Construction of a Full-Length cDNA Library of Gossypium hirsutum L. and Identification of Two MADS-Box Genes

A full-length normalized cDNA library for the flower development stages of short-season cotton （Gossypium hirsutum L.） （CCRI36） was constructed. A total of 3 421 clones were randomly selected for sequencing, with a total of 3 175 effective sequences obtained after removal of empty-carriers and low-quality sequences. Clustering the 3 175 high-quality expressed sequence tags （ESTs） resulted in a set of 2 906 non-redundant sequences comprised of 233 contigs and 2 673 singletons. Comparative analyses indicated that 913 （43.6%） of the unigenes had homologues with function-known genes or functionassumed genes in the National Center for Biotechnology Information. In addition, 763 （36.4%） of the unigenes were functionally classified using Gene Ontology hierarchy. Through EST alignment and the screening method, the full-length cDNA of two MADS-box genes viz., GhMADSll and GhMADS12 were acquired. These genes may play a role in flower development. Phylogenetie analysis indicated that GhMADS11 and GhMADS12 had high homology and close evolutionary relationship with AGL2/SEP-type and PI-type genes, respectively. The expression of both GhMADSll and GhMADS12, genes was high in reproductive organs. In floral organs, GhMADSll expression was high in petals （whor12） and ovules, while GhMADS12 expression was high in petals （whor12） and stamens （whor13）. Results show that the EST strategy based on a normalized cDNA library is an effective method for gene identification. The study provides more insights for future molecular research on the regulation mechanism of cotton flower development.

A MADS-box gene is involved in soybean resistance to multiple Soybean mosaic virus strains

Soybean mosaic virus(SMV)is a member of the genus Potyvirus that extensively impairs global soybean production.The full-length coding sequence of the MADS-box transcription factor Gm CAL was cloned from the SMV-resistant soybean cultivar Kefeng 1.SMV-induced expression analysis indicated that Gm CAL responded quickly to SMV-SC8 infection in Kefeng 1 but not in NN1138-2.Gm CAL was expressed at high levels in flowers and pods but at lower levels in leaves.The gene was localized to the nucleus by subcellular localization assay.Virus-induced gene silencing did not increase the accumulation of SMV in Gm CAL-silenced Kefeng 1 plants(with silencing efficiency~80%)after SC8 inoculation.Gm CAL-silencing plants still conferred resistance to SC8 that might be owing to incomplete silencing of genes with lower expression.SMV content decreased significantly in Gm CAL-overexpressing NN1138-2 plants after SMVSC3,SMV-SC7,and SMV-SC8 inoculation in comparison with a vector control,showing that overexpression of Gm CAL conferred broad-spectrum resistance to multiple SMV strains.These results confirm that Gm CAL,a key regulator but not a specific SC8 resistance gene(Rsc8),is a positive regulatory transcription factor involved in soybean resistance to SMV.

Characterization of the Promoter of a Homolog of Maize MADS-Box Gene m18

Maize （Zea mays L.） is one of the world’s major food crops, and often suffers from tremendous yield loss caused by abiotic stresses. The MADS-box genes are known to play versatile roles in plants, controlling plant responses to multiple abiotic stresses. However, understanding of regulation of their expressions by the conventional loss-of-function approach is very dififcult. So far, regulation of MADS-box gene expression is little known. The best approach to retrieve expression regulation of this category of genes is to characterize expression of their promoters. In this study, the promoter of a homolog （GenBank accession no. EC864166） of maize MADS-box gene m18 was cloned by way of genome-walking PCR, named Pro66. Predicative analysis indicated that Pro66 contains more than one TATA box and multiple cis-acting environmental conditions-responsive elements （ECREs）. Pro66 could drive expression of theβ-glucuronidase （GUS）-encoding gene in maize, and heterologous expression of GUS in red pepper stressed by water deifcit, salt, copper, iron deifciency, heat, cold, and grown under short and long photoperiods, echoing predicative ECREs. Conclusively, maize MADS-box gene m18 likely plays versatile functions in maize response to multiple abiotic stresses due to the promoter with multiple cis-acting elements. The complex arrangement of multiple cis-acting elements in the promoter features meticulously regulated expression of m18. The results give informative clues for heterologous utilisation of the promoters in monocot and dicot species. The copy of the ECREs and heterologous expression of the promoter in dicot species are also discussed.

The Influence of Photoperiod on the Regulation of Root and Callus Initiation of Perle Noir (<i>V. vinifera</i>L.): Expression of MADS-Box Gene

To study the influence of photoperiod on roots differentiation in the Tunisian grapevine (Vitis vinifera L.) cultivar Perle noir, roots and callus initiation were analyzed under three different conditions of day length: long day (LD), short day (SD) and darkness (D). The photoperiod influenced the number of callus and roots per cuttings;it has a significant effect on the roots and callus initiation. Expression profile analysis of six MADS-box genes (VTM8, VSEP2, VAG12, VAG17-1, VAG17-2 and VSOC1.3) during root and callus development is in agreement with the above-mentioned observation. The expression of the MADS-box genes during root and callus development fluctuated in a tissue-dependent manner. These data suggest that all genes are expressed in roots under three photoperiods. Total darkness gives the number of the most important root per cutting compared to the other two conditions. This photoperiodic condition gave the most important expression of the studied genes VAG12, VAG17-2, VAG17-1, VTM8 and VSEP2 transcripts were not found in callus grown in the dark or in LD conditions, respectively. VSOC1.3 transcripts were not found in callus grown in the dark or in SD conditions, respectively. Transcript abundance of VTM8 and VSOC1 was highest in LD.

板栗MADS-box蛋白基因(CmMADS3)的克隆和表达分析

根据MADS-box基因保守区结构,设计简并引物,从板栗(Castaneamollissima)中分离出花特异表达基因的cDNA片段。并通过5’RACE方法获得了全长cDNA,命名为CmMADS3。该片段全长1016bp,包含一个729bp的开放阅读框,推导的氨基酸序列(243个氨基酸)与拟南芥的SEP1、SEP2和SEP33类MADS-box蛋白有很高的序列相似性。系统进化分析同样将CmMADS3基因归入MADS-box基因家族的AGL2组。RT-PCR分析显示,该基因在板栗的花和幼果中表达丰度高,在茎中有微弱的表达,在叶中不表达,研究结果表明CmMADS3基因是板栗花器官发育中具有E功能的功能基因。

GmNMH7, a MADS-box transcription factor, inhibits root development and nodulation of soybean(Glycine max [L.] Merr.)

As an important food crop and oil crop, soybean(Glycine max [L.] Merr.) is capable of nitrogen-fixing by root nodule. Previous studies showed that GmNMH7, a transcription factor of MADS-box family, is associated with nodule development, but its specific function remained unknown. In this study, we found that GmN MH7 was specifically expressed in root and nodule and the expression pattern of GmNMH7 was similar to several genes involved in early development of nodule(GmENOD40-1, GmENOD40-2, GmNFR1 a, GmNFR5 a, and GmNIN) after rhizobia inoculation. The earlier expression peak of GmNMH7 compared to the other genes(GmENOD40-1, GmENOD40-2, GmNFR1 a, GmNFR5 a, and GmNIN) indicated that the gene is related to the nod factor(NF) signaling pathway and functions at the early development of nodule. Over-expression of GmNMH7 in hairy roots significantly reduced the nodule number and the root length. In the transgenic hairy roots, overexpression of GmN MH7 significantly down-regulated the expression levels of GmE NOD40-1, GmE NOD40-2, and GmN FR5α. Moreover, the expression of GmNMH7 could respond to abscisic acid(ABA) and gibberellin(GA_3) treatment in the root of Zigongdongdou seedlings. Over-expressing GmNMH7 gene reduced the content of ABA, and increased the content of GA_3 in the positive transgenic hairy roots. Therefore, we concluded that GmNMH7 might participate in the NF signaling pathway and negatively regulate nodulation probably through regulating the content of GA_3.

Identification and validation of a major gene for kernel length at the P1 locus in Triticum polonicum

Kernel size, one of the traits that determine wheat yield, is controlled by multiple quantitative trait loci.Polish wheat(Triticum polonicum) has elongated and plump kernel and is a valuable material for breeding high-yielding wheat cultivars. However, genes or loci determining kernel length(KL) in Polish wheat are unknown. We identified and validated a major KL gene, KL-PW, at the P1 locus in Polish wheat. KL-PW is VRT-A2, which encodes a MIKC-type MADS-box protein(MADS55). An insertion/deletion mutation in intron 1 of VRT-A2;led to an alternatively spliced transcript, VRT-A2;. Quantitative PCR analysis showed that VRT-A2;was more highly expressed in developing seeds than was VRT-A2 Ailanmai.Brassinosteroid(BR) sensitivity experiment and the expression of BR-related genes indicated that VRTA2;functions as a positive regulator of BR responses. VRT-A2;significantly increased KL of wheat.These findings not only reveal the molecular basis of KL-PW in controlling KL, but also provide a valuable genetic resource for increasing kernel size in wheat.

Duplication and Divergence of Floral MADS-Box Genes in Grasses： Evidence for the Generation and Modification of Novel Regulators

The process of flowering is controlled by a hierarchy of floral genes that act as flowering time genes, inflorescence/floral meristem Identity genes, and/or floral organ-identity genes. The most important and well-characterized floral genes are those that belong to the MADS-box family of transcription factors. Compelling evidence suggests that floral MADS-box genes have experienced a few large-scale duplication events. In particular, the precore eudicot duplication events have been considered to correlate with the emergence and diversification of core eudicots. Duplication of floral MADS-box genes has also been documented in monocots, particularly In grasses, although a systematic study is lacking. In the present study, by conducting extensive phylogenetlc analyses, we identified pre-Poaceae gene duplication events in each of the AP1, P1, AG, AGL11, AGL2/3/4, and AGL9gene lineages. Comparative genomic studies further indicated that some of these duplications actually resulted from the genome doubling event that occurred 66-70 million years ago （MYA）. In addition, we found that after gene duplication, exonization （of intron sequences） and pseudoexonization （of exon sequences） have contributed to the divergence of duplicate genes in sequence structure and, possibly, gene function.

Gene Duplication and the Evolution of Plant MADS-box Transcription Factors

Since the first MADS-box transcription factor genes were implicated in the establishment of floral organ identity in a couple of model plants, the size and scope of this gene family has begun to be appreciated in a much wider range of species. Over the course of millions of years the number of MADS-box genes in plants has increased to the point that the Arabidopsis genome contains more than 100. The understanding gained from studying the evolution, regulation and function of multiple MADS-box genes in an increasing set of species, makes this large plant transcription factor gene family an ideal subject to study the processes that lead to an increase in gene number and the selective birth, death and repurposing of its component members. Here we will use examples taken from the MADS-box gene family to review what is known about the factors that influence the loss and retention of genes duplicated in different ways and examine the varied fates of the retained genes and their associated biological outcomes.

Genome Sequencing Reveals the Role of MADS-box Gene Families in the Floral Morphology Evolution of Orchids

Orchid origin and evolution are common topics in evolutionary biology. Orchidaceae have approximately 30 000 orchid species distributed in diverse habitats and account for approximately 10% of the flowering plant species worldwide. Orchids provide us with materials to explore coevolution and organic evolution. In this review, we highlighted the genome study progress of orchids. In addition, we revealed the role of MADS-box gene families in the floral morphology and evolution of orchids. Genomics studies confirmed that all five subfamilies of existing orchids evolved from a common ancestor. Loss of Mβ MADS-box genes resulted in the endosperm from the seed of all existing orchids being absent. Perianth reversion to the ancestral state occurred because Apostasia and Apostasioideae lost B-AP3 and E class paralogous genes. Loss of P-subclade members of MIKC*-Type in Phalaenopsis equestris, Dendrobium catenatum, and Epidendroideae caused the formation of pollinium.In addition, the combined loss of AGL12 and contraction of ANR1 gave orchids the ability to be successfully epiphytic on trees or rocks and to develop a unique root system. Both pollinium and epiphytic production on trees are beneficial for orchid adaptations, and Epidendroideae evolved more species(~ 20 000) than Apostasioideae(16 species). Genome studies shed new light on determining the evolutionary history of orchids and understanding the genetic mechanisms of orchid morphological evolution.

Functional Analysis of the ZAG2 Promoter from Maize in Transgenic Tobaccos

The function of the 3 040 bp sequence at the upstream translation starting site （ATG） of the ZAG2 gene, isolated from the maize genome, was studied. The sequence analysis showed that the sequence contained a typical class C MADS-box gene regulatory element. The 5′ UTR region of the gene contains a 1 299-bp intron that might have important regulatory functions. To study the sequence function, deletion derivatives of promoter-reporter （uidA） gene fusions were generated and transformed into tobaccos. The GUS staining and fluorescence quantification results showed that the GUS activity was detected only in the third and fourth whorl floral organs of the transgenic tobaccos under driving the promoter including the first intron, while detected in all the organs and was stronger under driving the promoter without the first intron. However, the GUS activity was just detected in one whorl of the fourth or third floral organs under driving of the 35S promoter. These results suggested that the first intron of the ZAG2 gene contains functional regulatory elements, which turned out to be important for gene expression in the heterologous systems. Moreover, the GUS activity was decreased when the reporter gene driven by the promoters with 5′-deletions, respectively, from -1 606 to -951 and -951 to -426 nts, which indicates that positive regulatory elements are present in these two sequence stretches.

Identification and Expression of Floral Organ Homeotic Genes from Alpinia oblongifolia (Zingiberaceae)

Current understanding of the classical ABC model of floral development has provided a new set of characters to evaluate floral evolution. However, what is still lacking is a clear assessment of this genetic program across monocots. Here, to investigate the evolution of members of class A and B genes in monocots, we report the sequence characteristic and transcript expression of three new MADS-box genes in Alpinia oblongifolia Hayata. Sequence and phylogenetic analysis reveals that these genes are FUL-like and AP3-1ike. Therefore, they were termed AoFL1, AoFL2 and AoAP3. AoFL1 contains the FUL motif, but AoFL2 lacks this motif. Their expression revealed by in situ hybridization may reflect the ancestral function of FUL-like genes in the specification of inflorescence and floral meristems. The AoAP3 gene contains two conserved motifs, the PI-derived and paleoAP3 motifs. The AoAP3 transcripts located to the corolla and stamen, and hybridization signals were detected in the central whorl. These expression patterns suggest that the functions of homologous organ identity genes are diversified in A. oblongifolia. The implications of these findings on the conservation of homologous gene function are discussed.

Angiosperm-wide analysis of fruit and ovary evolution aided by a new nuclear phylogeny supports association of the same ovary type with both dry and fleshy fruits

Fruit functions in seed protection and dispersal and belongs to many dry and fleshy types,yet their evolutionary pattern remains unclear in part due to uncertainties in the phylogenetic relationships among several orders and families.Thus we used nuclear genes of 502 angiosperm species representing 231 families to reconstruct a well supported phylogeny,with resolved relationships for orders and families with previously uncertain placements.Using this phylogeny as a framework,molecular dating supports a Triassic origin of the crown angiosperms,followed by the emergence of most orders in the Jurassic and Cretaceous and their rise to ecological dominance during the Cretaceous Terrestrial Revolution.The robust phylogeny allowed an examination of the evolutionary pattern of fruit and ovary types,revealing a trend of parallel carpel fusions during early diversifications in eudicots,monocots,and magnoliids.Moreover,taxa in the same order or family with the same ovary type can develop either dry or fleshy fruits with strong correlations between specific types of dry and fleshy fruits;such associations of ovary,dry and fleshy fruits define several ovaryfruit"modules"each found in multiple families.One of the frequent modules has an ovary containing multiple ovules,capsules and berries,and another with an ovary having one or two ovules,achenes(or other single-seeded dry fruits)and drupes.This new perspective of relationships among fruit types highlights the closeness of specific dry and fleshy fruit types,such as capsule and berry,that develop from the same ovary type and belong to the same module relative to dry and fleshy fruits of other modules(such as achenes and drupes).Further analyses of gene families containing known genes for ovary and fruit development identified phylogenetic nodes with multiple gene duplications,supporting a possible role of whole-genome duplications,in combination with climate changes and animal behaviors,in angiosperm fruit and ovary diversification.

Nuclear phylogenomics of Asteraceae with increased sampling provides new insights into convergent morphological and molecular evolution

Convergent morphological evolution is widespread in flowering plants,and understanding this phenomenon relies on well-resolved phylogenies.Nuclear phylogenetic reconstruction using transcriptome datasets has been successful in various angiosperm groups,but it is limited to taxa with available fresh materials.Asteraceae,which are one of the two largest angiosperm families and are important for both ecosystems and human livelihood,show multiple examples of convergent evolution.Nuclear Asteraceae phylogenies have resolved relationships among most subfamilies and many tribes,but many phylogenetic and evolutionary questions regarding subtribes and genera remain,owing to limited sampling.Here,we increased the sampling for Asteraceae phylogenetic reconstruction using transcriptomes and genome-skimming datasets and produced nuclear phylogenetic trees with 706 species representing two-thirds of recognized subtribes.Ancestral character reconstruction supports multiple convergent evolutionary events in Asteraceae,with gains and losses of bilateral floral symmetry correlated with diversification of some subfamilies and smaller groups,respectively.Presence of the calyx-related pappus may have been especially important for the success of some subtribes and genera.Molecular evolutionary analyses support the likely contribution of duplications of MADS-box and TCP floral regulatory genes to innovations in floral morphology,including capitulum inflorescences and bilaterally symmetric flowers,potentially promoting the diversification of Asteraceae.Subsequent divergences and reductions in CYC2 gene expression are related to the gain and loss of zygomorphic flowers.This phylogenomic work with greater taxon sampling through inclusion of genome-skimming datasets reveals the feasibility of expanded evolutionary analyses using DNA samples for understanding convergent evolution.

XAANTAL2 （AGL14） Is an Important Component of the Complex Gene Regulatory Network that Underlies Arabidopsis Shoot Apical Meristem Transitions

In Arabidopsis thaliana, multiple genes involved in shoot apical meristem （SAM） transitions have been char- acterized, but the mechanisms required for the dynamic attainment of vegetative, inflorescence, and floral meristem （VM, IM, FM） cell fates during SAM transitions are not well understood. Here we show that a MADS-box gene, XAANTAL2 （XAL2/AGL14）, is necessary and sufficient to induce flowering, and its regula- tion is important in FM maintenance and determinacy, xal2 mutants are late flowering, particularly under short-day （SD） condition, while XAL2 overexpressing plants are early flowering, but their flowers have vege- tative traits. Interestingly, inflorescences of the latter plants have higher expression levels of LFY, AP1, and TFL1 than wild-type plants. In addition we found that XAL2 is able to bind the TFL1 regulatory regions. On the other hand, the basipetal carpels of the 35S：：XAL2 lines lose determinacy and maintain high levels of WUS expression under SD condition. To provide a mechanistic explanation for the complex roles of XAL2 in SAM transitions and the apparently paradoxical phenotypes of XAL2 and other MADS-box （SOCl, AGL24） over- expressors, we conducted dynamic gene regulatory network （GRN） and epigenetic landscape modeling. We uncovered a GRN module that underlies VM, IM, and FM gene configurations and transition patterns in wild- type plants as well as loss and gain of function lines characterized here and previously. Our approach thus provides a novel mechanistic framework for understanding the complex basis of SAM development.

Isolation and Characterization of a Putative Class E Gene from Taihangia rupestris

Studies In model plants showed that SEPALLATA （SEP） genes are required for the Identification of floral organs and the determination of floral meristems In Arabidopsis. In this paper a SEP homolog, TrSEP3, was Isolated from a China-specific species, Taihangla rupestrisi Yü et LI. Phylogenetlc analysis showed that the gene belongs to the SEP3-clade of SEP （previous AGL2） subfamily. In situ hybridization was used to reveal the potential functional specification, and the results showed that TrSEP3 expression was first observed in floral meristems and then confined to the floral primordla of the three inner whorls. In the matured flower, TrSEP3 was strongly expressed In the tips of pistils and weak In stamens and petals. The evolution force analysis shows that TrSEP3 might undergo a relaxed negative selection. These results suggested that TrSEP3 may not only function In determining the identity of floral merlstems and the primordia of three inner whorls, but also function In matured reproductive organs.

Ectopic expression of a hyacinth AGL6 homolog caused earlier flowering and homeotic conversion in Arabidopsis

MADS-box genes are involved in floral organ development.Here we report thatan AGL6(Agamous-like 6)-like MADS-box gene,H0AGL6,was isolated from Hyacinthus orientalisL.Expression pattern analysis demonstrated that H0AGL6 transcript was detected in inflorescencebuds,tepals,carpels and ovules,but not in stamina,leaves or scales.Transgenic Arabidopsis plantsectopically expressing H0AGL6 exhibited novel phenotypes of significantly reduced plantsize,extremely early flowering,and losing inflorescence indeterminacy.In addition,wide homeoticconversion of sepals,petals,and leaves into carpel-like or ovary structures,and disappearance ornumber reduction of stamens in 35S::HoAGL6 Arabidopsis plants were also observed.RT-PCR analysisindicated that the expressions of flowering time gene SOC1 and flower meristem identity gene LFYwere significantly up-regulated in 35S::Ho4GL6transgenic Arabidopsis plants,and the expressionlevels of floral organ identity genes AG and SEP1 in leaves were also elevated.These resultsindicated that H0AGL6 was involved in the regulation of flower transition and flower organformation.

Temporal and Spatial Requirement of EMF1 Activity for Arabidopsis Vegetative and Reproductive Development

EMBRYONIC FLOWER （EMF） genes are required to maintain vegetative development via repression of flower homeotic genes in Arabidopsis. Removal of EMF gene function caused plants to flower upon germination, producing abnormal and sterile flowers. The pleiotropic effect of ernfl mutation suggests its requirement for gene programs involved in diverse developmental processes. Transgenic plants harboring EMF1 promoter：：glucuronidase （GUS） reporter gene were generated to investigate the temporal and spatial expression pattern of EMF1. These plants displayed differential GUS activity in vegetative and flower tissues, consistent with the role of EMF1 in regulating multiple gene programs. EMFI：：GUS expression pattern in emf mutants suggests organ-specific auto-regulation. Sense- and antisense （as） EMF1 cDNA were expressed under the control of stage- and tissue-specific promoters in transgenic plants. Characterization of these transgenic plants showed that EMF1 activity is required in meristematic as well as differentiating tissues to rescue emf mutant phenotype. Temporal removal or reduction of EMF1 activity in the embryo or shoot apex of wild-type seedlings was sufficient to cause early flowering and terminal flower formation in adult plants. Such reproductive cell memory is reflected in the flower MADS-box gene activity expressed prior to flowering in these early flowering plants. However, temporal removal of EMF1 activity in flower meristem did not affect flower development. Our results are consistent with EMF1＇s primary role in repressing flowering in order to allow for vegetative growth.

Cloning and characterization of PhPI9 involved in floral development from Phalaenopsis Orchid

In the attempt to discover new genes involved in the floral development in monocotyledonousin species,we have cloned and characterized the homologous PISTALLATA-like(PI-like)gene from Phalaenopsis hybrid cultivar named PhPI9(Phalaenopsis PI STILLATA#9).The cDNA of PhPI9 has a fragment of 834 bp and has 60%identity with the PISTILATA from Arabidopsis.The deduced amino acid sequence of PhPI9 had the typical PI-motif.It also formed a subclade with other monocot PI-type genes in phylogenetic analysis.Southern analysis showed that PhPI9 was present in the Phalaenopsis orchid genome as a single copy.Furthermore,it was expressed only in the lip of the Phalaenopsis flower and no expression was detected in vegetative organs.Thus,as a B-function MADS-box gene,PhPI9 specifies floral organ identity in orchids.