem formation stage (TF_1) while some floral organ development related genes were up-regulated significantly in floral organs determination stage (TF_2) and floral organs maturation (TF_3) stage, implying the essential roles of these DEGs in flower induction and maturation of Lei bamboo. Additionally, a total of 25 MADS-box unigenes were identified. Based on the expression profile, B, C/D and E clade genes were more related to floral organs development compared with A clade genes in Lei bamboo. CONCLUSIONS: This transcriptome data presents fundamental information about the genes and pathways involved in flower induction and development of Lei bamboo. Moreover, a critical sampling method is provided which could be benefit for bamboo flowering mechanism study.

Identification and characterization of the MADS-box genes highly expressed in the laticifer cells of Hevea brasiliensis.

MADS-box transcription factors possess many functions in plant reproduction and development. However, few MADS-box genes related to secondary metabolites regulation have been identified. In Hevea brasiliensis, natural rubber is a representative cis-polyisoprenoids in secondary metabolism which occurs in the rubber laticifer cells, the molecular regulation basis of natural rubber biosynthesis is not clear. Here, a total of 24 MADS-box genes including 4 type I MADS-box genes and 20 type II MADS-box genes were identified in the transcriptome of rubber tree latex. The phylogenetic analysis was performed to clarify the evolutionary relationships of all the 24 rubber tree MADS-box proteins with MADS-box transcription factors from Arabidopsis thaliana and Oryza sativa. Four type I MADS-box genes were subdivided into Mα (3 genes) and Mß (1 gene). Twenty type II MADS-box genes were subclassified into MIKC* (8 genes) and MIKCc (12 genes). Eight MADS-box genes (HblMADS3, 5, 6, 7, 9, 13, 23, 24) were predominant expression in laticifers. ABA up-regulated the expression of HblMADS9, and the expression of HblMADS3, HblMADS5, HblMADS24 were up-regulated by MeJA. The function of HblMADS24 was elucidated. HblMADS24 bound HbFPS1 promoter in yeast and HblMADS24 activated HbFPS1 promoter in tobacco plants. Moreover, we proposed that HblMADS24 is a transcription activator of HbFPS1 which taking part in natural rubber biosynthesis.

Genome-wide Analysis of the MADS-Box Gene Family in Watermelon.

MADS-box genes comprise a family of transcription factors that function in the growth and development of plants. To obtain insights into their evolution in watermelon (Citrullus lanatus), we carried out a genome-wide analysis and identified 39 MADS-box genes. These genes were classified into MIKCc (25), MIKC*(3), Mα (5), Mß (3), and Mγ (3) clades according to their phylogenetic relationship with Arabidopsis thaliana and Cucumis sativus; moreover, these 25 genes in the MIKC clade could be classified into 13 subfamilies, and the Flowering Locus C (FLC) subfamily is absent in watermelon. Analysis of the conserved gene motifs showed similar motifs among clades. Continuing chromosomal localizations analysis indicated that MADS-box genes were distributed across 11 chromosomes in watermelon, and these genes were conditioned to be differentially expressed during plant growth and development. This research provides information that will aid further investigations into the evolution of the MADS-box gene family in plants.

Genome-wide identification and classification of MIKC-type MADS-box genes in Streptophyte lineages and expression analyses to reveal their role in seed germination of orchid.

BACKGROUND: MADS-box genes play crucial roles in plant floral organ formation and plant reproductive development. However, there is still no information on genome-wide identification and classification of MADS-box genes in some representative plant species. A comprehensive investigation of MIKC-type genes in the orchid Dendrobium officinale is still lacking. RESULTS: Here we conducted a genome-wide analysis of MADS-box proteins from 29 species. In total, 1689 MADS-box proteins were identified. Two types of MADS-box genes, termed type I and II, were found in land plants, but not in liverwort. The SQUA, DEF/GLO, AG and SEP subfamilies existed in all the tested flowering plants, while SQUA was absent in the gymnosperm Ginkgo biloba, and no genes of the four subfamilies were found in a charophyte, liverwort, mosses, or lycophyte. This strongly corroborates the notion that clades of floral organ identity genes led to the evolution of flower development in flowering plants. Nine subfamilies of MIKCC genes were present in two orchids, D. officinale and Phalaenopsis equestris, while the TM8, FLC, AGL15 and AGL12 subfamilies may be lost. In addition, the four clades of floral organ identity genes in both orchids displayed a conservative and divergent expression pattern. Only three MIKC-type genes were induced by cold stress in D. officinale while 15 MIKC-type genes showed different levels of expression during seed germination. CONCLUSIONS: MIKC-type genes were identified from streptophyte lineages, revealing new insights into their evolution and development relationships. Our results show a novel role of MIKC-type genes in seed germination and provide a useful clue for future research on seed germination in orchids.

Asymmetric birth and death of type I and type II MADS-box gene subfamilies in the rubber tree facilitating laticifer development.

The rubber tree (Hevea brasiliensis Muell. Arg.) is a rubber producing crop and contains specialized laticifers. MADS-box genes are a family of transcription factor genes that regulate plant development, especially floral organ and gametophyte development. 97 MADS-box genes were identified in the rubber tree through transcriptomes and genome mining. 93.8% of the genes were mapped onto the genome scaffolds in correspondence to the coverage (93.8%) of current version of sequenced genome. Phylogenetic analysis indicates that type II MADS-box genes have been more actively duplicated than their orthologous genes in Arabidopsis and rice, so that most (70, 72.2%) of the MADS-box genes in the rubber tree belong to type II subfamily. This is a high percentage compared to those in Arabidopsis (43.7%) and rice (56.8%). Moreover, 69 out of 70 type II genes in the rubber tree are transcribed, and they are mostly predominantly expressed in flowers, but some genes are predominantly expressed in laticifers, suggesting their roles in both flower and laticifer development. The number of type I genes in the rubber tree is only 27 (27.8%), a much smaller number compared to their orthologous genes in Arabidopsis (56.3%) and rice (43.2%). At the same time, most of the type I genes (55.6%, 15) in the rubber tree are silent and are probably pseudogenes. The high birth rate and low death rate of type II genes and low birth rate and high death rate of type I genes may corresponds to special developmental requirements in the rubber tree, e.g. the development of laticifer system for biosynthesis of cis-polyisoprene, the rubber. Moreover, atypical MIKC* factors (e.g. HbMADS1 in S-clade, and HbMADS20 in P-clade) are identified. These genes are diverged to typical MIKC* genes in sequences and facilitate functions required in laticifer development and rubber biosynthesis, which is not necessary in Arabidopsis and rice.

B and E MADS-box genes determine the perianth formation in Cymbidium goeringii Rchb.f.

Cymbidium goeringii Rchb.f. is an important ornamental plant with a striking well-differentiated lip. Its complex floral architecture presents an exciting opportunity to examine perianth development. In flowering plants, class A, B and E floral homeotic genes play key roles in the specification of perianth identity. In this study, we used a cDNA library of wild-type C. goeringii flower buds for transcriptome sequencing. Eighteen candidate class A, B and E genes (including AP1/FUL-, AP2-, DEF-, GLO-, SEP- and AGL6-like genes) were identified. Quantitative real time polymerase chain reaction (qRT-PCR) results showed that CgDEF1, CgSEP2 and CgAGL6-1 were strongly detected only in the sepals and petals and were significantly downregulated in the lips. CgDEF3, CgDEF4 and CgAGL6-3 were highly expressed in the lips and lip-like petals but were only minimally detected in the sepals. Yeast two-hybrid analysis indicated that CgDEF1 and CgGLO formed a heterodimer. CgAGL6-1/CgSEP2 and CgDEF1 formed higher-order protein complexes with the assistance of the CgGLO protein, and both CgAGL6-1 and CgSEP2 formed a heterodimer. CgDEF3/CgDEF4 could interact independently with CgGLO and CgAGL6-3, respectively, while CgDEF3 and CgDEF4 also formed heterodimers with the assistance of the CgGLO. Based on a comprehensive analysis relating these gene expression patterns to protein interaction profiles, the mechanism of sepal/petal/lip determination was studied in C. goeringii. Furthermore, a hypothesis explaining the sepal/petal/lip determination of C. goeringii is proposed. The lip-quartet (CgDEF3/CgDEF4/CgAGL6-3/CgGLO) promoted lip formation, whereas the sepal/petal-quartet (CgDEF1/CgAGL6-1/CgSEP2/CgGLO) promoted sepal/petal formation. These results enrich the current knowledge regarding the mechanism and pathways of perianth formation in orchids.

Phylogenomic Synteny Network Analysis of MADS-Box Transcription Factor Genes Reveals Lineage-Specific Transpositions, Ancient Tandem Duplications, and Deep Positional Conservation.

Conserved genomic context provides critical information for comparative evolutionary analysis. With the increase in numbers of sequenced plant genomes, synteny analysis can provide new insights into gene family evolution. Here, we exploit a network analysis approach to organize and interpret massive pairwise syntenic relationships. Specifically, we analyzed synteny networks of the MADS-box transcription factor gene family using 51 completed plant genomes. In combination with phylogenetic profiling, several novel evolutionary patterns were inferred and visualized from synteny network clusters. We found lineage-specific clusters that derive from transposition events for the regulators of floral development (APETALA3 and PI) and flowering time (FLC) in the Brassicales and for the regulators of root development (AGL17) in Poales. We also identified two large gene clusters that jointly encompass many key phenotypic regulatory Type II MADS-box gene clades (SEP1, SQUA, TM8, SEP3, FLC, AGL6, and TM3). Gene clustering and gene trees support the idea that these genes are derived from an ancient tandem gene duplication that likely predates the radiation of the seed plants and then expanded by subsequent polyploidy events. We also identified angiosperm-wide conservation of synteny of several other less studied clades. Combined, these findings provide new hypotheses for the genomic origins, biological conservation, and divergence of MADS-box gene family members.

Functional characterization of AGAMOUS-subfamily members from cotton during reproductive development and in response to plant hormones.

KEY MESSAGE: Expression analysis of the AG -subfamily members from G. hirsutum during flower and fruit development. Reproductive development in cotton, including the fruit and fiber formation, is a complex process; it involves the coordinated action of gene expression regulators, and it is highly influenced by plant hormones. Several studies have reported the identification and expression of the transcription factor family MADS-box members in cotton ovules and fibers; however, their roles are still elusive during the reproductive development in cotton. In this study, we evaluated the expression profiles of five MADS-box genes (GhMADS3, GhMADS4, GhMADS5, GhMADS6 and GhMADS7) belonging to the AGAMOUS-subfamily in Gossypium hirsutum. Phylogenetic and protein sequence analyses were performed using diploid (G. arboreum, G. raimondii) and tetraploid (G. barbadense, G. hirsutum) cotton genomes, as well as the AG-subfamily members from Arabidopsis thaliana, Petunia hybrida and Antirrhinum majus. qPCR analysis showed that the AG-subfamily genes had high expression during flower and fruit development in G. hirsutum. In situ hybridization analysis also substantiates the involvement of AG-subfamily members on reproductive tissues of G. hirsutum, including ovule and ovary. The effect of plant hormones on AG-subfamily genes expression was verified in cotton fruits treated with gibberellin, auxin and brassinosteroid. All the genes were significantly regulated in response to auxin, whereas only GhMADS3, GhMADS4 and GhMADS7 genes were also regulated by brassinosteroid treatment. In addition, we have investigated the GhMADS3 and GhMADS4 overexpression effects in Arabidopsis plants. Interestingly, the transgenic plants from both cotton AG-like genes in Arabidopsis significantly altered the fruit size compared to the control plants. This alteration suggests that cotton AG-like genes might act regulating fruit formation. Our results demonstrate that members of the AG-subfamily in G. hirsutum present a conserved expression profile during flower development, but also demonstrate their expression during fruit development and in response to phytohormones.

Characterization and Functional Analysis of Five MADS-Box B Class Genes Related to Floral Organ Identification in Tagetes erecta.

According to the floral organ development ABC model, B class genes specify petal and stamen identification. In order to study the function of B class genes in flower development of Tagetes erecta, five MADS-box B class genes were identified and their expression and putative functions were studied. Sequence comparisons and phylogenetic analyses indicated that there were one PI-like gene-TePI, two euAP3-like genes-TeAP3-1 and TeAP3-2, and two TM6-like genes-TeTM6-1 and TeTM6-2 in T. erecta. Strong expression levels of these genes were detected in stamens of the disk florets, but little or no expression was detected in bracts, receptacles or vegetative organs. Yeast hybrid experiments of the B class proteins showed that TePI protein could form a homodimer and heterodimers with all the other four B class proteins TeAP3-1, TeAP3-2, TeTM6-1 and TeTM6-2. No homodimer or interaction was observed between the euAP3 and TM6 clade members. Over-expression of five B class genes of T. erecta in Nicotiana rotundifolia showed that only the transgenic plants of 35S::TePI showed altered floral morphology compared with the non-transgenic line. This study could contribute to the understanding of the function of B class genes in flower development of T. erecta, and provide a theoretical basis for further research to change floral organ structures and create new materials for plant breeding.

Ancestral and more recently acquired syntenic relationships of MADS-box genes uncovered by the Physcomitrella patens pseudochromosomal genome assembly.

KEY MESSAGE: The Physcomitrella pseudochromosomal genome assembly revealed previously invisible synteny enabling realisation of the full potential of shared synteny as a tool for probing evolution of this plant's MADS-box gene family. Assembly of the sequenced genome of Physcomitrella patens into 27 mega-scaffolds (pseudochromosomes) has confirmed the major predictions of our earlier model of expansion of the MADS-box gene family in the Physcomitrella lineage. Additionally, microsynteny has been conserved in the immediate vicinity of some recent duplicates of MADS-box genes. However, comparison of non-syntenic MIKC MADS-box genes and neighbouring genes indicates that chromosomal rearrangements and/or sequence degeneration have destroyed shared synteny over longer distances (macrosynteny) around MADS-box genes despite subsets comprising two or three MIKC genes having remained syntenic. In contrast, half of the type I MADS-box genes have been transposed creating new syntenic relations with MIKC genes. This implies that conservation of ancient ancestral synteny of MIKC genes and of more recently acquired synteny of type I and MIKC genes may be selectively advantageous. Our revised model predicts the birth rate of MIKC genes in Physcomitrella is higher than that of type I genes. However, this difference is attributable to an early tandem duplication and an early segmental duplication of MIKC genes prior to the two polyploidisations that account for most of the expansion of the MADS-box gene family in Physcomitrella. Furthermore, this early segmental duplication spawned two chromosomal lineages: one with a MIKC (C) gene, belonging to the PPM2 clade, in close proximity to one or a pair of MIKC* genes and another with a MIKC (C) gene, belonging to the PpMADS-S clade, characterised by greater separation from syntenic MIKC* genes. Our model has evolutionary implications for the Physcomitrella karyotype.

Genome-wide identification and characterization of MADS-box family genes related to organ development and stress resistance in Brassica rapa.

BACKGROUND: MADS-box transcription factors (TFs) are important in floral organ specification as well as several other aspects of plant growth and development. Studies on stress resistance-related functions of MADS-box genes are very limited and no such functional studies in Brassica rapa have been reported. To gain insight into this gene family and to elucidate their roles in organ development and stress resistance, we performed genome-wide identification, characterization and expression analysis of MADS-box genes in B. rapa. RESULTS: Whole-genome survey of B. rapa revealed 167 MADS-box genes, which were categorized into type I (Mα, Mß and Mγ) and type II (MIKC(c) and MIKC*) based on phylogeny, protein motif structure and exon-intron organization. Expression analysis of 89 MIKC(c) and 11 MIKC* genes was then carried out. In addition to those with floral and vegetative tissue expression, we identified MADS-box genes with constitutive expression patterns at different stages of flower development. More importantly, from a low temperature-treated whole-genome microarray data set, 19 BrMADS genes were found to show variable transcript abundance in two contrasting inbred lines of B. rapa. Among these, 13 BrMADS genes were further validated and their differential expression was monitored in response to cold stress in the same two lines via qPCR expression analysis. Additionally, the set of 19 BrMADS genes was analyzed under drought and salt stress, and 8 and 6 genes were found to be induced by drought and salt, respectively. CONCLUSION: The extensive annotation and transcriptome profiling reported in this study will be useful for understanding the involvement of MADS-box genes in stress resistance in addition to their growth and developmental functions, which ultimately provides the basis for functional characterization and exploitation of the candidate genes for genetic engineering of B. rapa.

Evolutionary and expression analysis of a MADS-box gene superfamily involved in ovule development of seeded and seedless grapevines.

MADS-box transcription factors are involved in many aspects of plant growth and development, such as floral organ determination, fruit ripening, and embryonic development. Yet not much is known about grape (Vitis vinifera) MADS-box genes in a relatively comprehensive genomic and functional way during ovule development. Accordingly, we identified 54 grape MADS-box genes, aiming to enhance our understanding of grape MADS-box genes from both evolutionary and functional perspectives. Synteny analysis indicated that both segmental and tandem duplication events contributed to the expansion of the grape MADS-box family. Furthermore, synteny analysis between the grape and Arabidopsis genomes suggested that several grape MADS-box genes arose before divergence of the two species. Phylogenetic analysis and comparisons of exon-intron structures provided further insight into the evolutionary relationships between the genes, as well as their putative functions. Based on phylogenetic tree analysis, grape MADS-box genes were divided into type I and type II subgroups. Tissue-specific expression analysis suggested roles in both vegetative and reproductive tissue development. Expression analysis of the MADS-box genes following gibberellic acid (GA3) treatment revealed their response to GA3 treatment and that seedlessness caused by GA3 treatment underwent a different mechanism from that of normal ovule abortion. Expression profiling of MADS-box genes from six cultivars suggests their function in ovule development and may represent potential ovule identity genes involved in parthenocarpy. The results presented provide a few candidate genes involved in ovule development for future study, which may be useful in seedlessness-related molecular breeding programs.

GmFULa, a FRUITFULL homolog, functions in the flowering and maturation of soybean.

KEY MESSAGE: A FRUITFULL homolog GmFULa was cloned and found to play roles in the flowering and maturation of soybean. Soybean varieties exhibit great diversity in terms of flowering and maturation due to differences in their photoperiodic responses. The underlying mechanism remains unclear despite the fact that some upstream flowering genes have been studied. FRUITFULL (FUL) genes are one group of downstream flowering genes known to have major roles in reproductive transition, floral meristem identity, and floral organ identity. However, FUL homologs and their functions are poorly understood in soybean. Here, a soybean FUL homolog was cloned from the late-maturing photoperiod-sensitive soybean variety Zigongdongdou (ZGDD) and designated GmFULa. In ZGDD, GmFULa exhibited a terminal-preferential expression pattern, with higher expression in the root and shoot apices than in the middle parts. Diurnal rhythm analysis revealed that photoperiod regulates the GmFULa expression level but does not alter its diurnal rhythm. ZGDD was maintained under different photoperiod conditions (long day, LD; short day, SD; LD after 13 short days, SD13-LD) to assess GmFULa expression in newly expanded leaves and in the shoot apex. From this analysis, GmFULa expression was detected in the floral meristem, floral organs and their primordia; trifoliate leaves; and the inflorescence meristem, with the expression levels induced by SD and inhibited by LD. GmFULa expression was also associated with maturity in seven soybean varieties with different photoperiod sensitivities. Therefore, photoperiod conditions affect the expression level of GmFULa but not its diurnal rhythm. The gene plays pleiotropic roles in reproductive transition, flowering, and leaf development and is associated with maturity in soybean.

The pineapple AcMADS1 promoter confers high level expression in tomato and Arabidopsis flowering and fruiting tissues, but AcMADS1 does not complement the tomato LeMADS-RIN (rin) mutant.

A previous EST study identified a MADS box transcription factor coding sequence, AcMADS1, that is strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses place the AcMADS1 protein in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of ethylene in climacteric tomato. LeMADS-RIN has been proposed to be a global ripening regulator shared among climacteric and non-climacteric species, although few functional homologs of LeMADS-RIN have been identified in non-climacteric species. AcMADS1 shares 67 % protein sequence similarity and a similar expression pattern in ripening fruits as LeMADS-RIN. However, in this study AcMADS1 was not able to complement the tomato rin mutant phenotype, indicating AcMADS1 may not be a functionally conserved homolog of LeMADS-RIN or has sufficiently diverged to be unable to act in the context of the tomato network of interacting proteins. The AcMADS1 promoter directed strong expression of the GUS reporter gene to fruits and developing floral organs in tomato and Arabidopsis thaliana, suggesting AcMADS1 may play a role in flower development as well as fruitlet ripening. The AcMADS1 promoter provides a useful molecular tool for directing transgene expression, particularly where up-regulation in developing flowers and fruits is desirable.

DEF- and GLO-like proteins may have lost most of their interaction partners during angiosperm evolution.

BACKGROUND AND AIMS: DEFICIENS (DEF)- and GLOBOSA (GLO)-like proteins constitute two sister clades of floral homeotic transcription factors that were already present in the most recent common ancestor (MRCA) of extant angiosperms. Together they specify the identity of petals and stamens in flowering plants. In core eudicots, DEF- and GLO-like proteins are functional in the cell only as heterodimers with each other. There is evidence that this obligate heterodimerization contributed to the canalization of the flower structure of core eudicots during evolution. It remains unknown as to whether this strict heterodimerization is an ancient feature that can be traced back to the MRCA of extant flowering plants or if it evolved later during the evolution of the crown group angiosperms. METHODS: The interactions of DEF- and GLO-like proteins of the early-diverging angiosperms Amborella trichopoda and Nuphar advena and of the magnoliid Liriodendron tulipifera were analysed by employing yeast two-hybrid analysis and electrophoretic mobility shift assay (EMSA). Character-state reconstruction, including data from other species as well, was used to infer the ancestral interaction patterns of DEF- and GLO-like proteins. KEY RESULTS: The yeast two-hybrid and EMSA data suggest that DEF- and GLO-like proteins from early-diverging angiosperms both homo- and heterodimerize. Character-state reconstruction suggests that the ability to form heterodimeric complexes already existed in the MRCA of extant angiosperms and that this property remained highly conserved throughout angiosperm evolution. Homodimerization of DEF- and GLO-like proteins also existed in the MRCA of all extant angiosperms. DEF-like protein homodimerization was probably lost very early in angiosperm evolution and was not present in the MRCA of eudicots and monocots. GLO-like protein homodimerization might have been lost later during evolution, but very probably was not present in the MRCA of eudicots. CONCLUSIONS: The flexibility of DEF- and GLO-like protein interactions in early-diverging angiosperms may be one reason for the highly diverse flower morphologies observed in these species. The results strengthen the hypothesis that a reduction in the number of interaction partners of DEF- and GLO-like proteins, with DEF-GLO heterodimers remaining the only DNA-binding dimers in core eudicots, contributed to developmental robustness, canalization of flower development and the diversification of angiosperms.

MADS goes genomic in conifers: towards determining the ancestral set of MADS-box genes in seed plants.

BACKGROUND AND AIMS: MADS-box genes comprise a gene family coding for transcription factors. This gene family expanded greatly during land plant evolution such that the number of MADS-box genes ranges from one or two in green algae to around 100 in angiosperms. Given the crucial functions of MADS-box genes for nearly all aspects of plant development, the expansion of this gene family probably contributed to the increasing complexity of plants. However, the expansion of MADS-box genes during one important step of land plant evolution, namely the origin of seed plants, remains poorly understood due to the previous lack of whole-genome data for gymnosperms. METHODS: The newly available genome sequences of Picea abies, Picea glauca and Pinus taeda were used to identify the complete set of MADS-box genes in these conifers. In addition, MADS-box genes were identified in the growing number of transcriptomes available for gymnosperms. With these datasets, phylogenies were constructed to determine the ancestral set of MADS-box genes of seed plants and to infer the ancestral functions of these genes. KEY RESULTS: Type I MADS-box genes are under-represented in gymnosperms and only a minimum of two Type I MADS-box genes have been present in the most recent common ancestor (MRCA) of seed plants. In contrast, a large number of Type II MADS-box genes were found in gymnosperms. The MRCA of extant seed plants probably possessed at least 11-14 Type II MADS-box genes. In gymnosperms two duplications of Type II MADS-box genes were found, such that the MRCA of extant gymnosperms had at least 14-16 Type II MADS-box genes. CONCLUSIONS: The implied ancestral set of MADS-box genes for seed plants shows simplicity for Type I MADS-box genes and remarkable complexity for Type II MADS-box genes in terms of phylogeny and putative functions. The analysis of transcriptome data reveals that gymnosperm MADS-box genes are expressed in a great variety of tissues, indicating diverse roles of MADS-box genes for the development of gymnosperms. This study is the first that provides a comprehensive overview of MADS-box genes in conifers and thus will provide a framework for future work on MADS-box genes in seed plants.

Identification and characterization of FaSOC1, a homolog of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 from strawberry.

A MADS-box gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) integrates multiple flowering signals to regulate floral transition in Arabidopsis. Strawberry (Fragaria spp.) is an economically important fruit crop, but its molecular control of flowering is largely unknown. In this study, a SOC1-like gene, FaSOC1, was isolated and characterized from strawberry. The open reading frame of FaSOC1 was 648bp, encoding a protein of 215 amino acids. Sequence alignment and phylogenetic analysis showed that the FaSOC1 protein contained a highly conserved MADS domain and a SOC1 motif, and that it was a member of the SOC1-like genes of dicots. The FaSOC1 protein mainly localized in the cytoplasm of onion epidermal cells and Arabidopsis protoplasts, and showed no transcriptional activation activity in yeast cells. Under the floral induction conditions, the expression of FaSOC1 increased during the first 2weeks of short-day treatment, but declined dramatically during three to 4weeks. FaSOC1 was highly expressed in reproductive organs, including shoot apices, floral buds, flowers, stamens and sepals. Overexpression of FaSOC1 in wild-type Arabidopsis caused early flowering and upregulated the expression of flowering time genes LFY and AP1. In addition, the yeast two-hybrid and BiFC assays confirmed that FaSOC1 could interact with AGL24. In conclusion, these results suggest that FaSOC1 is a flowering promoter in strawberry.