MADS-box Research Articles

Orchid Bsister gene PeMADS28 displays conserved function in ovule integument development

The ovules and egg cells are well developed to be fertilized at anthesis in many flowering plants. However, ovule development is triggered by pollination in most orchids. In this study, we characterized the function of a Bsister gene, named PeMADS28, isolated from Phalaenopsis equestris, the genome-sequenced orchid. Spatial and temporal expression analysis showed PeMADS28 predominantly expressed in ovules between 32 and 48 days after pollination, which synchronizes with integument development. Subcellular localization and protein–protein interaction analyses revealed that PeMADS28 could form a homodimer as well as heterodimers with D-class and E-class MADS-box proteins. In addition, ectopic expression of PeMADS28 in Arabidopsis thaliana induced small curled rosette leaves, short silique length and few seeds, similar to that with overexpression of other species’ Bsister genes in Arabidopsis. Furthermore, complementation test revealed that PeMADS28 could rescue the phenotype of the ABS/TT16 mutant. Together, these results indicate the conserved function of BsisterPeMADS28 associated with ovule integument development in orchid.

MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

Osteoporosis is a metabolic bone disease with dysregulated coupling between bone resorption and bone formation, which results in decreased bone mineral density. The MEF2C locus, which encodes the transcription factor MADS box transcription enhancer factor 2, polypeptide C (MEF2C), is strongly associated with adult osteoporosis and osteoporotic fractures. Although the role of MEF2C in bone and cartilage formation by osteoblasts, osteocytes, and chondrocytes has been studied, the role of MEF2C in osteoclasts, which mediate bone resorption, remains unclear. In this study, we identified MEF2C as a positive regulator of human and mouse osteoclast differentiation. While decreased MEF2C expression resulted in diminished osteoclastogenesis, ectopic expression of MEF2C enhanced osteoclast generation. Using transcriptomic and bioinformatic approaches, we found that MEF2C promotes the RANKL-mediated induction of the transcription factors c-FOS and NFATc1, which play a key role in osteoclastogenesis. Mechanistically, MEF2C binds to FOS regulatory regions to induce c-FOS expression, leading to the activation of NFATC1 and downstream osteoclastogenesis. Inducible deletion of Mef2c in mice resulted in increased bone mass under physiological conditions and protected mice from bone erosion by diminishing osteoclast formation in K/BxN serum induced arthritis, a murine model of inflammatory arthritis. Our findings reveal direct regulation of osteoclasts by MEF2C, thus adding osteoclasts as a cell type in which altered MEF2C expression or function can contribute to pathological bone remodeling.

Distance-based measurement determines the coexistence of B protein hetero- and homodimers in lily tepal and stamen tetrameric complexes.

The floral quartet model proposes that plant MADS box proteins function as higher order tetrameric complexes. However, in planta evidence for MADS box tetramers remains scarce. Here, we applied a strategy using in vivo fluorescence resonance energy transfer (FRET) based on thedistance change and distance symmetry of stable tetrameric complexes in tobacco (Nicotiana benthamiana) leaf cells to improve the accuracy of the estimation of heterotetrameric complex formation. This measuring system precisely verified the stable state of Arabidopsis petal (AP3/PI/SEP3/AP1) and stamen (AP3/PI/SEP3/AG) complexes and showed that the lily (Lilium longiflorum) PI co-orthologs LMADS8 and LMADS9 likely formed heterotetrameric petal complexes with Arabidopsis AP3/SEP3/AP1, which rescued petal defects of pi mutants. However, L8/L9 did not form heterotetrameric stamen complexes with Arabidopsis AP3/SEP3/AG to rescue the stamen defects of the pi mutants. Importantly, this system was applied successfully to find complicated tepal and stamen heterotetrameric complexes in lily. We found that heterodimers of B function AP3/PI orthologs (L1/L8) likely coexist with the homodimers of PI orthologs (L8/L8, L9/L9) to form five (two most stable and three stable) tepal- and four (one most stable and three stable) stamen-related heterotetrameric complexes with A/E and C/E function proteins in lily. Among these combinations, L1 preferentially interacted with L8 to form the most stable heterotetrameric complexes, and the importance of the L8/L8 and L9/L9 homodimers in tepal/stamen formation in lily likely decreased to a minor part during evolution. The system provides substantial improvements for successfully estimating the existence of unknown tetrameric complexes in plants.

Heterogeneous Expression and Purification of the Wheat VRN1 K-Box Domain Suggest the Formation of a Tetramer of the VRN1 Protein

In cereal species such as wheat (Ttiticum aestivum) and barley (Hordeum vulgare), many studies have indicated that VERNALIZATION 1 (VRN1) functions as a flowering promoter, which activates florigen gene expression. The wheat florigen gene Wheat FLOWERING LOCUS T (WFT, which is identical to VRN3) is an integrator of the vernalization, photoperiod and autonomous pathways in wheat flowering, and the WFT expression is correlated with the VRN1 expression. VRN1 encodes an APETALA1/FRUITFULL-like MADS-box transcription factor which expression is induced by vernalization, leading to flowering thorough up-regulation of WFT. In Arabidopsis, it has been reported that protein-protein interactions are keys for MADS-box protein function and MADS-box transcription factors must dimerize to bind to the target gene. In this study, by using gel permeation chromatography (GPC) with purified VRN1 protein, we indicated the possibility that VRN1 protein exists as tetramer-like as flowering homeotic MADS-box proteins in Arabidopsis.

Regulatory mechanisms underlying florigenesis in Vanilla planifolia Andrews: A study of MADS-box gene family

ABSTRACT Orchid flowers have unique floral organisation which make them indispensable to the floral industry. The MADS-box gene family encodes for transcription factors that are involved mainly in floral organisation. This study was conducted on Vanilla planifolia, which is an important orchid that is commercially exploited by the food industry for a highly valued flavour, vanillin. V. planifolia transcriptome was analysed and 33 MADS-box transcripts that encode putative MADS-box proteins, were identified. Phylogenetic analysis of the VpMADS proteins with other closely related plant species showed clear clustering into type I and type II. Protein characterisation, duplication events, and their evolutionary analysis were performed. The spatio-temporal expression profile for VpMADS genes at different plant developmental stages showed high expression of these genes in specific floral tissues. Secondary and tertiary structural analyses was also done for 13 MADS-box proteins representing each sub-class and their 3D structures were deduced. This study provides a basic understanding of the functioning of MADS-box gene family in V. planifolia and paves the path for functional characterisation of selected VpMADS genes in planta and in a heterologous system for managing the commercially important traits in this orchid.

FcMADS9 of fig regulates anthocyanin biosynthesis

MADS-box proteins have been shown to regulate fruit coloration, however, no information is available on MADS-box proteins in fig (Ficus carica L.). In this study, a full-length MADS-box gene was cloned from fig peels, namely FcMADS9, and its open reading frame was 759 bp, encoding 252 amino acids. Phylogenetic analysis suggested that it belonged to MIKCC family and SEPALLATA subfamily. Multiple sequence alignment showed that FcMADS9 contained the MADS-box, the Intervening, the Keratin and the C-terminal domains, and it composed a close relationship with MdMADS1, which regulates anthocyanin biosynthesis in apple. Overexpression of FcMADS9 upregulated the expressions of several anthocyanin biosynthetic genes and promoted anthocyanin accumulation in apple peels and calli, indicating its regulatory role in anthocyanin biosynthesis. The expression of FcMADS9 in fig peels was significantly enhanced by 8.24 times by 150 mg L−1 ethephon, a generator of ethylene, indicating that FcMADS9-induced anthocyanin accumulation may have a close relationship with ethylene. Then, anthocyanin accumulation in FcMADS9-overexpressed apple calli was further significantly increased by ethephon treatment, but was partially inhibited by 1-methylcyclopropene, an antagonist of ethylene for receptor binding sites, indicating that FcMADS9 regulates anthocyanin biosynthesis and ethylene is involved in this effect.

Phylogenetic and expression analysis of SEPALLATA-like gene in Brassica oleracea L. var. acephala

The E class MADS-box genes SEPALLATA (SEP)-like play critical roles in angiosperm reproductive growth, especially in floral organ differentiation. To analyze the sequence characteristics and spatio-temporal expression patterns of E-function MADS-box SEP-like genes during kale (Brassica oleracea L. var. acephala) flower development, BroaSEP1/2/3 (GenBank No. KC967957, KC967958, KC967960) homologues, three kale SEP MADS-box gene, were isolated from the kale variety 'Fourteen Line' using Rapid amplification of cDNA ends (RACE). Sequence and phylogenetic analysis indicated that these three SEP genes had a high degree of identity with SEP1, SEP2, SEP3 from Brassica oleracea var. oleracea, Brassica rapa, Raphanus sativus and Brassica napus, respectively. Alignment of the predicted amino acid sequences from these genes, along with previously published subfamily members, demonstrated that these genes comprise four regions of the typical MIKC-type MADS-box proteins: the MADS domain, intervening (I) domain and keratin-like (K) domain, and the C-terminal domain SEPⅠ and SEP Ⅱ motif. The longest open reading frame deduced from the cDNA sequences of BroaSEP1, BroaSEP2, and BroaSEP3 appeared to be 801 bp, 759 bp, 753 bp in length, respectively, which encoded proteins of 266, 252, and 250 amino acids respectively. Expression analyses using semi-quantitative RT-PCR and quantitative real-time PCR indicate that BroaSEP1/2/3 are specifically expressed in floral buds of kale during flower development process. The expression levels of the three genes are very different at different developmental stages, also in wild type, mutant flower with increased petals, and mutant flower with decreased petals. These different patterns of gene expression maybe cause the flowers to increase or decrease the petal number.

Genome-wide study of flowering-related MADS-box genes family in Cardamine hirsuta

MADS-box genes take part in diverse biological functions especially in development of reproductive structures and control of flowering time. Recently, Cardamine hirsuta has emerged as an exclusively powerful genetic system in comparative studies of development. Although the C. hirsuta genome sequence is available but a comprehensive analysis of its MADS-box family genes is still lacking. Here, we determined 50 Cardamine MADS-box genes through bioinformatics tools and classified them into 2 Mβ, 6 Mα and 2 Mγ and 40 MIKC-type (35 MIKCc and 5MIKC*) genes based on a phylogenetic analysis. The C. hirsuta MIKC subfamily could be further classified into 14 subgroups as Arabidopsis. However the number of MADS-box proteins was not equal among these subgroups. Based on the structural diversity among 50 MADS-box genes, 2 lineages were obtained, type I and type II. The lowest number of introns (0 or 1) was found in the Mα, Mβ, and Mγ groups of the type I genes. The most Cardamine MADS-box genes were randomly distributed on only three chromosomes. C. hirsuta had a relatively lower number of flowering MADS-box genes than A. thaliana and probably tandem duplication event resulted in the expansion of FLC, SQUA and TM3 family members in Arabidopsis. Moreover among the conserved motifs, ChMADS5 of SQUA, ChMADS34 of TM3 and ChMADS51 of AGL15 families had no K-domain. This study provides a basis for further functional investigation of MADS-box genes in C. hirsuta.

Identification and analysis of micro-exons in AP2/ERF and MADS gene families.

Micro-exons are a set of ultrashort exons with lengths≤51 nucleotides. Our previous study revealed that micro-exons were enriched in AP2 domains and K-box domains, which are crucial components of AP2/ERF (APETALA2/ethylene-responsive element-binding protein) and MADS-box (an acronym of MCM1, AGAMOUS, DEFICIENS and SRF) genes, respectively. In this study, we analyzed micro-exons in the AP2/ERF family from 63 species and demonstrated that 76.8% of micro-exons are concentrated in AP2 domains. Most micro-exons appeared in the AP2 subfamily of all the terrestrial plants, but not algae. In addition, micro-exons and AP2 domains are conserved and under negative selection. The MIKC gene is a typical MADS-box gene family in terrestrial plants and includes one MADS-box domain and one K-box domain. A total of 92.3% of micro-exons were observed in K-box domains, and two micro-exons usually encoded a region of K-box domain, which is the key to MADS-box protein polymerization. Furthermore, the micro-exons of the K-box domain had higher ratios of nonsynonymous mutations than those of the AP2 domains. Overall, here we explored the relationships and differences among micro-exons in AP2/ERF and MADS families, and revealed potential functional roles of micro-exons in these domains.

Potentials and Challenges of Genomics for Breeding Cannabis Cultivars.

Cannabis (Cannabis sativa L.) is an influential yet controversial agricultural plant with a very long and prominent history of recreational, medicinal, and industrial usages. Given the importance of this species, we deepened some of the main challenges—along with potential solutions—behind the breeding of new cannabis cultivars. One of the main issues that should be fixed before starting new breeding programs is the uncertain taxonomic classification of the two main taxa (e.g., indica and sativa) of the Cannabis genus. We tried therefore to examine this topic from a molecular perspective through the use of DNA barcoding. Our findings seem to support a unique species system (C. sativa) based on two subspecies: C. sativa subsp. sativa and C. sativa subsp. indica. The second key issue in a breeding program is related to the dioecy behavior of this species and to the comprehension of those molecular mechanisms underlying flower development, the main cannabis product. Given the role of MADS box genes in flower identity, we analyzed and reorganized all the genomic and transcriptomic data available for homeotic genes, trying to decipher the applicability of the ABCDE model in Cannabis. Finally, reviewing the limits of the conventional breeding methods traditionally applied for developing new varieties, we proposed a new breeding scheme for the constitution of F1 hybrids, without ignoring the indisputable contribution offered by genomics. In this sense, in parallel, we resumed the main advances in the genomic field of this species and, ascertained the lack of a robust set of SNP markers, provided a discriminant and polymorphic panel of SSR markers as a valuable tool for future marker assisted breeding programs.

FRUITFULL Is a Repressor of Apical Hook Opening in Arabidopsis thaliana.

Plants adjust their architecture to a constantly changing environment, requiring adaptation of differential growth. Despite their importance, molecular switches, which define growth transitions, are largely unknown. Apical hook development in dark grown Arabidopsis thaliana (A. thaliana) seedlings serves as a suitable model for differential growth transition in plants. Here, we show that the phytohormone auxin counteracts the light-induced growth transition during apical hook opening. We, subsequently, identified genes which are inversely regulated by light and auxin. We used in silico analysis of the regulatory elements in this set of genes and subsequently used natural variation in gene expression to uncover correlations between underlying transcription factors and the in silico predicted target genes. This approach uncovered that MADS box transcription factor AGAMOUS-LIKE 8 (AGL8)/FRUITFULL (FUL) modulates apical hook opening. Our data shows that transient FUL expression represses the expression of growth stimulating genes during early phases of apical hook development and therewith guards the transition to growth promotion for apical hook opening. Here, we propose a role for FUL in setting tissue identity, thereby regulating differential growth during apical hook development.

Transcriptome sequencing and metabolite profiling analyses provide comprehensive insight into molecular mechanisms of flower development in Dendrobium officinale (Orchidaceae).

This research provides comprehensive insight into the molecular networks and molecular mechanisms underlying D. officinale flower development. Flowers are complex reproductive organs and play a crucial role in plant propagation, while also providing sustenance for insects and natural bioactive metabolites for humans. However, knowledge about gene regulation and floral metabolomes in flowers is limited. In this study, we used an important orchid species (Dendrobium officinale), whose flowers can be used to make herbal tea, to perform transcriptome sequencing and metabolic profiling of early- and medium-stage flower buds, as well as opened flowers, to provide comprehensive insight into the molecular mechanisms underlying flower development. A total of 8019 differentially expressed genes (DEGs) and 239 differentiated metabolites were found. The transcription factors that were identified and analyzed belong exclusively to the MIKC-type MADS-box proteins and auxin responsive factors that are known to be involved in flower development. The expression of genes involved in chlorophyll and carotenoid biosynthesis strongly matched the metabolite accumulation patterns. The genes related to flavonoid and polysaccharide biosynthesis were active during flower development. Interestingly, indole-3-acetic acid and abscisic acid, whose trend of accumulation was inverse during flower development, may play an important role in this process. Collectively, the identification of DEGs and differentiated metabolites could help to illustrate the regulatory networks and molecular mechanisms important for flower development in this orchid.

Evolutionary Variation in MADS Box Dimerization Affects Floral Development and Protein Abundance in Maize.

Interactions between MADS box transcription factors are critical in the regulation of floral development, and shifting MADS box protein-protein interactions are predicted to have influenced floral evolution. However, precisely how evolutionary variation in protein-protein interactions affects MADS box protein function remains unknown. To assess the impact of changing MADS box protein-protein interactions on transcription factor function, we turned to the grasses, where interactions between B-class MADS box proteins vary. We tested the functional consequences of this evolutionary variability using maize (Zea mays) as an experimental system. We found that differential B-class dimerization was associated with subtle, quantitative differences in stamen shape. In contrast, differential dimerization resulted in large-scale changes to downstream gene expression. Differential dimerization also affected B-class complex composition and abundance, independent of transcript levels. This indicates that differential B-class dimerization affects protein degradation, revealing an important consequence for evolutionary variability in MADS box interactions. Our results highlight complexity in the evolution of developmental gene networks: changing protein-protein interactions could affect not only the composition of transcription factor complexes but also their degradation and persistence in developing flowers. Our results also show how coding change in a pleiotropic master regulator could have small, quantitative effects on development.

Evolution and development of three highly specialized floral structures of bee-pollinated Phalaenopsis species

BackgroundVariation in shape and size of many floral organs is related to pollinators. Evolution of such organs is driven by duplication and modification of MADS-box and MYB transcription factors. We applied a combination of micro-morphological (SEM and micro 3D-CT scanning) and molecular techniques (transcriptome and RT-PCR analysis) to understand the evolution and development of the callus, stelidia and mentum, three highly specialized floral structures of orchids involved in pollination. Early stage and mature tissues were collected from flowers of the bee-pollinated Phalaenopsis equestris and Phalaenopsis pulcherrima, two species that differ in floral morphology: P. equestris has a large callus but short stelidia and no mentum, whereas P. pulcherrima has a small callus, but long stelidia and a pronounced mentum.ResultsOur results show the stelidia develop from early primordial stages, whereas the callus and mentum develop later. In combination, the micro 3D-CT scan analysis and gene expression analyses show that the callus is of mixed petaloid-staminodial origin, the stelidia of staminodial origin, and the mentum of mixed sepaloid-petaloid-staminodial origin. SEP clade 1 copies are expressed in the larger callus of P. equestris, whereas AP3 clade 1 and AGL6 clade 1 copies are expressed in the pronounced mentum and long stelidia of P. pulcherrima. AP3 clade 4, PI-, AGL6 clade 2 and PCF clade 1 copies might have a balancing role in callus and gynostemium development. There appears to be a trade-off between DIV clade 2 expression with SEP clade 1 expression in the callus, on the one hand, and with AP3 clade 1 and AGL6 clade 1 expression in the stelidia and mentum on the other.ConclusionsWe detected differential growth and expression of MADS box AP3/PI-like, AGL6-like and SEP-like, and MYB DIV-like gene copies in the callus, stelidia and mentum of two species of Phalaenopsis, of which these floral structures are very differently shaped and sized. Our study provides a first glimpse of the evolutionary developmental mechanisms driving adaptation of Phalaenopsis flowers to different pollinators by providing combined micro-morphological and molecular evidence for a possible sepaloid–petaloid–staminodial origin of the orchid mentum.

Technical benefit on apple fruit of controlled atmosphere influenced by 1-MCP at molecular levels.

The apple is a highly perishable fruit after harvesting and, therefore, several storage technologies have been studied to provide the consumer market with a quality product with a longer shelf life. However, little is known about the apple genome that is submitted to the storage, and even less with the application of ripening inhibitors. Due to these factors, this study sought to elucidate the transcriptional profile of apple cultivate Gala stored in a controlled atmosphere (AC) treated and not treated with 1-methyl cyclopropene (1-MCP). Through the genetic mapping of the apple, applying the microarray technique, it was possible to verify the action of treatments on transcripts related to photosynthesis, carbohydrate metabolism, response to hormonal stimuli, nucleic acid metabolism, reduction of oxidation, regulation of transcription and metabolism of cell wall and lipids. The results showed that the transcriptional profile in the entire genome of the fruit showed significant differences in the relative expression of the gene, this in response to CA in the presence and absence of 1-MCP. It should be noted that the transcription genes involved in the anabolic pathway were only maintained after six months in fruits treated with 1-MCP. The data in this work suggests that the apple in the absence of 1-MCP begins to prepare its metabolism to mature, even during the storage period in AC. Meanwhile, in the presence of the inhibitor, the transcriptional profile of the fruit is similar to that at the time of harvest. It was also found that a set of genes that code for ethylene receptors, auxin homeostasis, MADS Box, and NAC transcription factors may be involved in the regulation of post-harvest ripening after storage and in the absence of 1-MCP.

GmNMHC5, A Neoteric Positive Transcription Factor of Flowering and Maturity in Soybean.

The soybean (Glycine max (L.) Merr.) is an important oil and food crop. Its growth and development is regulated by complex genetic networks, and there are still many genes with unknown functions in regulation pathways. In this study, GmNMHC5, a member of the MADS-box protein family, was found to promote flowering and maturity in the soybean. Gene expression profiling in transgenic plants confirmed that the 35S:GmNMHC5 T3 generation had early flowering and precocity. We used CRISPR-Cas9 to edit GmNMHC5 and found that late flowering and maturity occurred in Gmnmhc5 lines with stable inheritance. Remarkably, in the 35S:GmNMHC5 plants, the expression of flowering inhibitors GmFT1a and GmFT4 was inhibited. In addition, overexpression of GmNMHC5 in ft-10 (a late flowering Arabidopsis thaliana mutant lacking Flowering Locus T (FT) function) rescued the extremely late-flowering phenotype of the mutant A. thaliana. These results suggest that GmNMHC5 is a positive transcription factor of flowering and maturity in the soybean, which has a close relationship with FT homologs in the flowering regulation pathway. This discovery provides new ideas for the improvement of the flowering regulation network, and can also provide guidance for future breeding work.

Characterization of an APETALA1 and a FRUITFUL-like homolog in chrysanthemum

MADS box transcription factors encoded by APETALA1/FRUITFUL (AP1/FUL-like) genes are key regulators of flowering in angiosperms. Here, two such genes were isolated from the ornamental species Chrysanthemum morifolium cultivar ‘Jinba’. One of the genes (CmAP1L1) was shown to belong to the AP1 clade, and the other (CmFL2) to the FUL clade. Despite their phylogenetic difference, their transcriptional behavior was similar: in plants prior to flowering, both genes were strongly transcribed in the leaves, while in plants which had reached the reproductive stage, their transcription was most abundant in young buds (2–4 mm in diameter) and was at only a low level in the mature inflorescence. Both proteins were localized in the nucleus as well as in the cell membrane. Their over-expression in both cases accelerated flowering, which was also the case when they were constitutively expressed in Arabidopsis thaliana. While the transcription of CmAP1L1 followed an atypical circadian rhythm, that of CmFL2 was arrhythmic. This work suggested that the AP1/FUL-like genes might play a conserved function in promoting flowering in different species.

BcAP3, a MADS box gene, controls stamen development and male sterility in Pak-choi (Brassica rapa ssp. chinensis)

Stamen development is an important developmental process controlled by multiple internal and external factors. Developmental abnormalities of stamens can disrupt the structure and function of anthers, and then result in male sterility. As well known, APETELA 3 (AP3) has a clear function in regulating stamen development, which may impact in male sterility. However, the mechanisms of stamen development and male sterility controlled by AP3 are still not very clear, particular in Pak-choi (Brassica rapa ssp. chinensis). In this work, BcAP3 encoded a protein containing a MADS-box domain, which was a homolog of AtAP3, was identified in Pak-choi. Sequence alignments and phylogenetic analysis indicated that BcAP3 was highly similar to AtAP3. BcAP3 was shown to be localized to the nucleus and exhibited the potential of transcription factor. The transcript of BcAP3 was only expressed in flowers of Pak-choi, indicating that it may act in flower development. Overexpression of BcAP3 in Arabidopsis resulted in developmental abnormalities of anther wall and low vigor pollen, which were associated with the phenotype of male sterility. Expression levels of NST1 and NST2, involved in secondary wall thickening in anther walls, were significantly higher in the BcAP3-transgenic plants than in control plants, suggesting that BcAP3 may affect anther wall development by regulating NST1 and NST2. Taken together, our study demonstrated that BcAP3 could play an essential role in stamen development and male sterility.

Transcriptional repression specifies the central cell for double fertilization

Double fertilization is a key innovation for the evolutionary success of angiosperms by which the two fertilized female gametes, the egg cell and central cell, generate the embryo and endosperm, respectively. The female gametophyte (embryo sac) enclosed in the sporophyte is derived from a one-celled haploid cell lineage. It undergoes successive events of mitotic divisions, cellularization, and cell specification to give rise to the mature embryo sac, which contains the two female gametes accompanied by two types of accessory cells, namely synergids and antipodals. How the cell fate of the central cell is specified has long been equivocal and is further complicated by the structural diversity of female gametophyte across plant taxa. Here, MADS-box protein AGL80 was verified as a transcriptional repressor that directly suppresses the expression of accessory cell-specific genes to specify the central cell. Further genetic rescue and phylogenetic assay of the AGL80 orthologs revealed a possible conserved mechanism in the Brassicaceae family. Results from this study provide insight into the molecular determination of the second female gamete cell in Brassicaceae.

Large-Volume Vascularized Muscle Grafts Engineered From Groin Adipose Tissue in Perfusion Bioreactor Culture.

Muscle tissue engineering still remains a major challenge. An axial vascular pedicle and a perfusion bioreactor are necessary for the development and maintenance of a large-volume engineered muscle tissue to provide circulation within the construct. This study aimed to determine whether large-volume vascularized muscle-like constructs could be made from rat groin adipose tissue in a perfusion bioreactor. Epigastric adipofascial flaps based on the inferior superficial epigastric vessels were elevated bilaterally in male Lewis rats and connected to the bioreactor. The system was run using a cable pump and filled with myogenic differentiation medium in the perfusion bioreactor for 1, 3, 5, or 7 weeks. The resulting tissue constructs were characterized with respect to the morphology and muscle-related expression of genes and proteins. The histological examination demonstrated intact muscle-like tissue fibers; myogenesis was verified by the expression of myosin, MADS box transcription enhancer factor 2 D, desmin-a disintegrin and metalloproteinase domain (ADAM) 12-and M-cadherin using reverse transcription-polymerase chain reaction. Western blot analysis for desmin, MyoD1, N-cadherin, and ADAM12 was performed to verify the myogenic phenotype of the extracted differentiated tissue and prove the formation of muscle-like constructs. A large-volume vascularized muscle tissue could be engineered in a perfusion bioreactor. The resulting tissue had muscle-like histological features and expressed muscle-related genes and proteins, indicating that the trans-differentiation of adipose tissue into muscle tissue occurred.