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International audience; Dianthus chinensis is widely used as a landscaping plant in rock gardens and has high commercial value. However, few studies of this plant's regeneration and genetic transformation have been reported. In this study, cotyledons of D. chinensis were used as explants for regeneration and infection with Agrobacterium. The results indicated that the efficiency of regeneration was markedly improved by using 1-naphthylcetic acid (NAA). The cotyledons were cocultured with Agrobacterium tumefaciens EHA105 containing pK7GWIWG2-GFP plasmids for 15-20 min. Transgenic regenerated plants were obtained by kanamycin (80 mg.L-1) screening. qRT-PCR results confirmed the expression of GFP and the DcIPT1 (which is related to cell division) gene in the transgenic DcIPT1 plants of D. chinensis. The results show that the proliferation ability of transgenic DcIPT1 plants is enhanced. Overall, we report the successful establishment of a reliable and efficient method for cotyledon transformation and plant regeneration in D. chinensis based on the combination of infection with Agrobacterium and plant regeneration. This work will contribute to functional gene research and genetic improvement of Dianthus species plants.

An SNP mutation in the miR172 target site of the DcAP2L gene is associated with a double flower phenotype in Dianthus chinensis., The double flower is a highly important breeding trait that affects the ornamental value in many flowering plants. To get a better understanding of the genetic mechanism of double flower formation in Dianthus chinensis, we have constructed a high-density genetic map using 140 F2 progenies derived from a cross between a single flower genotype and a double flower genotype. The linkage map was constructed using double-digest restriction site-associated DNA sequencing (ddRAD-seq) with 2353 single nucleotide polymorphisms (SNPs). Quantitative trait locus (QTL) mapping analysis was conducted for 12 horticultural traits, and major QTLs were identified for nine of the 12 traits. Among them, two major QTLs accounted for 20.7% and 78.1% of the total petal number variation, respectively. Bulked segregant RNA-seq (BSR-seq) was performed to search accurately for candidate genes associated with the double flower trait. Integrative analysis of QTL mapping and BSR-seq analysis using the reference genome of Dianthus caryophyllus suggested that an SNP mutation in the miR172 cleavage site of the A-class flower organ identity gene APETALA2 (DcAP2L) is responsible for double flower formation in Dianthus through regulating the expression of DcAG genes.

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International audience; Floral scent is one of the most important characters in horticultural plants. Roses (Rosa spp.) have been cultivated for their scent since antiquity. However, probably by selecting for cultivars with long vase life, breeders have lost the fragrant character in many modern roses, especially the ones bred for the cut flower market. The genetic inheritance of scent characters has remained elusive so far. In-depth knowledge of this quantitative trait is thus very much needed to breed more fragrant commercial cultivars. Furthermore, rose hybrids harbor a composite genomic structure, which complexifies quantitative trait studies. To understand rose scent inheritance, we characterized a segregating population from two diploid cultivars, Rosa × hybrida cv H190 and Rosa wichurana, which have contrasting scent profiles. Several quantitative trait loci for the major volatile compounds in this progeny were identified. One among these loci contributing to the production of 2-phenylethanol, responsible for the characteristic odor of rose, was found to be colocalized with a candidate gene belonging to the 2-phenylethanol biosynthesis pathway: the PHENYLACETALDEHYDE SYNTHASE gene RhPAAS. An in-depth allele-specific expression analysis in the progeny demonstrated that only one allele was highly expressed and was responsible for the production of 2-phenylethanol. Unexpectedly, its expression was found to start early during flower development, before the production of the volatile 2-phenylethanol, leading to the accumulation of glycosylated compounds in petals.; Analyse des composés du parfum dans une population en ségrégation qui montre que la synthèse du2-phenylethanol est liée à l'expression d'un allèle deu gène de laphenylacetaldehyde synthase dans les pétales

International audience; Dianthus is a large genus containing many species with high ornamental economic value. Extensive breeding strategies permitted an exploration of an improvement in the quality of cultivated carnation, particularly in flowers. However, little is known on the molecular mechanisms of flower development in carnation. Here, we report the identification and description of MADS-box genes in carnation (DcaMADS) with a focus on those involved in flower development and organ identity determination. In this study, 39 MADS-box genes were identified from the carnation genome and transcriptome by the phylogenetic analysis. These genes were categorized into four subgroups (30 MIKCc, two MIKC*, two M alpha, and five M gamma). The MADS-box domain, gene structure, and conserved motif compositions of the carnation MADS genes were analysed. Meanwhile, the expression of DcaMADS genes were significantly different in stems, leaves, and flower buds. Further studies were carried out for exploring the expression of DcaMADS genes in individual flower organs, and some crucial DcaMADS genes correlated with their putative function were validated. Finally, a new expression pattern of DcaMADS genes in flower organs of carnation was provided: sepal (three class E genes and two class A genes), petal (two class B genes, two class E genes, and one SHORT VEGETATIVE PHASE (SVP)), stamen (two class B genes, two class E genes, and two class C), styles (two class E genes and two class C), and ovary (two class E genes, two class C, one AGAMOUS-LIKE 6 (AGL6), one SEEDSTICK (STK), one B sister, one SVP, and one Ma). This result proposes a model in floral organ identity of carnation and it may be helpful to further explore the molecular mechanism of flower organ identity in carnation.

International audience; Cultivated roses have a very ancient history. Artificial crossing led to what is today perceived as modern rose cultivars. Impressively, these modern rose cultivars were established from less than 10 species, which have contributed to the origin of the about 35,000 existing rose cultivars. Roses exhibit an extraordinary diversity of traits, both of economic and scientific importance. Several recent studies have been marked as important milestones on the journey towards deeply understanding the molecular and genetic mechanisms that govern these traits, yet we still lack information on the genome sequences of rose species and cultivars, especially those that heavily participated to rose domestication and breeding programs.

Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions.

The Translationally Controlled Tumor Protein (TCTP) is a conserved protein which expression was associated with several biochemical and cellular functions. Loss-of-function mutants are lethal both in animals and in plants, making the identification of its exact role difficult. Recent data using the model plant Arabidopsis thaliana provided the first viable adult knockout for TCTP and helped addressing the biological role of TCTP during organ development and the functional conservation between plants and animals. This chapter summarizes our up to date knowledge about the role of TCTP in plants and discuss about conserved functions and mechanisms between plants and animals.

Aquaporins (AQPs) are essential channel proteins that execute multi-functions throughout plant growth and development, including water transport, uncharged solutes uptake, stress response, and so on. Here, we report the first genome-wide identification and characterization AQP (BvAQP) genes in sugar beet (Beta vulgaris), an important crop widely cultivated for feed, for sugar production and for bioethanol production. Twenty-eight sugar beet AQPs (BvAQPs) were identified and assigned into five subfamilies based on phylogenetic analyses: seven of plasma membrane (PIPs), eight of tonoplast (TIPs), nine of NOD26-like (NIPs), three of small basic (SIPs), and one of x-intrinsic proteins (XIPs). BvAQP genes unevenly mapped on all chromosomes, except on chromosome 4. Gene structure and motifs analyses revealed that BvAQP have conserved exon-intron organization and that they exhibit conserved motifs within each subfamily. Prediction of BvAQPs functions, based on key protein domains conservation, showed a remarkable difference in substrate specificity among the five subfamilies. Analyses of BvAQPs expression, by mean of RNA-seq, in different plant organs and in response to various abiotic stresses revealed that they were ubiquitously expressed and that their expression was induced by heat and salt stresses. These results provide a reference base to address further the function of sugar beet aquaporins and to explore future applications for plants growth and development improvements as well as in response to environmental stresses.

International audience; Roses hold high symbolic value and great cultural importance in different societies throughout human history. They are widely used as garden ornamental plants, as cut flowers, and for the production of essential oils for the perfume and cosmetic industries. Domestication of roses has a long and complex history, and the rose species have been hybridized across vast geographic areas such as Europe, Asia, and the Middle East. The domestication processes selected several flower characters affecting floral quality, such as recurrent flowering, double flowers, petal colours, and fragrance. The molecular and genetic events that determine some of these flower characters cannot be studied using model species such as Arabidopsis thaliana, or at least only in a limited manner. In this review, we comment on the recent development of genetic, genomic, and transcriptomic tools for roses, and then focus on recent advances that have helped unravel the molecular mechanisms underlying several rose floral traits.

International audience; Phyllody is a flower abnormality in which leaf-like structures replace flower organs in all whorls. Here, we investigated the origin and the molecular mechanism of phyllody phenotype in Rosa chinensis cv. Viridiflora, an ancient naturally occurring Chinese mutant cultivar. Reciprocal grafting experiments and microscopy analyses, demonstrated that the phyllody phenotype in Viridiflora is not associated with phytoplasmas infection. Transcriptome comparisons by the mean of RNA-Seq identified 672 up regulated and 666 down-regulated genes in Viridiflora compared to its closely related genotype R. chinensis cv. Old Blush. A fraction of these genes are putative homologs of genes known to be involved in flower initiation and development. We show that in flower whorl 2 of Viridiflora, a down-regulation of the floral organ identity genes RcPISTILLATA (RcPI), RcAPETALA3 (RcAP3) and RcSEPALLATA3 (RcSEP3), together with an up -regulation of the putative homolog of the gene SUPPRESSOR of OVEREXPRESSION of CONSTANS1 (RcSOC1) are likely at the origin of the loss of petal identity and leaf -like structures formation. In whorl 3 of Viridiflora, ectopic expression of RcAPETALA2 (RcAP2) along with the down regulation of RcPI, RcAP3, and RcSEP3 is associated with loss of stamens identity and leaf -like structures formation. In whorl 4, the ectopic expression of RcAP2 associated with a down -regulation of RcSEP3 and of the C-class gene RcAGAMOUS correlate with loss of pistil identity. The latter also suggested the antagonist effect between the A and C class genes in the rose. Together, these data suggest that modified expression of the ABCE flower organ identity genes is associated with the phyllody phenotype in the rose Viridiflora and that these genes are important for normal flower organs development.

*† ‡ SUMMARY In Arabidopsis, four homeotic gene classes, A, B, C and E, are required for the patterning of floral organs. However, very little is known about how the activity of these master genes is translated into regulatory processes leading to specific growth patterns and the formation of organs with specific shapes and sizes. Previously we showed that the transcript variant BPEp encodes a bHLH transcription factor that is involved in limiting petal size by controlling post-mitotic cell expansion. Here we show that the phytohormone jasmonate is required for control of BPEp expression. Expression of BPEp was negatively regulated in opr3 mutant flowers that are deficient in jasmonate synthesis. Moreover, the expression of BPEp was restored in opr3 flowers following exogenous jasmonate treatments. Expression of the second transcript variant BPEub, which originates from the same gene as BPEp via an alternative splicing event, was not affected, indicating that BPEp accumulation triggered by jasmonate occurs at the post-transcriptional level. Consistent with these data, opr3 exhibited an increase in petal size as a result of increased cell size, as well as a modified vein pattern, phenotypes that are similar to those of thebpe-1 mutant. Furthermore, exogenous treatments with jasmonate rescued petal phenotypes associated with loss of function of OPR3. Our data demonstrate that jasmonate signaling downstream of OPR3 is involved in the control of cell expansion and in limiting petal size, and that BPEp is a downstream target that functions as a component mediating jasmonate signaling during petal growth.

Stop to smell the roses Some roses smell beautiful, yet others only look beautiful. Magnard et al. leveraged this distinction to study the biosynthesis of geraniol, a monoterpene alcohol in rose scent (see the Perspective by Tholl and Gershenzon). Enzymes known for geraniol synthesis in other plants, such as basil, did not seem to provide that function for roses. Instead, a diphosphohydrolase, which functions in the cytoplasm of cells in rose petals, generates the geraniol emitted by fragrant roses. Identification of the enzyme and its gene enables marker-assisted breeding to put the perfume back into beauty. Science , this issue p. 81 ; see also p. 28

Background For centuries roses have been selected based on a number of traits. Little information exists on the genetic and molecular basis that contributes to these traits, mainly because information on expressed genes for this economically important ornamental plant is scarce. Results Here, we used a combination of Illumina and 454 sequencing technologies to generate information on Rosa sp. transcripts using RNA from various tissues and in response to biotic and abiotic stresses. A total of 80714 transcript clusters were identified and 76611 peptides have been predicted among which 20997 have been clustered into 13900 protein families. BLASTp hits in closely related Rosaceae species revealed that about half of the predicted peptides in the strawberry and peach genomes have orthologs in Rosa dataset. Digital expression was obtained using RNA samples from organs at different development stages and under different stress conditions. qPCR validated the digital expression data for a selection of 23 genes with high or low expression levels. Comparative gene expression analyses between the different tissues and organs allowed the identification of clusters that are highly enriched in given tissues or under particular conditions, demonstrating the usefulness of the digital gene expression analysis. A web interface ROSAseq was created that allows data interrogation by BLAST, subsequent analysis of DNA clusters and access to thorough transcript annotation including best BLAST matches on Fragaria vesca, Prunus persica and Arabidopsis. The rose peptides dataset was used to create the ROSAcyc resource pathway database that allows access to the putative genes and enzymatic pathways. Conclusions The study provides useful information on Rosa expressed genes, with thorough annotation and an overview of expression patterns for transcripts with good accuracy.

International audience; Cultivated for centuries, the varieties of rose have been selected based on a number of flower traits. Understanding the genetic and molecular basis that contributes to these traits will impact on future improvements for this economically important ornamental plant. In this study, we used scanning electron microscopy and sections of meristems and flowers to establish a precise morphological calendar from early rose flower development stages to senescing flowers. Global gene expression was investigated from floral meristem initiation up to flower senescence in three rose genotypes exhibiting contrasted floral traits including continuous versus once flowering and simple versus double flower architecture, using a newly developed Affymetrix microarray (Rosa1_Affyarray) tool containing sequences representing 4765 unigenes expressed during flower development. Data analyses permitted the identification of genes associated with floral transition, floral organs initiation up to flower senescence. Quantitative real time PCR analyses validated the mRNA accumulation changes observed in microarray hybridizations for a selection of 24 genes expressed at either high or low levels. Our data describe the early flower development stages in Rosa sp, the production of a rose microarray and demonstrate its usefulness and reliability to study gene expression during extensive development phases, from the vegetative meristem to the senescent flower.

Plant organ growth and final size are determined by coordinated cell proliferation and expansion. The BIGPETALp (BPEp) basic helix-loop-helix (bHLH) transcription factor was shown to limit Arabidopsis thaliana petal growth by influencing cell expansion. We demonstrate here that BPEp interacts with AUXIN RESPONSE FACTOR8 (ARF8) to affect petal growth. This interaction is mediated through the BPEp C-terminal domain (SDBPEp) and the C-terminal domain of ARF8. Site-directed mutagenesis identified an amino acid consensus motif in SDBPEp that is critical for mediating BPEp-ARF8 interaction. This motif shares sequence similarity with motif III of ARF and AUXIN/INDOLE-3-ACETIC ACID proteins. Petals of arf8 mutants are significantly larger than those of the wild type due to increased cell number and increased cell expansion. bpe arf8 double mutant analyses show that during early petal development stages, ARF8 and BPEp work synergistically to limit mitotic growth. During late stages, ARF8 and BPEp interact to limit cell expansion. The alterations in cell division and cell expansion observed in arf8 and/or bpe mutants are associated with a change in expression of early auxin-responsive genes. The data provide evidence of an interaction between an ARF and a bHLH transcription factor and of its biological significance in regulating petal growth, with local auxin levels likely influencing such a biological function.

Background Roses have been cultivated for centuries and a number of varieties have been selected based on flower traits such as petal form, color, and number. Wild-type roses have five petals (simple flowers), whereas high numbers of petals (double flowers) are typical attributes of most of the cultivated roses. Here, we investigated the molecular mechanisms that could have been selected to control petal number in roses. Methodology/Principal Findings We have analyzed the expression of several candidate genes known to be involved in floral organ identity determination in roses from similar genetic backgrounds but exhibiting contrasting petal numbers per flower. We show that the rose ortholog of AGAMOUS (RhAG) is differentially expressed in double flowers as compared to simple flowers. In situ hybridization experiments confirm the differential expression of RhAG and demonstrate that in the double-flower roses, the expression domain of RhAG is restricted toward the center of the flower. Conversely, in simple-flower roses, RhAG expression domain is wider. We further show that the border of RhAG expression domain is labile, which allows the selection of rose flowers with increased petal number. Double-flower roses were selected independently in the two major regions for domestication, China and the peri-Mediterranean areas. Comparison of RhAG expression in the wild-type ancestors of cultivated roses and their descendants both in the European and Chinese lineages corroborates the correlation between the degree of restriction of RhAG expression domain and the number of petals. Our data suggests that a restriction of RhAG expression domain is the basis for selection of double flowers in both the Chinese and peri-Mediterranean centers of domestication. Conclusions/Significance We demonstrate that a shift in RhAG expression domain boundary occurred in rose hybrids, causing double-flower phenotype. This molecular event was selected independently during rose domestication in Europe/Middle East and in China.

The growth of an organism and its size determination require the tight regulation of cell proliferation and cell growth. However, the mechanisms and regulatory networks that control and integrate these processes remain poorly understood. Here, we address the biological role of Arabidopsis translationally controlled tumor protein (AtTCTP) and test its shared functions in animals and plants. The data support a role of plant AtTCTP as a positive regulator of mitotic growth by specifically controlling the duration of the cell cycle. We show that, in contrast to animal TCTP , plant AtTCTP is not implicated in regulating postmitotic growth. Consistent with this finding, plant AtTCTP can fully rescue cell proliferation defects in Drosophila loss of function for dTCTP. Furthermore, Drosophila dTCTP is able to fully rescue cell proliferation defects in Arabidopsis tctp knockouts. Our data provide evidence that TCTP function in regulating cell division is part of a conserved growth regulatory pathway shared between plants and animals. The study also suggests that, although the cell division machinery is shared in all multicellular organisms to control growth, cell expansion can be uncoupled from cell division in plants but not in animals.

International audience; Somatic embryogenesis was induced from in vitro-derived leaf explants of Rosa chinensis cultivar (cv) Old Blush. Calli producing embryos with expanded cotyledons (RcOBType1 embryos) were obtained. Further refinements of the callus maintenance medium generated a more typical rose embryogenic callus (RcOBType2) displaying high levels of secondary embryogenesis and embryos with limited cotyledon expansion Agrobacterium tumefaciens-mediated transformation assays using β-glucuronidase (GUS) reporter gene showed that both types of embryos were competent for transformation. Under selection conditions, transformed RcOBType1 explants produced non chimaeric transformed embryos, from which shoots could be adventitiously regenerated. In contrast to RcOBType1, transformed RcOBType2 embryos directly yielded transformed shoots when repeatedly cultured in selective regeneration conditions. Transformation efficiency ranged between three to nine percent and shoots suitable for rooting were obtained within 6-8 months. Transgenic plants were transferred into the greenhouse and molecularly confirmed. The availability of transformation methods in a diploid rose, R. chinensis cv. Old Blush, will be useful for gene functional studies.

In Arabidopsis, APETALA1, PISTILLATA, APETALA3 and SEPALLATA interact to form multimeric protein complexes required to specify petal identity. However, the downstream events that lead to petal specific shape and size remain largely unknown. Organ final size can be influenced by cell number or cell expansion or both. To date, no gene that specifically limits petal size by controlling postmitotic cell expansion has been identified. Here we have identified a novel petal-expressed, basic helix-loop-helix encoding gene (BIGPETAL, BPE) that is involved in the control of petal size. BPE is expressed via two mRNAs derived from an alternative splicing event. The BPEub transcript is expressed ubiquitously, whereas the BPEp transcript is preferentially expressed in petals. We demonstrate that BPEp is positively regulated downstream of APETALA3, PISTILLATA, APETALA1 and PISTILLATA3 and is negatively regulated downstream of AGAMOUS. Plants that lack the petal-expressed variant BPEp have larger petals as a result of increased cell size, showing that BPEp interferes with postmitotic cell expansion. BPEp is therefore a part of the network that links the patterning genes to final morphogenesis.

International audience; Single-pass sequences were obtained from the 5P P-ends of a total of 1794 rose petal cDNA clones. Cluster analysis identified 242 groups of sequences and 635 singletons indicating that the database represents a total of 877 genes. Putative functions could be assigned to 1151 of the transcripts. Expression analysis indicated that transcripts of several of the genes identified accumulated specifically in petals and stamens. The cDNA library and expressed sequence tag database described here represent a valuable resource for future research aimed at improving economically important rose characteristics such as flower form, longevity and scent. ß

Changing shape is changing structure. Deciphering the mechanical contribution of the structural elements of the cells in shape changes is thus crucial to link the mechanical control of growth with development. Many measurements on plant and animal cells rather stress the differences in mechanical properties between both kingdoms. However, this conclusion relies on independent measurements, with very different set-ups and tissues, thus impairing any quantitative comparison. Here we took advantage of a previously described micro-rheometer to compare animal and plant single cell rheology with the same set-up. Using this method, we were able to quantitatively assess the dominant elastic behavior of plant cells in different conditions, and compare it with the viscoelastic behavior of animal cells. Surprisingly, we found that wall-less plant cells exhibit the same rheology as animal cells. This suggests that, despite the main structural differences between animal and plant cells, they also share a common mechanical core. Further investigations revealed that microtubules were the main responsible for the rheological behavior of wall-less plant cells whereas the mechanical properties of animal cells were mainly dependent on the actin network. Thus, wall-less plant cells and animal cells may have developed different strategies to converge to the same mechanical behavior.

Tobacco mosaic virus (TMV) coat protein (CP) in absence of RNA self-assembles into several different structures depending on pH and ionic strength. Transgenic plants that produce self-assembling CP are resistant to TMV infection, a phenomenon referred to as coat-protein-mediated resistance (CP-MR). The mutant CP Thr42Trp (CP(T42W)) produces enhanced CP-MR compared to wild-type CP. To establish the relationship between the formation of 20S CP aggregates and CP-MR, virus-like particles (VLPs) produced by TMV variants that yield high levels of CP-MR were characterized. We demonstrate that non-helical structures are found in VLPs formed in vivo by CP(T42W) but not by wild-type CP and suggest that the mutation shifts the intracellular equilibrium of aggregates from low to higher proportions of non-helical 20S aggregates. A similar shift in equilibrium of aggregates was observed with CP(D77R), another mutant that confers high level of CP-MR. The mutant CP(D50R) confers a level of CP-MR similar to wild-type CP and aggregates in a manner similar to wild-type CP. We conclude that increased CP-MR is correlated with a shift in intracellular equilibrium of CP aggregates, including aggregates that interfere with virus replication.