The expression and phylogenetic analysis of four AP3-like paralogs in the stamens, carpels, and single-whorl perianth of the paleoherb Asarum caudigerum.

The paleoherb species Asarum caudigerum (Aristolochiaceae) is important for research into the origin and evolution of angiosperm flowers due to its basal position in the angiosperm phylogeny. In this study, four MADS-box-containing transcripts were isolated from A. caudigerum by rapid amplification of cDNA ends (RACE). Sequence comparisons and phylogenetic analyses indicated that they possess high homology to AP3 subfamily genes, which have been shown previously to be involved in petal and stamen development in eudicots. Reverse-transcription quantitative PCR (RT-qPCR) and in situ hybridization analyses showed AcAP3-A expression mainly in the second whorl (stamens) and AcAP3-B expression in whorls 1 and 3 (perianth and carpels). Compared with eudicot AP3 homologs, premature translation termination codons were caused by an insertion in the K1 domain of AcAP3-C, and by a deletion in the 7th exon of AcAP3-D. Sequence analyses suggested that the A. caudigerum AP3 lineage had undergone gene duplication and subfunctionalization, diverging in expression patterns during perianth, stamen, and carpel development. Based on comparative genomic and phylogenetic analyses, we concluded that subfunctionalization has likely contributed to the persistence of two functional AP3 paralogs, that two other copies may have become pseudogenes, and that these AP3 duplication and subfunctionalization events may have contributed to the evolution of the unusual floral morphology of A. caudigerum.

Developmental genetics of the perianthless flowers and bracts of a paleoherb species, Saururus chinensis.

Saururus chinensis is a core member of Saururaceae, a perianthless (lacking petals or sepals) family. Due to its basal phylogenetic position and unusual floral composition, study of this plant family is important for understanding the origin and evolution of perianthless flowers and petaloid bracts among angiosperm species. To isolate genes involved in S. chinensis flower development, subtracted floral cDNA libraries were constructed by using suppression subtractive hybridization (SSH) on transcripts isolated from developing inflorescences and seedling leaves. The subtracted cDNA libraries contained a total of 1,141 ESTs and were used to create cDNA microarrays to analyze transcript profiles of developing inflorescence tissues. Subsequently, qRT-PCR analyses of eight MADS-box transcription factors and in situ hybridizations of two B-class MADS-box transcription factors were performed to verify and extend the cDNA microarray results. Finally, putative phylogenetic relationships within the B-class MADS-box gene family were determined using the discovered S. chinensis B-class genes to compare K-domain sequences with B genes from other basal angiosperms. Two hundred seventy-seven of the 1,141 genes were found to be expressed differentially between S. chinensis inflorescence tissues and seedling leaves, 176 of which were grouped into at least one functional category, including transcription (14.75%), energy (12.59%), metabolism (9.12%), protein-related function (8.99%), and cellular transport (5.76%). qRT-PCR and in situ hybridization of selected MADS-box genes supported our microarray data. Phylogenetic analysis indicated that a total of six B-class MADS-box genes were isolated from S. chinensis. The differential regulation of S. chinensis B-class MADS-box transcription factors likely plays a role during the development of subtending bracts and perianthless flowers. This study contributes to our understanding of inflorescence development in Saururus, and represents an initial step toward understanding the formation of petaloid bracts in this species.

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA.

BACKGROUND: Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level. CONCLUSIONS: Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.

De novo sequencing and characterization of the floral transcriptome of Dendrocalamus latiflorus (Poaceae: Bambusoideae).

BACKGROUND: Transcriptome sequencing can be used to determine gene sequences and transcript abundance in non-model species, and the advent of next-generation sequencing (NGS) technologies has greatly decreased the cost and time required for this process. Transcriptome data are especially desirable in bamboo species, as certain members constitute an economically and culturally important group of mostly semelparous plants with remarkable flowering features, yet little bamboo genomic research has been performed. Here we present, for the first time, extensive sequence and transcript abundance data for the floral transcriptome of a key bamboo species, Dendrocalamus latiflorus, obtained using the Illumina GAII sequencing platform. Our further goal was to identify patterns of gene expression during bamboo flower development. RESULTS: Approximately 96 million sequencing reads were generated and assembled de novo, yielding 146,395 high quality unigenes with an average length of 461 bp. Of these, 80,418 were identified as putative homologs of annotated sequences in the public protein databases, of which 290 were associated with the floral transition and 47 were related to flower development. Digital abundance analysis identified 26,529 transcripts differentially enriched between two developmental stages, young flower buds and older developing flowers. Unigenes found at each stage were categorized according to their putative functional categories. These sequence and putative function data comprise a resource for future investigation of the floral transition and flower development in bamboo species. CONCLUSIONS: Our results present the first broad survey of a bamboo floral transcriptome. Although it will be necessary to validate the functions carried out by these genes, these results represent a starting point for future functional research on D. latiflorus and related species.

Colored nectar as an honest signal in plant-animal interactions.

Many flowering plants obtain the services of pollinators by using their floral traits as signals to advertise the rewards they offer to visitors-such as nectar, pollen and other food resources. Some plants use colorful pigments to draw pollinators' attention to their nectar, instead of relying on the appeal of nectar taste. Although this rare floral trait of colored nectar was first recorded by the Greek poet Homer in the Odyssey, it has only recently received the attention of modern science. This mini-review focuses on recent findings about some of the species that use colored nectar; topics include its function as an honest signal for pollinators, as well as the pigments responsible for the nectar coloration. Such research of the ecology and physiology of colored nectar expands our understanding of the role and evolution of pollinator signaling in plants.

Dark purple nectar as a foraging signal in a bird-pollinated Himalayan plant.

• Some plants secrete coloured nectar to attract pollinators, but little is known about the chemical origins of nectar colouration and its ecological function. Leucosceptrum canum stands out as the only plant with coloured nectar recorded in the Himalayas. Here, we focused on the compound associated with the dark colour of the nectar, as well as its secretion dynamics during the flowering season and its relationship to pollinators. • Fresh nectar was analysed by semi-preparative reversed-phase high-performance liquid chromatography (HPLC), LC-MS and HRESIMS (high resolution electronspray ionization mass spectroscopy) to determine which compound causes the nectar colouration. Behavioural experiments were conducted with birds and honeybees to elucidate the effect of the nectar colour and volume on pollinators. • We identified a purple anthocyanidin, 5-hydroxyflavylium, as a natural nectar product for the first time. Two short-billed birds were found to pollinate this plant, which employs two nectar-based mechanisms to direct bird pollinators to reproductively active flowers, controlling nectar palatability and presenting a foraging signal for birds by altering nectar volume and colour in a developmental stage-specific manner. • 5-Hydroxyflavylium was found to be the cause of the nectar colouration, the function of which is to act as a foraging signal to increase pollination efficiency through nectar visibility and palatability.

FZR2/CCS52A1 mediated endoreduplication in Arabidopsis development.

Plant organogenesis generally involves three basic processes: cell division, cell expansion and cell differentiation. Endoreduplication, a process of genome replication without intervening mitosis, often occurs during cell expansion and cell differentiation. The switch from the mitotic cell cycle to the endocycle, however, is still poorly understood in plants. We have recently demonstrated that FIZZY-RELATED2 (FZR2) is a factor controlling endoreduplication in Arabidopsis. fzr2 mutants lacked gross morphological defects but showed a general decrease of endoploidy level in trichomes and other leaf cells, while expression of FZR2 under constitutive or tissue specific promoters induced extra or ectopic endoreduplication in all tissues examined. We also showed that decrease of leaf cell size in fzr2 mutants could be compensated by increased cell proliferation. In this addendum, we discuss additional phenotypes of FZR2 misexpression, including apparent mosaic leaf sectors in which local cell overexpansion due to 35S::FZR2 appears to be compensated by reduced cell expansion in neighboring tissues.

FZR2/CCS52A1 expression is a determinant of endoreduplication and cell expansion in Arabidopsis.

Endoreduplication, a modified cell cycle that allows cells to increase ploidy without subsequent cell division, is a key component of plant growth and development. In this work, we show that some, but not all, of the endoreduplication of Arabidopsis (Arabidopsis thaliana) is mediated by the expression of a WD40 gene, FIZZY-RELATED2 (FZR2). Loss-of-function alleles show reduced endoreduplication and reduced expansion in trichomes and other leaf cells. Misexpression of FZR2 is sufficient to drive ectopic or extra endoreduplication in leaves, roots, and flowers, leading to alteration of cell sizes and, sometimes, organ size and shape. Our data, which suggest that reduced cell size can be compensated by increased cell proliferation to allow normal leaf morphology, are discussed with respect to the so-called compensation mechanism of plant development.