Stages Of Floral Development Research Articles

Identification of Suitable Reference Genes for Gene Expression Normalization in the Quantitative Real-Time PCR Analysis of Sweet Osmanthus (Osmanthus fragrans Lour.)

Quantitative real-time PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Several studies examining the selection of reference genes have been performed in ornamental plants but none in sweet osmanthus (Osmanthus fragrans Lour.). Based on transcriptomic sequencing data from O. fragrans buds at four developmental stages, six reference genes (OfACT, OfEF1α, OfIDH, OfRAN1, OfTUB, and OfUBC2) with stable expression (0.5 to 2 fold change in expression levels between any two developmental stages), as well as the commonly used reference gene Of18S, were selected as candidates for gene expression normalization in the RT-qPCR analysis of O. fragrans. For the normalization of RT-qPCR with two dyes, SYBR Green and EvaGreen, the expressional stability of seven candidate reference genes in 43 O. fragrans samples was analyzed using geNorm, NormFinder and BestKeeper. For RT-qPCR using SYBR Green, OfRAN1 and OfUBC2 were the optimal reference genes for all samples and different cultivars, OfACT and OfEF1α were suitable for different floral developmental stages, and OfACT was the optimal reference gene for different temperature treatments. The geometric mean values of the optimal reference gene pairs for the normalization of RT-qPCR are recommended to be used for all samples, different cultivars and different floral developmental stages in O. fragrans. For RT-qPCR using EvaGreen, OfUBC2 was the optimal reference gene for all samples and different cultivars, and OfACT was the optimal reference gene for different floral developmental stages and different temperature treatments. As the worst reference gene, Of18S should not be used as a reference gene in O. fragrans in the future. Our results provide a reference gene application guideline for O. fragrans gene expression characterization using RT-qPCR.

Photoperiod affects floral ontogeny in strawberry (Fragaria × ananassa Duch.) plug plants

Strawberry (Fragaria×ananassa Duch.) plug plants of the short day (SD) cultivars ‘Earliglow’, ‘Seneca’, ‘Jewel’, ‘Chandler’ and ‘Cavendish’, and the long day (LD) cultivars ‘Seascape’, ‘Fern’ and ‘Selva’ were exposed to 0, 1, 2 or 4 weeks of short-days (8h) at 15°C followed by forcing in a long-day (16h) greenhouse. Leaf, runner and flower growth were monitored weekly. Floral ontogeny and plant phenology of three week old plug plants was sensitive to pre-forcing short-day conditioning and the degree of sensitivity varied with cultivar and parameter examined. In all SD cultivars except ‘Chandler’ conditioning hastened flowering. The length of conditioning needed to enhance precocity and intensity of enhancement varied with cultivar. Conditioning did not alter the time of flowering for LD cultivars. SD conditioning enhanced leaf production under LD forcing in some cultivars. Each cultivar exhibited its own characteristic response to photoperiod for runner production regardless of the traditional photoperiodic category to which it belonged. The general classification for enhanced runner production of SD cultivars under LD and LD cultivars under SD is questionable since runner production responses of cultivars did not follow this categorization: runner production was enhanced by SD conditioning in the SD cultivar ‘Chandler’ and inhibited by SD in the LD cultivar ‘Fern’. Runner and inflorescence production are not necessarily antagonistic processes in short-day cultivars, as sometimes suggested. Reduced runner formation was observed with a concomitant increase in inflorescence production in some cultivars. The reduction in runner production in other cultivars was a direct inhibitory response to short days rather than an inhibition caused by flowering. Runner production was reduced but flowering was not enhanced with short day conditioning. In addition, SD enhanced flowering in ‘Chandler’ did not inhibit runner production. Cultivars did not fall into specific photoperiod response categories (SD or LD) with respect to flowering either. All SD and one of three LD cultivars were vegetative at the start of the experiment. Inflorescence production occurred during long-day forcing in non-conditioned controls indicating that floral initiation occurred during the long days in the greenhouse. Nevertheless, altered flowering under LD forcing of SD conditioned plugs compared to controls in several cultivars indicated that floral ontogeny under long days was altered with pre-forcing, short-day conditioning. All cultivars except ‘Seascape’ and ‘Cavendish’ exhibited enhanced initiation and differentiation following SD conditioning. Each stage of floral development (induction/initiation, development and differentiation) was differentially affected by photoperiod and as many of these stages as possible should be evaluated to provide a clear description of any response. The only general description of flowering response to photoperiod in strawberry is that SD exposure enhances differentiation in both SD and LD cultivars. General categorizations for initiation and development should not be made as responses to photoperiod for these stages are cultivar specific.

Floral biology of indigenous pummelo genotypes

Flower morphology and bud development of pummelo accessions CG-1, CG-18 and CG-151 were studied at the Pummelo Orchard of Regional Agricultural Research Station, BARI, Akbarpur, Moulvibazar and the Horticulture Laboratory of Bangabandhu Sheikh Mujibur Rahman Agricultural University during 2008-2009. Pummelo flowers were bisexual, bore singly on leaf axils or in clusters with or without leaf on stem in all accessions, and colour were white. Calyx diameter varied from 0.94 in CG-1 to 1.02 in CG-18. Number of petals per flower ranged from 4.0 to 4.5. Anthers were yellow in colour and only CG- 151 produced few rudimentary styles. Diameter of stigma varied from 0.39 mm to 0.49 mm. Number of locules per ovary was in between 14.6 to16.0 and number of ovules per locules varied from 4.0 to 9.0. Stages of floral bud development from initiation to anthesis were divided into 9 distinct stages. In pummelo, a total of 27.7 to 31.2 days were required from a bud initiation to reach its fully developed stage. Suitable time for emasculation of pummelo flowers was found within 26 days from flower bud initiation. Between 3:00am to 5:00am, about 76% flowers were found to be opened and between 4:00pm to 5:00pm in all the three accessions, dehiscence of pollens was recorded. Abscission of stamen, petal and style started after 50.8, 76.4 and 162.3 hrs and completed after 128.4, 137.9 and 228.3 hrs of anthesis, respectively.Bangladesh J. Agril. Res. 40(2): 177-188, June 2015

Differential selection on pollen and pistil traits in relation to pollen competition in the context of a sexual conflict over timing of stigma receptivity.

Sexual conflict and its evolutionary consequences are understudied in plants, but the theory of sexual conflict may help explain how selection generates and maintains variability. Here, we investigated selection on pollen and pistil traits when pollen arrives sequentially to partially receptive pistils in relation to pollen competition and a sexual conflict over timing of stigma receptivity in the mixed-mating annual Collinsia heterophylla (Plantaginaceae). In this species the conflict is generated by early fertilizing pollen that reduces seed production, which is counteracted by delaying receptivity in the recipient. We performed sequential two-donor pollinations at early floral developmental stages involving two pollen deposition schedules (with or without a time lag of 1 day), using only outcross or self and outcross pollen. We investigated pollen and pistil traits in relation to siring success (male fitness) and seed production (female fitness). In contrast to previous findings in receptive pistils in C. heterophylla and in other species, last arriving pollen donors showed highest siring success in partially receptive pistils. The last male advantage was weaker when self pollen was the first arriving donor. Two measures of germination rate (early and late) and pollen tube growth rate of first arriving donors were important for siring success in crosses with a time lag, while only late germination rate had an effect in contemporary crosses. Curiously, late stigma receptivity was negatively related to seed production in our contemporary crosses, which was opposite to expectation. Our results in combination with previous studies suggest that pollen and pistil traits in C. heterophylla are differentially advantageous depending on stage of floral development and varying pollen deposition schedules. Variation in success of these traits over floral development time may result from sexually antagonistic selection.

Variation of floret fertility in hexaploid wheat revealed by tiller removal.

Grain number per spike, which is greatly influenced by floret fertility, is an important trait of wheat (Triticum aestivum L.) yield. Maximum floret primordia, fertile floret, and final grain number per spikelet are three crucial factors of floret fertility. Floral degradation plays a critical role in determining these three floret fertility-related traits. Twelve hexaploid spring wheat genotypes were selected to investigate the influence of detillering on floral degradation and floret fertility-related traits in the field and greenhouse. Notably, the green anther stage was found to consistently have the maximum floret primordia number. Visible floral degradation, however, was observed to occur at several floral developmental stages, specifically from green anther stage to anthesis. Detillering was able to delay floral degradation in most cases and was evidently highly associated with increased maximum floret primordia, fertile floret, and final grain number per spikelet, with only a few exceptions. Thermal time required for each floral developmental stage was overall not influenced by detillering. These data hereby reveal a predominant spikelet fertility pattern along the spike in which the number of fertile florets per spikelet at anthesis becomes developmentally confined.

Flower and Microspore Development in ‘Campbell Early’ (Vitis labruscana) and ‘Tamnara’ (V. spp.) Grapes

Abstract: The majority of cultivated varieties of grape have perfect flowers that are clustered in an individual inflorescence. Grape flower has a single pistil, five stamens, a protective flower cap (calyptra), and a calyx. After fertilization, an indivi dual flower develops into a single berry. Although there are a number of reported studies focusing on berry formation, berry enlar gement, and sugar accumulation in grape, the morphological studies of flower, including gametophyte morphogenesis and structural chan ge in floral organs, have not yet been studied in detail. In this study, we investigated the flower structure and development char acteristics of grape using microscopy and defined the floral development stages 9 to 13 based on microspore or male gametophyte development stage from tetrad to mature pollen. We used seeded diploid table grapes ‘Campbell Early’ ( Vitis labruscana) and ‘Tamnara’ (V. spp.) as plant materials. At floral development stage 9, pollen mother cells develop to tetrads. During floral development sta ges 10 to 11, unicellular microspore develop to mid bicellular pollen. At the end of floral stage 12, male gametophyte develops to mature tricelluar pollen. In floral stage 13, the flower cap falls off and flower bud opens. During floral development sta ges 9 to 12, there were no major changes in calyx length, whereas the length of the flower cap continuously increased. The flower cap-to-calyx length ratio was 2.0, 3.0, 4.5, and 6.5 at floral stages 9, 10, 11, and 12, respectively. The flower cap-to-calyx length ratio was consistent in the two grape cultivars, suggesting that the ratio is a morphological character representing floral dev elopment stage. This study provides a reference for determining floral development stage of the two grape cultivars. It will be useful for the determination of optimum time for microspore culture needed to generate doubled haploid lines and appropriate gibberellic acid treatment needed to induce parthenocarpic fruit development in ‘Tamnara’ grape.Additional key words:

Sex-Biased Temporal Gene Expression in Male and Female Floral Buds of Seabuckthorn (Hippophae rhamnoides).

Seabuckthorn is an economically important dioecious plant in which mechanism of sex determination is unknown. The study was conducted to identify seabuckthorn homologous genes involved in floral development which may have role in sex determination. Forty four putative Genes involved in sex determination (GISD) reported in model plants were shortlisted from literature survey, and twenty nine seabuckthorn homologous sequences were identified from available seabuckthorn genomic resources. Of these, 21 genes were found to differentially express in either male or female flower bud stages. HrCRY2 was significantly expressed in female flower buds only while HrCO had significant expression in male flowers only. Among the three male and female floral development stages (FDS), male stage II had significant expression of most of the GISD. Information on these sex-specific expressed genes will help in elucidating sex determination mechanism in seabuckthorn.

Variance components, heritability and correlation analysis of anther and ovary size during the floral development of bread wheat.

Anther and ovary development play an important role in grain setting, a crucial factor determining wheat (Triticum aestivum L.) yield. One aim of this study was to determine the heritability of anther and ovary size at different positions within a spikelet at seven floral developmental stages and conduct a variance components analysis. Relationships between anther and ovary size and other traits were also assessed. The thirty central European winter wheat genotypes used in this study were based on reduced height (Rht) and photoperiod sensitivity (Ppd) genes with variable genetic backgrounds. Identical experimental designs were conducted in a greenhouse and field simultaneously. Heritability of anther and ovary size indicated strong genetic control. Variance components analysis revealed that anther and ovary sizes of floret 3 (i.e. F3, the third floret from the spikelet base) and floret 4 (F4) were more sensitive to the environment compared with those in floret 1 (F1). Good correlations were found between spike dry weight and anther and ovary size in both greenhouse and field, suggesting that anther and ovary size are good predictors of each other, as well as spike dry weight in both conditions. Relationships between spike dry weight and anther and ovary size at F3/4 positions were stronger than at F1, suggesting that F3/4 anther and ovary size are better predictors of spike dry weight. Generally, ovary size showed a closer relationship with spike dry weight than anther size, suggesting that ovary size is a more reliable predictor of spike dry weight.

Selection of Reference Genes for Real-time Quantitative Polymerase Chain Reaction Analysis of Light-dependent Anthocyanin Biosynthesis in Chrysanthemum

The light-dependent coloration of the vital organs of horticultural crops affects multiple parts of production and sales. The simplicity of the metabolic pathways of anthocyanins and the characteristics of light-dependent coloration make chrysanthemum (Chrysanthemum ×morifolium) an ideal subject for studying the mechanism of light-regulated anthocyanin biosynthesis. In this study, real-time quantitative reverse transcription–polymerase chain reaction (PCR) was used in the analysis of the expression levels of anthocyanin biosynthesis genes in C. ×morifolium ‘Reagan’. The reference genes selected were those assumed to remain at constant levels in three flower color lines at five floral developmental stages and at two light conditions. Using digital gene expression technology, we selected nine reference genes with moderate expression in the chrysanthemum ray florets at various floral developmental stages under illuminated and dark conditions as the candidate reference genes for further study. After comprehensively analyzing the stability of gene expression with three distinct statistical algorithms, geNorm, NormFinder, and qBase plus, we found that F-box and PP2A were the most stable genes in all of the samples. In addition, we analyzed the relative expression level of the CmF3H gene in different samples to verify the reference genes that we selected. This study provides a consensus list of validated reference genes that will benefit future studies of the expression of chrysanthemum genes involved in anthocyanin biosynthesis and floral development under various light conditions. Moreover, this information will also promote the molecular breeding of horticultural crops for their color modification.

Temporal characterization of 2-phenylethanol in strongly and weakly scented genotypes of damask rose.

The molecular and physiological properties of 2-phenylethanol (2-PE) in the strongly scented genotype (SSG) and a weakly scented genotype (WSG) of damask rose at six floral developmental stages were investigated. The chemical compositions of volatile emissions were determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analysis of the floral headspace. In both genotypes, the relative percentage of 2-PE increased more in SSG than WSG, as flowers developed. In the petals of damask rose the relative transcript levels of phenyl acetaldehyde reductase (PAR) were higher at stages 3 and 4 in SSG and WSG, respectively. Also, the expression pattern of PAR indicated a significant difference between two genotypes during flower developmental stages. In this study, enzymatic activity leading to the synthesis of 2-PE from the phenyl acetaldehyde (PAld) moderately increased during flower development up to stage 5 in SSG. However, high level of PAR enzymatic activity was observed in stage 3 of WSG. These results indicated that the pattern activity of PAR was different in two used genotypes of damask rose. For SSG, PAR activities were low in early stage of flower development and then gradually increased reaching its highest value at full bloom stage. In WSG, no significant change in enzyme activity was seen after stage 3.

Ectopic expression of a Catalpa bungei (Bignoniaceae) PISTILLATA homologue rescues the petal and stamen identities in Arabidopsis pi-1 mutant

PISTILLATA (PI) plays crucial roles in Arabidopsis flower development by specifying petal and stamen identities. To investigate the molecular mechanisms underlying organ development of woody angiosperm in Catalpa, we isolated and identified a PI homologue, referred to as CabuPI (C. bungei PISTILLATA), from two genetically cognate C. bungei (Bignoniaceae) bearing single and double flowers. Sequence and phylogenetic analyses revealed that the gene is closest related to the eudicot PI homologues. Moreover, a highly conserved PI-motif is found in the C-terminal regions of CabuPI. Semi-quantitative and quantitative real time PCR analyses showed that the expression of CabuPI was restricted to petals and stamens. However, CabuPI expression in the petals and stamens persisted throughout all floral development stages, but the expression levels were different. In 35S::CabuPI transgenic homozygous pi-1 mutant Arabidopsis, the second and the third whorl floral organs produced normal petals and a different number of stamens, respectively. Furthermore, ectopic expression of the CabuPI in transgenic wild-type or heterozygote pi-1 mutant Arabidopsis caused the first whorl sepal partially converted into a petal-like structure. These results clearly reveal the functional conservation of PI homologues between C. bungei and Arabidopsis.

Pattern and process in the evolution of the sole dioecious member of Brassicaceae.

BackgroundLepidium sisymbrioides, a polyploid New Zealand endemic, is the sole dioecious species in Brassicaceae and therefore the closest dioecious relative of the model plant Arabidopsis thaliana. The attractiveness of developing this system for future studies on the genetics of sex determination prompted us to investigate historical and developmental factors surrounding the evolution of its unisexual flowers. Our goal was to determine the evolutionary pattern of polyploidization of L. sisymbrioides and the timing and process of flower reproductive organ abortion. To that end, we used a combination of phylogenetics to place this species within the complex history of polyploidization events in Lepidium and histology to compare its floral ontogeny to that of its closest hermaphroditic relatives and to A. thaliana.ResultsUsing a nuclear locus (PISTILLATA), we reconstructed the gene tree among Lepidium taxa and applied a phylogenetic network analysis to identify ancestral genomes that contributed to the evolution of L. sisymbrioides. Combining this phylogenetic framework with cytological and genome size data, we estimated L. sisymbrioides as an allo-octoploid resulting from three hybridization events. Our investigations of flower development showed that unisexual flowers appear to abort reproductive organs by programmed cell death in female flowers and by developmental arrest in male flowers. This selective abortion occurs at the same floral developmental stage in both males and females, corresponding to Arabidopsis stage nine.ConclusionsDioecy in Brassicaceae evolved once in L. sisymbrioides following several allopolyploidization events, by a process of selective abortion of reproductive organs at intermediate stages of flower development. Different developmental processes, but similar timing of abortions, affect male versus female flower development. An increased understanding of how and when reproductive organs abort in this species, combined with our estimates of ancestral genome contributions, ploidy and genome size, lay the foundation for future efforts to examine the genetic mechanisms involved in the evolution of unisexual flowers in the closest dioecious relative of the best studied model plant.Electronic supplementary materialThe online version of this article (doi:10.1186/2041-9139-5-42) contains supplementary material, which is available to authorized users.

Floral volatile alleles can contribute to pollinator-mediated reproductive isolation in monkeyflowers (Mimulus).

Pollinator-mediated reproductive isolation is a major factor in driving the diversification of flowering plants. Studies of floral traits involved in reproductive isolation have focused nearly exclusively on visual signals, such as flower color. The role of less obvious signals, such as floral scent, has been studied only recently. In particular, the genetics of floral volatiles involved in mediating differential pollinator visitation remains unknown. The bumblebee-pollinated Mimulus lewisii and hummingbird-pollinated Mimulus cardinalis are a model system for studying reproductive isolation via pollinator preference. We have shown that these two species differ in three floral terpenoid volatiles - d-limonene, β-myrcene, and E-β-ocimene - that are attractive to bumblebee pollinators. By genetic mapping and in vitro analysis of enzyme activity we demonstrate that these interspecific differences are consistent with allelic variation at two loci, LIMONENE-MYRCENE SYNTHASE (LMS) and OCIMENE SYNTHASE (OS). Mimulus lewisii LMS (MlLMS) and OS (MlOS) are expressed most strongly in floral tissue in the last stages of floral development. Mimulus cardinalis LMS (McLMS) is weakly expressed and has a nonsense mutation in exon 3. Mimulus cardinalis OS (McOS) is expressed similarly to MlOS, but the encoded McOS enzyme produces no E-β-ocimene. Recapitulating the M.cardinalis phenotype by reducing the expression of MlLMS by RNA interference in transgenic M.lewisii produces no behavioral difference in pollinating bumblebees; however, reducing MlOS expression produces a 6% decrease in visitation. Allelic variation at the OCIMENE SYNTHASE locus is likely to contribute to differential pollinator visitation, and thus promote reproductive isolation between M.lewisii and M.cardinalis. OCIMENE SYNTHASE joins a growing list of 'speciation genes' ('barrier genes') in flowering plants.

The floral transcriptome of Eucalyptus grandis.

As a step toward functional annotation of genes required for floral initiation and development within the Eucalyptus genome, we used short read sequencing to analyze transcriptomes of floral buds from early and late developmental stages, and compared these with transcriptomes of diverse vegetative tissues, including leaves, roots, and stems. A subset of 4807 genes (13% of protein-coding genes) were differentially expressed between floral buds of either stage and vegetative tissues. A similar proportion of genes were differentially expressed among all tissues. A total of 479 genes were differentially expressed between early and late stages of floral development. Gene function enrichment identified 158 gene ontology classes that were overrepresented in floral tissues, including 'pollen development' and 'aromatic compound biosynthetic process'. At least 40 floral-dominant genes lacked functional annotations and thus may be novel floral transcripts. We analyzed several genes and gene families in depth, including 49 putative biomarkers of floral development, the MADS-box transcription factors, 'S-domain'-receptor-like kinases, and selected gene family members with phosphatidylethanolamine-binding protein domains. Expanded MADS-box gene subfamilies in Eucalyptus grandis included SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), SEPALLATA (SEP) and SHORT VEGETATIVE PHASE (SVP) Arabidopsis thaliana homologs. These data provide a rich resource for functional and evolutionary analysis of genes controlling eucalypt floral development, and new tools for breeding and biotechnology.

Floral development at multiple spatial scales in Polygonum jucundum (Polygonaceae), a distylous species with broadly open flowers.

Distyly, a special polymorph, has evolved in many groups of angiosperms and has attracted attention since Darwin’s time. Development studies on distylous taxa have helped us to understand the evolutionary process of this polymorph, but most of these studies focus on species with narrowly tubular corolla. Here, we studied the floral development of Polygonum jucundum, a distylous species with broadly open flowers, at multiple spatial scales. Results showed that the difference in stigma height between flowers of the two morphs was caused by differences in style growth throughout the entire floral development process. The observed difference in anther heights between the two morphs was because the filaments grew faster in short-styled (SS) than in long-styled (LS) flowers in the later stages of floral development. In addition, the longer styles in LS flowers than in SS flowers was because of faster cell division in the early stages of floral development. However, SS flowers had longer filaments than LS flowers primarily because of greater cell elongation. These results indicate that floral development in P. jucundum differs from that of distylous taxa with floral tubes shown in previous studies. Further, we conclude that the presence of distyly in species with open flowers is a result of convergent evolution.

Flower morphology and floral sequence in Artemisia annua (Asteraceae)1

• Artemisia annua produces phytochemicals possessing antimalarial, antitumor, anti-inflammatory, and anthelmintic activities. The main active ingredient, artemisinin, is extremely effective against malaria. Breeding to develop cultivars producing high levels of artemisinin can help meet worldwide demand for artemisinin and its derivatives. However, fundamental reproductive processes, such as the sequence of flowering and fertility, are not well understood and impair breeding and seed propagation programs.• Capitulum structure and floral sequence were studied using light and scanning electron microscopy to describe inflorescence architecture, floret opening, and seed set.• Florets are minute and born in capitula containing pistillate ray florets and hermaphroditic disk florets. Ray florets have elongated stigmatic arms that extend prior to disk floret opening. Disk florets exhibit protandry. During the staminate phase, pollen is released within a staminate tube and actively presented with projections at the tip of stigmas as the pistil elongates. During the pistillate phase, stigmatic arms bifurcate and reflex. Stigmas are of the dry type and stain positively for polysaccharides, lipids, and an intact cuticle. Floret numbers vary with genotype, and capitula are predominantly composed of disk florets. Both ray and disk florets produce filled seed.• Gynomonoecy, early opening of ray florets, and dichogamy of disk florets promote outcrossing in A. annua For breeding and seed development, flowering in genotypes can be synchronized under short days according to the floral developmental stages defined. Floret number and percentage seed fill vary with genotype and may be a beneficial selection criterion.

Specific duplication and dorsoventrally asymmetric expression patterns of Cycloidea-like genes in zygomorphic species of Ranunculaceae.

Floral bilateral symmetry (zygomorphy) has evolved several times independently in angiosperms from radially symmetrical (actinomorphic) ancestral states. Homologs of the Antirrhinum majus Cycloidea gene (Cyc) have been shown to control floral symmetry in diverse groups in core eudicots. In the basal eudicot family Ranunculaceae, there is a single evolutionary transition from actinomorphy to zygomorphy in the stem lineage of the tribe Delphinieae. We characterized Cyc homologs in 18 genera of Ranunculaceae, including the four genera of Delphinieae, in a sampling that represents the floral morphological diversity of this tribe, and reconstructed the evolutionary history of this gene family in Ranunculaceae. Within each of the two RanaCyL (Ranunculaceae Cycloidea-like) lineages previously identified, an additional duplication possibly predating the emergence of the Delphinieae was found, resulting in up to four gene copies in zygomorphic species. Expression analyses indicate that the RanaCyL paralogs are expressed early in floral buds and that the duration of their expression varies between species and paralog class. At most one RanaCyL paralog was expressed during the late stages of floral development in the actinomorphic species studied whereas all paralogs from the zygomorphic species were expressed, composing a species-specific identity code for perianth organs. The contrasted asymmetric patterns of expression observed in the two zygomorphic species is discussed in relation to their distinct perianth architecture.

Floral ontogeny in Dipterygeae (Fabaceae) reveals new insights into one of the earliest branching tribes in papilionoid legumes

Flowers of Dipterygeae (Fabaceae, Papilionoideae) exhibit an unusual petaloid calyx. The two adaxial sepals are large and petaloid, and the three abaxial sepals form a three-toothed lobe. The goal of this study was to elucidate the ontogenetic pathways of this peculiar calyx in light of the floral development of the three genera that comprise the tribe. Floral buds of Dipteryx alata, Pterodon pubescens and Taralea oppositifolia were analysed using scanning electron microscopy and light microscopy. The order of bracteole and sepal initiation varies among the species. The androecium is asymmetric. The carpel cleft is positioned to the right or to the left, and is opposite the adaxial antepetalous stamen. The peculiarity of the calyx becomes noticeable in the intermediate stages of floral development. It results from the differential growth of the sepal primordia, in which the abaxial and lateral primordia remain diminutive during floral development, compared with the adaxial ones that enlarge and elongate. Bracteoles, abaxial sepals, petals and anthers are appendiculate, except in T. oppositifolia, in which the appendices were not found in bracteoles or anthers. These appendices comprise secretory canals or cavities. Considering that the ontogenetic pathway for the formation of the petaloid calyx is similar and exclusive for Dipterygeae, it might be a potential synapomorphy for the group, with the presence of secretory canals in the appendices of abaxial and lateral sepals and petals. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 529–550.

Spontaneous Multi‐Pistil Mutantmp1in Alfalfa: Floral Anatomy and Embryo Sac Development

A spontaneous mutant with two to three pistils,mp1,was identified from ovule fertility investigations in a natural alfalfa (Medicago sativaL.) population. No cytological investigations of pistil development and seeding mechanisms in reduced‐fertility multi‐pistil mutants have been performed to date. In this study, floral ontogeny, embryonic development, and pollen tube guidance were compared between alfalfamp1and wild‐type flowers. The results indicated that pod‐setting ofmp1was significantly lower (8.18%) than that of wild‐type plants (71.43%), which indicates thatmp1was partial‐female‐sterile. In the late stage of floral development that forms stamen and pistil organs, an extra carpel was visible inmp1in place of the vexillary stamen. Normal patterns of megasporogenesis forming a functional chalazal megaspore were observed both inmp1and wild‐type plants. However,mp1showed abnormal development during megagametogenesis for more than one egg cell and two synergids in mature embryo sacs. In comparison with a fertile flower, the stigma ofmp1has no honeycomb‐like cuticle and less exudate to adhere to pollen grains. Furthermore, pollen tubes ofmp1grew in a disordered fashion and could not penetrate into ovules through micropyles, while pollen tubes from fertile plants were attracted to the micropyle in the “N” way and grew in a highly organized manner. This study is the first report of a spontaneous multi‐pistil mutation inM. sativa. Our results reveal thatmp1is a new floral homeotic mutant with partial female sterility due to abnormal female gametogenesis, stigma development, and pollen tube guidance.

Cloning and Expression of Two Soluble Acid Invertase Gene Isoforms from Rhododendron

Soluble acid invertase [SAI (Enzyme Commission 3.2.1.26)] plays an important role in catalyzing the hydrolysis of sucrose into hexoses and regulates floral development. Full-length cDNAs encoding RhSAI1 and RhSAI2 isoforms were cloned from Rhododendron hybrid ‘Yuqilin’ and they exhibited high amino acid sequence identity (89%) to each other. The protein sequences contain highly conserved motifs present in all SAIs, including the β-fructosidase motif N-D-P-(D/N), a putative active site W-E-C-(I/V)-D, and R-D-P. The expression of RhSAI1 and RhSAI2 genes was under spatial and temporal control. Expression of both RhSAI1 and RhSAI2 genes was most abundant in stems, and expression was lowest in roots and leaves, respectively. The expression of RhSAI2 was significantly lower than that of RhSAI1 in all organs. During floral development, RhSAI1 was highly expressed at the earliest stage (Stage I), decreased until Stage III, and increased again at the terminal stage. The pattern of RhSAI2 expression was distinctly different, showing a continuous increase during floral development. Consistent with the levels of RhSAI1 expression, SAI activity decreased during floral development and was inversely correlated with the soluble sugar content. Abundant expression of RhSAI1 at the transcriptional level in addition to high SAI activity during the initial stages of floral development may play a vital role in supplying the energy needed for rapid cell division and growth of flowers.