Stamen Development Research Articles

Aberrant Stamen Development is Associated with Parthenocarpic Fruit Set Through Up-Regulation of Gibberellin Biosynthesis in Tomato.

Parthenocarpy, a process in which fruit set occurs without fertilization, leads to the production of seedless fruit. A number of floral homeotic mutants with abnormal stamen development exhibit parthenocarpic fruit set. Flower development is thought to repress ovary growth before anthesis. However, the mechanism of parthenocarpic fruit development caused by aberrant flower formation is poorly understood. To investigate the molecular mechanism of parthenocarpic fruit development in floral homeotic mutants, we performed functional analysis of Tomato APETALA3 (TAP3) by loss-of-function approaches. Organ-specific promoter was used to induce organ-specific loss of function in stamen and ovary/fruit. We observed increased cell expansion in tap3 mutants and TAP3-RNAi lines during parthenocarpic fruit growth. These were predominantly accompanied by the up-regulation of GA biosynthesis genes, including SlGA20ox1, SlGA20ox2, and SlGA20ox3, as well as reduced expression of the GA-inactivating gene SlGA2ox1 and the auxin signaling gene SlARF7 involved in a crosstalk between GA and auxin. These transcriptional profiles are in agreement with the GA levels in these lines. These results suggest that stamen development negatively regulates fruit set by repressing the GA biosynthesis.

Modulation of Auxin Levels in Pollen Grains Affects Stamen Development and Anther Dehiscence in Arabidopsis.

Auxin regulates diverse aspects of flower development in plants, such as differentiation of the apical meristem, elongation of the stamen, and maturation of anthers and pollen. It is known that auxin accumulates in pollen, but little information regarding the biological relevance of auxin in this tissue at different times of development is available. In this work, we manipulated the amount of free auxin specifically in developing pollen, using transgenic Arabidopsis lines that express the bacterial indole-3-acetic acid-lysine synthetase (iaaL) gene driven by a collection of pollen-specific promoters. The iaaL gene codes for an indole-3-acetic acid-lysine synthetase that catalyzes the conversion of free auxin into inactive indole-3-acetyl-l-lysine. The transgenic lines showed several abnormalities, including the absence of short stamina, a diminished seed set, aberrant pollen tubes, and perturbations in the synchronization of anther dehiscence and stamina development. This article describes the importance of auxin accumulation in pollen and its role in stamina and anther development.

The sex-determining gene CitACS4 is a pleiotropic regulator of flower and fruit development in watermelon (Citrullus lanatus).

In the species of the Cucurbitaceae family, the occurrence of separate male and female flowers in the same plant (monoecy) is controlled by an ethylene biosynthesis ACS gene, which specifically suppresses the development of stamen in the female flower. In watermelon, a mutation of loss of function in CitACS4 promotes the conversion of female into hermaphrodite flowers, and of monoecious into andromonoecious plants. We have studied whether the ethylene produced by CitACS4 enzyme could also be involved in other ethylene-regulated traits, including pistillate flowering transition and the number of female flowers per plant, the development of floral organs other than stamens, as well as fruit and seed set, and fruit development. A linkage analysis approach was performed in three independent F2 populations segregating for the two alleles of the gene (M, monoecious; m, andromonoecious), and the different traits under study. The CitACS4m allele not only cosegregated with andromonoecy, but also with earlier pistillate transition, an increased number of pistillate flowers per plant, and a slower growth and maturation of petals and carpels, which delayed anthesis time in hermaphrodite flowers. The m allele was also found to be linked to a reduced fruit set, which was not caused by a deficiency in pollination or fertilization. The gene also affected the longitudinal and transverse growth rates of the ovary and fruit, which means that fruits from andromonoecious plants (mm) were rounder than those from monoecious (MM) ones. Taken together, these data indicate that the locus defined by the ethylene biosynthesis and sex-determining gene CitACS4 acts as a pleiotropic regulator of the complete development of the pistillate flower and the earlier development of the fruit.

A new DNA marker for sex identification in purple asparagus

Garden asparagus (Asparagus officinalis L.) is an economically-important perennial crop. This plant is dioecious, as there are both male and female individuals; male individuals are preferred over females for agricultural production. To reduce the time required for garden asparagus breeding, various male-specific DNA markers are utilized. Male-specific DNA markers, such as Asp1-T7sp and MSSTS710, are currently available for sex identification in many asparagus cultivars. In the current study, we found that these markers are not suitable for sex identification in the purple asparagus cultivar ‘Pacific Purple’, as male-specific amplification of this marker was detected in some male individuals of this cultivar but not in other males. The Asp1-T7sp marker is suitable for use in sex identification in various Asparagus species related to A. officinalis, indicating that the region around this marker is conserved among these species. Thus, we isolated a DNA fragment around this marker by inverse PCR and produced a new DNA marker, MspHd, based on this sequence. However, like Asp1-T7sp and MSSTS710, MspHd was not suitable for sex identification in the cultivar ‘Pacific Purple’. Since all ‘Pacific Purple’ males have morphologically similar male flowers with functional stamens, we produced a new male-specific marker based on the sex determination gene, MSE1/AspMYB35/AspTDF1, which is responsible for stamen development. This marker, named AspMSD, is suitable for sex identification in ‘Pacific Purple’. In addition, this marker can be utilized for sex identification in various asparagus cultivars and some related Asparagus species.

Functional Specialization of Duplicated AGAMOUS Homologs in Regulating Floral Organ Development of Medicago truncatula

The C function gene AGAMOUS (AG) encodes for a MADS-box transcription factor required for floral organ identity and floral meristem (FM) determinacy in angiosperms. Unlike Arabidopsis, most legume plants possess two AG homologs arose by an ancient genome duplication event. Recently, two euAGAMOUS genes, MtAGa and MtAGb, were characterized and shown to fulfill the C function activity in the model legume Medicago truncatula. Here, we reported the isolation and characterization of a new mtaga allele by screening the Medicago Tnt1 insertion mutant collection. We found that MtAGa was not only required for controlling the stamen and carpel identity but also affected pod and seed development. Genetic analysis indicated that MtAGa and MtAGb redundantly control Medicago floral organ identity, but have minimal distinct functions in regulating stamen and carpel development in a dose-dependent manner. Interestingly, the stamens and carpels are mostly converted to numerous vexillum-like petals in the double mutant of mtaga mtagb, which is distinguished from Arabidopsis ag. Further qRT-PCR analysis in different mtag mutants revealed that MtAGa and MtAGb can repress the expression of putative A and B function genes as well as MtWUS, but promote putative D function genes expression in M. truncatula. In addition, we found that the abnormal dorsal petal phenotype observed in the mtaga mtagb double mutant is associated with the upregulation of CYCLOIDEA (CYC)-like TCP genes. Taken together, our data suggest that the redundant MtAGa and MtAGb genes of M. truncatula employ a conserved mechanism of action similar to Arabidopsis in determining floral organ identity and FM determinacy but may have evolved distinct function in regulating floral symmetry by coordinating with specific floral dorsoventral identity factors.

Evolutionary diversification of CYC/TB1-like TCP homologs andtheir recruitment for the control of branching and floral morphology in Papaveraceae (basal eudicots).

Angiosperms possess enormous morphological variation in plant architectures and floral forms. Previous studies in Pentapetalae and monocots have demonstrated the involvement of TCP domain CYCLOIDEA/TEOSINTE BRANCHED1-like (CYC/TB1) genes in the control of floral symmetry and shoot branching. However, how TCP/CYC-like (CYL) genes originated, evolved and functionally diversified remain unclear. We conducted a comparative functional study in Ranunculales, the sister lineage to all other eudicots, between Eschscholzia californica and Cysticapnos vesicaria, two species of Papaveraceae with actinomorphic and zygomorphic flowers, respectively. Phylogenetic analysis indicates that CYL genes in Papaveraceae form two paralogous lineages, PapaCYL1 and PapaCYL2. Papaveraceae CYL genes show highly diversified expression patterns as well as functions. Enhanced branching by silencing of EscaCYL1 suggests that the role of CYC/TB1-like genes in branching control is conserved in Papaveraceae. In contrast to the arrest of stamen development in Pentapetalae, PapaCYL genes promote stamen initiation and growth. In addition, we demonstrate that CyveCYLs are involved in perianth development, specifying sepal and petal identity in Cysticapnos by regulating the B-class floral organ identity genes. Our data also suggest the involvement of CyveCYL genes in the regulation of flower symmetry in Cysticapnos. Our work provides evidence of the importance of TCP/CYC-like genes in the promotion of morphological diversity across angiosperms.

Patterning the Asteraceae Capitulum: Duplications and Differential Expression of the Flower Symmetry CYC2-Like Genes.

There are several types of capitulum in the Asteraceae due to different combinations of florets varying in corolla shape and stamen development. Previous studies have shown that the formation of ray florets on a radiate capitulum may be related to the parallel co-option of CYC2-like genes among independent Asteraceae lineages. The present work tests that hypothesis and attempts to shed light on the pattern of evolution of the Asteraceae capitulum and floral heteromorphism under the regulation of CYC2-like genes. In this study, the evolutionary history of CYC2-like genes in the Asterales was reconstructed and their expression patterns were examined in species representing different capitulum types and several major Asteraceae lineages. To clarify the role of CYC2d clade genes in morphogenesis of ray flowers, overexpression of ClCYC2d was conducted in Chrysanthemum lavandulifolium. Our results show that there are six CYC2-like members in the Asteraceae; they are results of five duplication events starting from a single-copy gene in the common ancestor of the Goodeniaceae-Calyceraceae-Asteraceae group and completing before the divergence of the subfamily Carduoideae of Asteraceae. Spatial expression pattern of each of the Asteraceae CYC2-like members is conserved across the family. All the six members contribute to the development of the complexity of a capitulum: To form a ray floret, either CYC2c or CYC2g plays an essential role, while CYC2d represses the development of dorsal corolla lobes and stamens of the floret. In sum, the developmental program of making a ray flower is conserved involving functionally divergent CYC2-like genes. Based on extensive species sampling, this study provides an overview of the mode of regulation of CYC2-like genes that patterns the capitulum architectures and their transitions.

Differential methylation and expression of HUA1 ortholog in three sex types of papaya

Papaya is trioecious and an excellent system for studying sex determination and differentiation in plants. An ortholog of HUA1, CpHUA1, a gene controlling stamen and carpel development in Arabidopsis, was cloned and characterized in papaya. CpHUA1 consists of 12 exons with full genomic length of 19,313 bp in male AU9 and 19,312 bp in hermaphrodite SunUp, whereas the Arabidopsis HUA1 consists of 12 exons with full genomic length of 4300 bp. All the 324 SNPs between male and hermaphrodite varieties are in the 11th intron, which spans 8.5 kb. Quantitative RT-PCR revealed that CpHUA1 expression is highly elevated in carpels, suggesting that CpHUA1 may be involved in sex differentiation gene network. Southern blot analysis revealed a distinct restriction pattern in male AU9 compared to hermaphrodite Kapoho and SunUp, despite high DNA sequence identity and sharing of all but two EcoR I restriction sites in genomic CpHUA1 sequences of AU9 and SunUp. The methylation of cytosine at one restriction site in male but not in other two sex types may result in distinct restriction pattern of EcoR I in southern blot result. Bisulfite sequencing showed differential methylation of CpHUA1 among sex types, particularly the enrichment of sex-specific methylation in 9th and 11th intron. The methylation difference in cold stress induced male to hermaphrodite mutant mostly observed in the CHH context of CpHUA1, but no methylation difference detected in CHH context in other sex types, which may indicate the role of methylation in CHH context of CpHUA1 in temperature-related stress response and sex reversal.

The reproductive biology of two poorly known relatives of the fig (Ficus ) and insights into the evolution of the fig syconium

We conducted the first detailed investigation of the floral architecture and reproductive biology of two species from the genus Dorstenia, which are poorly known relatives of Ficus (Moraceae). Our aims were to extend and refine knowledge of the understudied genus Dorstenia and to explore possible insights into the evolution of the fig syconium. We characterised four key stages of floral development using light microscopy, scanning electron microscopy and histological staining. Reproductive biology was found to be complex and species-specific. Both study species are monoecious and produce an inflorescence of minute male and female flowers. Protogyny, associated with a spatial separation of male and female flowers and asynchronous stamen development, was species-specific, as was seed set. Our results reveal novel insights into the complex reproductive biology of an under-studied genus in the family Moraceae. We propose that exploring the reproductive biology of Dorstenia and other poorly known Ficus relatives will provide insights into the evolution of the fig syconium – the unique reproductive structure of this economically and ecologically important genus.

Functional Characterization of the Promoter and Second Intron of CUM1 During Flower Development in Cucumber (Cucumis sativus L.)

The characterization of flower specific promoter is critical during flower development by cucumber transformation technology. AGAMOUS (AG) is an organ identity gene that is required for carpel and stamen development in Arabidopsis. The promoter and second intron of AG contain multiple regulatory elements that confer proper spatial and temporal expression. Cucumber is an important vegetable with unisexual flowers. Cucumber MADS-box 1 (CUM1) is the AG homolog in cucumber, belonging to the euAG lineage along with AG. In situ hybridization showed that CUM1 was specifically expressed in the stamens and carpels of cucumber. GUS staining indicated that the second intron of CUM1 confers stamen-specific expression, while the promoter of CUM1 drives both stamen- and carpel-specific expression during the early stages of flower development, but is restricted to carpel- and connectivum-specific expression during the late stages of flower development. Furthermore, a yeast one-hybrid assay demonstrated that two auxin response factors (CsARF13 and CsARF17) had bound directly to the second intron of CUM1. Our data suggest that different regulatory circuits operate in AG homologs in plant species with distinct sex types.

Plant Glycine-Rich Proteins in Stress Response: An Emerging, Still Prospective Story.

Seed plants are sessile organisms that have developed a plethora of strategies for sensing, avoiding, and responding to stress. Several proteins, including the glycine-rich protein (GRP) superfamily, are involved in cellular stress responses and signaling. GRPs are characterized by high glycine content and the presence of conserved segments including glycine-containing structural motifs composed of repetitive amino acid residues. The general structure of this superfamily facilitates division of GRPs into five main subclasses. Although the participation of GRPs in plant stress response has been indicated in numerous model and non-model plant species, relatively little is known about the key physiological processes and molecular mechanisms in which those proteins are engaged. Class I, II, and IV members are known to be involved in hormone signaling, stress acclimation, and floral development, and are crucial for regulation of plant cells growth. GRPs of class IV [RNA-binding proteins (RBPs)] are involved in alternative splicing or regulation of transcription and stomatal movement, seed, pollen, and stamen development; their accumulation is regulated by the circadian clock. Owing to the fact that the overexpression of GRPs can confer tolerance to stress (e.g., some are involved in cold acclimation and may improve growth at low temperatures), these proteins could play a promising role in agriculture through plant genetic engineering. Consequently, isolation, cloning, characterization, and functional validation of novel GRPs expressed in response to the diverse stress conditions are expected to be growing areas of research in the coming years. According to our knowledge, this is the first comprehensive review on participation of plant GRPs in the response to diverse stress stimuli.

Characterization of the ZmbHLH122 transcription factor and its potential collaborators in maize male reproduction

OsEAT1 (DTD), a stamen-specific bHLH transcription factor, is a master regulator in rice male reproduction. To elucidate its functional mechanism in maize male reproduction, the maize orthologue ZmbHLH122 of OsEAT1 was isolated in this study. Sequence analysis revealed that the ZmbHLH122 coding sequence consists of 1422 bp which encode a protein of 473 amino acids. Bioinformatic analysis confirmed that ZmbHLH122 belongs to the bHLH TF family. The subcellular localization of ZmbHLH122-eGFP in rice protoplasts showed that ZmbHLH122 is a nuclear-localized protein. Yeast one-hybrid assays demonstrated that ZmbHLH122 exhibits weak transactivation ability. Genome-wide coexpression analysis identified 751 potential collaborators of ZmbHLH122, most of which are highly expressed in maize anthers and tassels, and sequence alignment analysis revealed that 43 coexpressed genes are homologous to Arabidopsis male fertility-related genes. Expression pattern analysis confirmed that ZmbHLH122 and its potential collaborators Ms23 (ZmbHLH16), ZmbHLH51, ZmbHLH66 (Ms32) and ZmMYB74 are preferentially expressed in the maize male reproductive organs, especially those at the uninucleate and early binucleate stages. Yeast two-hybrid assays revealed that ZmMYB74 directly interacts with two maize stamen development regulators, Ms23 (ZmbHLH16) and ZmbHLH51, and indirectly collaborates with ZmbHLH122. Our study lays the foundation for further understanding of the ZmbHLH122 molecular function in maize male reproduction.

Cyathia of Euphorbia spathulata (Euphorbiaceae) Possess Both Cleistogamous and Chasmogamous Modes of Pollination

ABSTRACT Cyathium morphology of Euphorbia spathulata from three populations in central Virginia was studied from early developmental stages through nominal anthesis, defined by exposure of the cyathium to the external environment by divergence of paired subtending cyathophylls. Observations were made via dissecting microscope, compound light microscope sections, and scanning electron microscopy. The plants studied revealed a consistent pattern of asynchronous stamen and pollen development as well as a series of predictable changes in style and stigma orientation. Initially, styles and stigmas of pistillate flowers maintain a near-axial (erect) position. Later, when still enclosed by subtending cyathophylls, styles reflex, placing stigmas near early developing anthers shedding pollen. Pollen grains observed on stigmas at this stage indicate cleistogamous pollination. Eventually, paired cyathophylls diverge, exposing the cyathium to the external environment; at this stage, styles and stigmas assume an asce...

Mapping and Identifying a Candidate Gene (Bnmfs) for Female-Male Sterility through Whole-Genome Resequencing and RNA-Seq in Rapeseed (Brassica napus L.).

In oilseed crops, carpel and stamen development play vital roles in pollination and rapeseed yield, but the genetic mechanisms underlying carpel and stamen development remain unclear. Herein, a male- and female-sterile mutant was obtained in offspring of a (Brassica napus cv. Qingyou 14) × (Qingyou 14 × B. rapa landrace Dahuang) cross. Subsequently, F2–F9 populations were generated through selfing of the heterozygote plants among the progeny of each generation. The male- and female-sterility exhibited stable inheritance in successive generations and was controlled by a recessive gene. The mutant kept the same chromosome number (2n = 38) as B. napus parent but showed abnormal meiosis for male and female. One candidate gene for the sterility was identified by simple sequence repeat (SSR) and insertion deletion length polymorphism (InDel) markers in F7–F9 plants, and whole-genome resequencing with F8 pools and RNA sequencing with F9 pools. Whole-genome resequencing found three candidate intervals (35.40–35.68, 35.74–35.75, and 45.34–46.45 Mb) on chromosome C3 in B. napus and candidate region for Bnmfs was narrowed to approximately 1.11-Mb (45.34–46.45 M) by combining SSR and InDel marker analyses with whole-genome resequencing. From transcriptome profiling in 0–2 mm buds, all of the genes in the candidate interval were detected, and only two genes with significant differences (BnaC03g56670D and BnaC03g56870D) were revealed. BnaC03g56870D was a candidate gene that shared homology with the CYP86C4 gene of Arabidopsis thaliana. Quantitative reverse transcription (qRT)-PCR analysis showed that Bnmfs primarily functioned in flower buds. Thus, sequencing and expression analyses provided evidence that BnaC03g56870D was the candidate gene for male and female sterility in the B. napus mutant.

De novo transcriptome assembly from flower buds of dioecious, gynomonoecious and chemically masculinized female Coccinia grandis reveals genes associated with sex expression and modification

BackgroundCoccinia grandis (ivy gourd), is a dioecious member of Cucurbitaceae having heteromorphic sex chromosomes. Chromosome constitution of male and female plants of C. grandis is 22A + XY and 22A + XX respectively. Earlier we showed that a unique gynomonoecious form of C. grandis (22A + XX) also exists in nature bearing morphologically hermaphrodite flowers (GyM-H). Additionally, application of silver nitrate (AgNO3) on female plants induces stamen development leading to the formation of morphologically hermaphrodite flowers (Ag-H) despite the absence of Y-chromosome. Due to the unavailability of genome sequence and the slow pace at which sex-linked genes are identified, sex expression and modification in C. grandis are not well understood.ResultsWe have carried out a comprehensive RNA-Seq study from early-staged male, female, GyM-H, and Ag-H as well as middle-staged male and GyM-H flower buds. A de novo transcriptome was assembled using Trinity and annotated by BLAST2GO and Trinotate pipelines. The assembled transcriptome consisted of 467,233 ‘Trinity Transcripts’ clustering into 378,860 ‘Trinity Genes’. Female_Early_vs_Male_Early, Ag_Early_vs_Female_Early, and GyM-H_Middle_vs_Male_Middle comparisons exhibited 35,694, 3574, and 14,954 differentially expressed transcripts respectively. Further, qRT-PCR analysis of selected candidate genes validated digital gene expression profiling results. Interestingly, ethylene response-related genes were found to be upregulated in female buds compared to male buds. Also, we observed that AgNO3 treatment suppressed ethylene responses in Ag-H flowers by downregulation of ethylene-responsive transcription factors leading to stamen development. Further, GO terms related to stamen development were enriched in early-staged male, GyM-H, and Ag-H buds compared to female buds supporting the fact that stamen growth gets arrested in female flowers.ConclusionsSuppression of ethylene responses in both male and Ag-H compared to female buds suggests a probable role of ethylene in stamen suppression similar to monoecious cucurbits such as melon and cucumber. Also, pollen fertility associated GO terms were depleted in middle-staged GyM-H buds compared to male buds indicating the necessity of Y-chromosome for pollen fertility. Overall, this study would enable identification of new sex-biased genes for further investigation of stamen arrest, pollen fertility, and AgNO3-mediated sex modification.

Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development.

This study aimed at elucidating regulatory components behind floral organ identity determination and tissue development. It remains unclear how organ identity proteins facilitate development of organ primordia into tissues with a determined identity, even though it has long been accepted that floral organ identity is genetically determined by interaction of identity genes according to the ABC model. Using the chromatin immunoprecipitation sequencing technique, we identified OsTGA10, encoding a bZIP transcription factor, as a target of the MADS box protein OsMADS8, which is annotated as an E-class organ identity protein. We characterized the function of OsTGA10 using genetic and molecular analyses. OsTGA10 was preferentially expressed during stamen development, and mutation of OsTGA10 resulted in male sterility. OsTGA10 was required for tapetum development and functioned by interacting with known tapetum genes. In addition, in ostga10 stamens, the hallmark cell wall thickening of the endothecium was defective. Our findings suggest that OsTGA10 plays a mediator role between organ identity determination and tapetum development in rice stamen development, between tapetum development and microspore development, and between various regulatory components required for tapetum development. Furthermore, the defective endothecium in ostga10 implies that cell wall thickening of endothecium is dependent on tapetum development.

Identification and expression profiling of microRNAs involved in the stigma exsertion under high-temperature stress in tomato

BackgroundAutogamy in cultivated tomato varieties is a derived trait from wild type tomato plants, which are mostly allogamous. However, environmental stresses can cause morphological defects in tomato flowers and hinder autogamy. Under elevated temperatures, tomato plants usually exhibit the phenotype of stigma exsertion, with severely hindered self-pollination and fruit setting, whereas the inherent mechanism of stigma exsertion have been hitherto unknown. Numerous small RNAs (sRNAs) have been shown to play significant roles in plant development and stress responses, however, none of them have been studied with respect to stamen and pistil development under high-temperature conditions. We investigated the associations between stigma exsertion and small RNAs using high-throughput sequencing technology and molecular biology approaches.ResultsSixteen sRNA libraries of Micro-Tom were constructed from plants stamen and pistil samples and sequenced after 2 d and 12 d of exposure to heat stress, respectively, from which a total of 110 known and 84 novel miRNAs were identified. Under heat stress conditions, 34 known and 35 novel miRNAs were differentially expressed in stamens, and 20 known and 10 novel miRNAs were differentially expressed in pistils. GO and KEGG pathway analysis showed that the predicted target genes of differentially expressed miRNAs were significantly enriched in metabolic pathways in both stamen and pistil libraries. Potential miRNA-target cleavage cascades that correlated with the regulation of stigma exsertion under heat stress conditions were found and validated through qRT-PCR and RLM-5′ RACE.ConclusionOverall, a global spectrum of known and novel miRNAs involved in tomato stigma exsertion and induced by high temperatures were identified using high-throughput sequencing and molecular biology approaches, laying a foundation for revealing the miRNA-mediated regulatory network involved in the development of tomato stamens and pistils under high-temperature conditions.

Decreased sorbitol synthesis leads to abnormal stamen development and reduced pollen tube growth via an MYB transcription factor, MdMYB39L, in apple (Malus domestica).

Sugars produced by photosynthesis not only fuel plant growth and development, but may also act as signals to regulate plant growth and development. This work focuses on the role of sorbitol, a sugar alcohol, in flower development and pollen tube growth of apple (Malus domestica). Transgenic 'Greensleeves' apple trees with decreased sorbitol synthesis had abnormal stamen development, a decreased pollen germination rate and reduced pollen tube growth, which were all closely related to lower sorbitol concentrations in stamens. RNA sequencing and quantitative RT-PCR analyses identified reduced transcript levels during stamen development and pollen tube growth in the transgenic trees of a stamen-specific MYB39-like transcription factor, MdMYB39L, and of its putative target genes involved in hexose uptake, cell wall formation and microsporogenesis. Suppressing MdMYB39L expression in pollen via antisense oligonucleotide transfection significantly reduced the expression of its putative target genes and pollen tube growth. Exogenous sorbitol application during flower development partially restored MdMYB39L expression, stamen development, and pollen germination and tube growth of the transgenic trees. Addition of sorbitol to the germination medium also partially restored pollen germination and tube growth of the transgenic trees. We conclude that sorbitol plays an essential role in stamen development and pollen tube growth via MdMYB39L in apple.

MiRNAs involved in the development and differentiation of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri

BackgroundSterile and fertile flowers are important evolutionary developmental phenotypes in angiosperm flowers. The development of floral organs, critical in angiosperm reproduction, is regulated by microRNAs (miRNAs). However, the mechanisms underpinning the miRNA regulation of the differentiation and development of sterile and fertile flowers remain unclear.ResultsHere, based on investigations of the morphological differences between fertile and sterile flowers, we used high-throughput sequencing to characterize the miRNAs in the differentiated floral organs of Viburnum macrocephalum f. keteleeri. We identified 49 known miRNAs and 67 novel miRNAs by small RNA (sRNA) sequencing and bioinformatics analysis, and 17 of these known and novel miRNA precursors were validated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, by comparing the sequencing results of two sRNA libraries, we found that 30 known and 39 novel miRNA sequences were differentially expressed, and 35 were upregulated and 34 downregulated in sterile compared with fertile flowers. Combined with their predicted targets, the potential roles of miRNAs in V. macrocephalum f. keteleeri flowers include involvement in floral organogenesis, cell proliferation, hormonal pathways, and stress responses. miRNA precursors and targets were further validated by quantitative real-time PCR (qRT-PCR). Specifically, miR156a-5p, miR156g, and miR156j expression levels were significantly higher in fertile flowers than in sterile flowers, while SPL genes displayed the opposite expression pattern. Considering that the targets of miR156 are predicted to be SPL genes, we propose that miR156 may be involved in the regulation of stamen development in V. macrocephalum f. keteleeri.ConclusionsWe identified miRNAs differentially expressed between fertile and sterile flowers in V. macrocephalum f. keteleeri and provided new insights into the important regulatory roles of miRNAs in the differentiation and development of fertile and sterile flowers.

Arabidopsis MYB24 Regulates Jasmonate-Mediated Stamen Development.

The phytohormone jasmonates (JAs) regulate various defense responses and diverse developmental processes including stamen development and fertility. Previous studies showed that JA induces CORONATINE INSENSITIVE 1-mediated degradation of JA ZIM-domain (JAZ) proteins, and activates the MYB transcription factors (such as MYB21 and MYB24) to regulate stamen development. In this study, we further uncover the mechanism underlying how MYB24 interacts with JAZs to control JA-regulated stamen development. We show that N-terminus of MYB21/24 interacts with 10 out of 12 JAZ proteins while both N-terminus and C-terminus of MYB24 are involved in dimerization of MYB21 and MYB24. Interestingly, male sterility of the JA-deficient mutant opr3 can be rescued by suitable level of the MYB24 overexpression but not by excessive high level of MYB24. Surprisingly, overexpression of MYB24NT, but not MYB24CT, could cause male sterility. These results provide new insights on MYB factors in JA-regulated stamen development.