Transcriptomic Signature of the SHATTERPROOF2 Expression Domain Reveals the Meristematic Nature of Arabidopsis Gynoecial Medial Domain.

Plant meristems, like animal stem cell niches, maintain a pool of multipotent, undifferentiated cells that divide and differentiate to give rise to organs. In Arabidopsis (Arabidopsis thaliana), the carpel margin meristem is a vital meristematic structure that generates ovules from the medial domain of the gynoecium, the female floral reproductive structure. The molecular mechanisms that specify this meristematic region and regulate its organogenic potential are poorly understood. Here, we present a novel approach to analyze the transcriptional signature of the medial domain of the Arabidopsis gynoecium, highlighting the developmental stages that immediately proceed ovule initiation, the earliest stages of seed development. Using a floral synchronization system and a SHATTERPROOF2 (SHP2) domain-specific reporter, paired with FACS and RNA sequencing, we assayed the transcriptome of the gynoecial medial domain with temporal and spatial precision. This analysis reveals a set of genes that are differentially expressed within the SHP2 expression domain, including genes that have been shown previously to function during the development of medial domain-derived structures, including the ovules, thus validating our approach. Global analyses of the transcriptomic data set indicate a similarity of the pSHP2-expressing cell population to previously characterized meristematic domains, further supporting the meristematic nature of this gynoecial tissue. Our method identifies additional genes including novel isoforms, cis-natural antisense transcripts, and a previously unrecognized member of the REPRODUCTIVE MERISTEM family of transcriptional regulators that are potential novel regulators of medial domain development. This data set provides genome-wide transcriptional insight into the development of the carpel margin meristem in Arabidopsis.

A MADS-box gene is specifically expressed in fibers of cotton (Gossypium hirsutum) and influences plant growth of transgenic Arabidopsis in a GA-dependent manner.

In this study, a cDNA, GhMADS14, encoding a typical MADS-box protein with 223 amino acids was isolated from a cotton cDNA library. Fluorescent microscopy indicated that the GhMADS14 protein was localized in the cell nucleus. GhMADS14 was specifically expressed in the elongating fibers, and its expression was gradually enhanced at early stages of fiber elongation and reached its peak in 9-10 DPA fibers. Overexpression of GhMADS14 in Arabidopsis hindered plant growth. Measurement and statistical analysis revealed that hypocotyl length of GhMADS14 transgenic seedlings was significantly reduced, and the height of the mature transgenic plants was remarkably less than that of the wild type. Furthermore, expression of GA 20-oxidase (AtGA20ox1 and AtGA20ox2) and GA 3-oxidase (AtGA3ox1 and AtGA3ox2) genes was remarkably reduced, whereas AtGA2ox1 and AtGA2ox8 were dramatically up-regulated in the transgenic plants, compared with the wild type. These results suggested that overexpression of GhMADS14 in Arabidopsis may alter expression levels of the genes related to GA biosynthetic and metabolic pathways, resulting in the reduction of endogenous GA amounts in cells. As a result, the transgenic plants grew slowly and display a GA-deficient phenotype.

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.

TrMADS3, a new MADS-box gene, from a perennial species Taihangia rupestris (Rosaceae) is upregulated by cold and experiences seasonal fluctuation in expression level.

In many temperate perennial plants, floral transition is initiated in the first growth season but the development of flower is arrested during the winter to ensure production of mature flowers in the next spring. The molecular mechanisms of the process remain poorly understood with few well-characterized regulatory genes. Here, a MADS-box gene, named as TrMADS3, was isolated from the overwintering inflorescences of Taihangia rupestris, a temperate perennial in the rose family. Phylogenetic analysis reveals that TrMADS3 is more closely related to the homologs of the FLOWERING LOCUS C lineage than to any of the other MIKC-type MADS-box lineages known from Arabidopsis. The TrMADS3 transcripts are extensively distributed in inflorescences, roots, and leaves during the winter. In controlled conditions, the TrMADS3 expression level is upregulated by a chilling exposure for 1 to 2 weeks and remains high for a longer period of time in warm conditions after cold treatment. In situ hybridization reveals that TrMADS3 is predominantly expressed in the vegetative and reproductive meristems. Ectopic expression of TrMADS3 in Arabidopsis promotes seed germination on the media containing relatively high NaCl or mannitol concentrations. These data indicate that TrMADS3 in a perennial species might have its role in both vegetative and reproductive meristems in response to cold.

IbMADS1 (Ipomoea batatas MADS-box 1 gene) is involved in tuberous root initiation in sweet potato (Ipomoea batatas).

BACKGROUND AND AIMS: The tuberization mechanism of sweet potato (Ipomoea batatas) has long been studied using various approaches. Morphological data have revealed that the tuberizing events result from the activation of the cambium, followed by cell proliferation. However, uncertainties still remain regarding the regulators participating in this signal-transduction pathway. An attempt was made to characterize the role of one MADS-box transcription factor, which was preferentially expressed in sweet potato roots at the early tuberization stage. METHODS: A differential expression level of IbMADS1 (Ipomoea batatas MADS-box 1) was detected temporally and spatially in sweet potato tissues. IbMADS1 responses to tuberization-related hormones were assessed. In order to identify the evolutionary significance, the expression pattern of IbMADS1 was surveyed in two tuber-deficient Ipomoea relatives, I. leucantha and I. trifida, and compared with sweet potato. In functional analyses, potato (Solanum tuberosum) was employed as a heterologous model. The resulting tuber morphogenesis was examined anatomically in order to address the physiological function of IbMADS1, which should act similarly in sweet potato. KEY RESULTS: IbMADS1 was preferentially expressed as tuberous root development proceeded. Its expression was inducible by tuberization-related hormones, such as jasmonic acid and cytokinins. In situ hybridization data showed that IbMADS1 transcripts were specifically distributed around immature meristematic cells within the stele and lateral root primordia. Inter-species examination indicated that IbMADS1 expression was relatively active in sweet potato roots, but undetectable in tuber-deficient Ipomoea species. IbMADS1-transformed potatoes exhibited tuber morphogenesis in the fibrous roots. The partial swellings along fibrous roots were mainly due to anomalous proliferation and differentiation in the xylem. CONCLUSIONS: Based on this study, it is proposed that IbMADS1 is an important integrator at the initiation of tuberization. As a result, the initiation and development of tuberous roots seems to be well regulated by a network involving a MADS-box gene in which such hormones as jasmonic acid and cytokinins may act as trigger factors.

Expression and DNA binding activity of the tomato transcription factor RIN (ripening inhibitor).

Recently, we have found that the accumulation of ripening inhibitor (RIN) protein increased gradually during tomato fruit ripening. Here, the recombinant protein was expressed in Escherichia coli and affinity-purified. The DNA binding activity of renatured RIN protein was tested by electrophoretic mobility shift assay. The results indicated that an optimal expression and purification system was suitable for obtaining active RIN with DNA binding activity.

Cloning of a MADS box gene (GhMADS3) from cotton and analysis of its homeotic role in transgenic tobacco.

A MADS box gene (GhMADS3) was cloned from cotton (Gossypium hirsutum L.) based on EST sequences. The predicted protein sequence of GhMADS3 showed 85%, 73%, and 62% identity with Theobroma cacao TcAG, Antirrhinum majus FAR, and Arabidopsis thaliana AG, respectively, and was grouped with AG homologues when the full length sequences excluding N-extensions were compared. GhMADS3 expressed in the wild type cotton flower primarily in stamens and carpels, which was comparable to AG in Arabidopsis. However, it was not expressed in floral buds of a homeotic cotton variant chv1. Ectopic expression of GhMADS3 in tobacco (Nicotiana tabacum L.) resulted in flowers with sepal-to-carpel and petal-to-stamen transformation. The carpelloid first whorl organs, with stigmatic tissue on their upper edges, had a white appearance when compared with the dark green color of the wild type sepals. At times, long filaments were observed at the fusion site of the first carpelloid oranges. The second whorl organs in staminoid were usually smaller than the wild type and the color was changed from pink to white. These results suggest that GhMADS3 has a homeotic role in flower development.

SUPPRESSOR OF FRIGIDA4, encoding a C2H2-Type zinc finger protein, represses flowering by transcriptional activation of Arabidopsis FLOWERING LOCUS C.

FLOWERING LOCUS C (FLC), a strong floral repressor, is one of the central regulators of flowering in Arabidopsis thaliana. The expression of FLC is increased by FRIGIDA (FRI) but decreased by vernalization, a long period of cold exposure that accelerates flowering. Although many aspects of FLC regulation have been reported, it is not known how FLC is transcriptionally activated by FRI at the molecular level. We isolated suppressor of FRIGIDA4 (suf4), a mutant that flowers early as a result of low FLC expression. SUF4 encodes a nuclear-localized protein with two C2H2-type zinc finger motifs and a Pro-rich domain. SUF4 protein interacts with FRI and FRIGIDA-LIKE1 (FRL1), two genes for which single mutations have the same phenotype as suf4. SUF4 also bound to the promoter of FLC in a chromatin immunoprecipitation assay, suggesting that SUF4 acts as a transcriptional activator of FLC after forming a complex with FRI and FRL1. In addition, suf4 suppresses luminidependens (ld), a late-flowering mutation that causes an increase of FLC, and SUF4 protein directly interacts with LD. Thus, we propose that LD binds to SUF4 to suppress its activity in the absence of FRI.

Spatiotemporal expression of duplicate AGAMOUS orthologues during floral development in Phalaenopsis.

The AGAMOUS (AG) family of MADS-box genes plays important roles in controlling the development of the reproductive organs of flowering plants. To understand the molecular mechanisms behind the floral development in the orchid, we isolated and characterized two AG-like genes from Phalaenopsis that we denoted PhalAG1 and PhalAG2. Phylogenetic analysis indicated that PhalAG1 and PhalAG2 fall into different phylogenetic positions in the AG gene family as they belong to the C- and D-lineages, respectively. Reverse transcription-polymerase chair reaction (RT-PCR) analyses showed that PhalAG1 and PhalAG2 transcripts were detected in flower buds but not in vegetative organs. Moreover, in situ hybridization experiments revealed that PhalAG1 and PhalAG2 hybridization signals were observed in the lip, column, and ovule during the floral development of Phalaenopsis, with little difference between the expression patterns of the two genes. These results suggest that both AG-like genes in Phalaenopsis act redundantly with each other in floral development.

Monitoring the interaction between DNA and a transcription factor (MEF2A) using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is an analytical method that allows distinguishing different populations of fluorescent probes in solution and provides data on their concentrations and their diffusion coefficients. FCS was used to characterize the interaction of the transcription factor (MEF2A) with its DNA target sequence. The myocyte enhancer factor 2 (MEF2) belongs to the MADS-box family and activates transcription of numerous muscle genes during myogenesis. Measurements were made using TAMRA-labelled oligonucleotide duplexes derived from a wild type (WT) or a mutated MEF2 target gene. Binding of the protein to the WT DNA resulted in significant changes of the diffusion. Specificity of the interaction was confirmed using the mutated DNA. Bound to free probe ratios were determined at different MEF2A concentrations and the apparent equilibrium dissociation constant K(D) for the full-length MEF2A was estimated.

Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid.

The complex flower organization of orchids offers an opportunity to discover new variant genes and different levels of complexity in the morphogenesis of flowers. In this study, four B-class Phalaenopsis DEF-like MADS-box genes were identified and characterized, including PeMADS2, PeMADS3, PeMADS4 and PeMADS5. Differential expression profiles of these genes were detected in the floral organs of P. equestris, suggesting distinctive roles in the floral morphogenesis of orchids. Furthermore, expressions of these genes were varied to different extents in the peloric mutants with lip-like petals. Expression of PeMADS4 was in lips and columns of wild type, and it extended to the lip-like petals in the peloric mutant. Expression of PeMADS5 was mainly in petals and to a lesser extent in columns in the wild type, whereas it was completely eliminated in the peloric mutant. Disruption of the PeMADS5 promoter region of the peloric mutant was detected at nucleotide +312 relative to the upstream of translational start codon, suggesting that a DNA rearrangement has occurred in the peloric mutant. Genomic structure analysis of the PeMADS5 showed that the exon length was conserved in exons 1-6, similar to DEF-like genes of other plants. Collectively, this is the first report that four DEF-like MADS genes were identified in a single monocotyledonous species and that they may play distinctive morphogenetic roles in the floral development of an orchid.

Isolation of MADS-box genes from sweet potato (Ipomoea batatas (L.) Lam.) expressed specifically in vegetative tissues.

New MADS-domain genes, IbMADS3 and IbMADS4, were isolated from pigmented and tuber-forming root tissue in sweet potato (Ipomoea batatas L.). Both genes were expressed preferentially in vegetative tissues, especially root tissues; white fibrous roots, pigmented roots, and developing tuberous roots. On sequence alignment, these genes fell into the STMADS group composed of SVP, STMADS11, STMADS16 and AGL24, which share high sequence similarity, similar expression patterns and similar function. Transcripts of these two genes in roots were found in the vascular cambium region. This particular expression pattern of these genes may lead to a higher proliferative potential of vegetative tissues, and may facilitate tuber initiation in sweet potato. These genes may lead to important information on the morphogenesis of vegetative structures.

PkMADS1 is a novel MADS box gene regulating adventitious shoot induction and vegetative shoot development in Paulownia kawakamii.

Direct regeneration of shoot buds in vitro is an important technique in plant genetic manipulation. We describe the isolation and functional characterization of a novel MADS box cDNA (PkMADS1) from Paulownia kawakamii leaf explants undergoing adventitious shoot regeneration. mRNA gel blot analysis confirmed the expression of PkMADS1 in the shoot-forming cultures, but no signal was observed in the callus-forming cultures. PkMADS1 transcripts were also detected in shoot apices, but not in root apices, initial leaf explants or the flower. In situ hybridization revealed that its expression was restricted to developing shoot primordia in the excised leaf cultures, suggesting a role for this gene in adventitious shoot formation. Transgenic Paulownia plants over-expressing the PkMADS1 gene showed some changes in phenotype, such as axillary shoot formation. In the antisense transformants, shoots were stunted and had altered phyllotaxy, and, in some lines, the shoot apical meristem appeared to have been used up early during shoot development. Leaf explants from the antisense transgenic plants showed a tenfold decrease in shoot regeneration compared with explants from sense transformants or wild-type. Our results show that PkMADS1 is a regulator of shoot morphogenesis.

Characterization of tobacco MADS-box genes involved in floral initiation.

MADS-box genes encode regulatory factors that are involved at various stages in plant development. These genes function not only during early floral meristem identity, but also when the fate of floral organ primordia is determined in a later step. Here, we screened a floral bud cDNA library to isolate a tobacco MADS-box gene, NtMADS4, using the rice MADS-box gene, OsMADS1, as a probe. We previously reported that OsMADS1 plays a critical role in flower development in rice. Ectopic expression of NtMADS4 caused phenotypes of extremely early flowering as well as dwarfism. Plant MADS proteins have a K domain that mediates the formation of dimers. This dimerization appears to be an essential step for a functional protein complex. NtMADS11 was isolated as an interacting partner of NtMADS4 by yeast two-hybrid screening. The latter was included in the AGAMOUS-like 2 (AGL2) family whereas the former was categorized in the SQUAMOSA (SQUA) family. While the transcript of NtMADS4 was detectable only in reproductive organs, that of NtMADS11 was seen in both reproductive and vegetative organs. Expression levels were high for both genes during early developmental stages. Ectopic expression of NtMADS11 and OsMADS14 was able to rescue the floral organ defects seen in the strong ap1-1 mutant. Roles of NtMADS4 and NtMADS11 in the floral initiation are discussed.