Published online Jul Received May 17; Accepted Jul 7. The use, distribution or reproduction in other forums is permitted, provided the original author s or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these terms. Abstract Vernicia fordii is a monoecious and diclinous species with male and female flowers on the same inflorescence.
Low female to male flower ratio is one of the main reasons for low yield in this species. However, little is known of its floral development and sex determination. Here, according to the results of scanning electron microscopy and histological analysis, the floral development of V. The male flowers are always unisexual, but the female flowers present bisexual characteristics, with sterile stamen staminode restricted to pre-meiosis of mother sporogenous cells and cell death occurring at later development stages.
To further elucidate the molecular mechanism underling sex determination at the divergence stage for male and female flowers, comparative transcriptome analysis was performed. In total, 56, unigenes were generated and genes were differentially expressed between male and female flowers, among which and DEGs differentially expressed genes showed high expression levels in males and females, respectively.
The transcriptome data showed that the sexual dimorphism of female flowers was affected by jasmonic acid, transcription factors, and some genes related to the floral meristem activity. In this study, we provide developmental characterization and transcriptomic information for better understanding of the development of unisexual flowers and the regulatory networks underlying the mechanism of sex determination in V.
Approximately half of these unisexual plants are monoecious species with separate male and female flowers on the same plant, such as maize Zea mays , cucumber Cucumis sativus , and melon Cucumis melo.
The other half are dioecious with male and female flowers on separate plants, such as white campion Silene latifolia , papaya Carica papaya , hemp Cannabis sativa , and annual mercury Mercurialis annua.
In flowering plant, sex determination is a developmental process that facilitates allogamy to support fitness and survival. In unisexual flowers, the process of sex determination is performed by the selective abortion or arrest of either male or female reproductive organs. The mechanisms of sex determination have been investigated in some model plants, such as maize and cucumber, and are mainly affected by genetics, plant hormones and environmental factors Bai and Xu, ; Li and Liu, For dioecious species, sex determination genes are often located in non-recombining regions of the sex chromosome Zhang et al.
In white campion, the Y chromosome is the largest chromosome in the genome and also the primary factor that determines floral sexuality, with at least one region suppressing female expression and another promoting male development Juarez and Banks, ; Zhang et al.
In papaya, sex determination is controlled by two types of Y chromosomes that control the male Y and the hermaphrodite Yh Ming et al. Sex determination genes have been identified and investigated in maize and cucurbits to improve the understanding of the regulatory network for sex determination. A sex determination model for melon was recently proposed in which the expression of CmWIP1, required for carpel abortion in the male flower, is dependent on the non-expression of CmACS It is known that plant hormones regulate plant growth and development and also affect sex differentiation in some monoecious and dioecious plants.
Ethylene is the primary hormone promoting female flower development in melon and cucumber, whereas gibberellins have opposite effects in these plants Yamasaki et al. In maize, gibberellins induce feminization while jasmonic acid and brassinosteroids induce masculinization Yamasaki et al.
Environmental factors also affect sex determination in many species, such as high temperature and long-day conditions, which can promote male flowers, whereas low temperature and short-day conditions induce feminization in cucumber Yamasaki et al. For the formation of unisexual flowers, the developmental fate of inappropriate sex organs is determined at different developmental stages in different plants Diggle et al. There are four stages of inappropriate sex organ abortion: In maize, the formation of unisexual flowers results in the abortion of pistil primordia in the tassel silkelets and the secondary florets of ear spikelets, mediated by programmed cell death PCD , and the arrest of stamen primordia in ear spikelets, which involves repression of the cell cycle Calderon-Urrea and Dellaporta, ; Kim et al.
In cucumber, the arrest of the stamen in female flowers is mainly restricted to the anther at stage 7 with DNA damage, which can be specifically detected by TUNEL assay. Moreover, the vestiges of a stamen and carpel are left in the female and male flowers respectively and remain metabolically active Hao et al. In contrast, hemp and annual mercury lack any vestiges of inappropriate sex organs and present as completely unisexual Diggle et al. Taken together, the transition from bisexual to unisexual features may occur in any cell specification stage of floral development with different processes for each diclinous species.
Vernicia fordii tung tree is a perennial deciduous woody oil plant in subtropical and tropical areas with tremendous potential as raw material to produce biodiesel Chang et al.
In recent years, the tung tree has been highlighted as an important woody bioenergy plant in China for resolving the increasing energy crisis.
Tung trees are monoecious and predominantly synoecious plants, the flowers of which are developed in panicled cymose inflorescences that are terminal and solitary on new branches. The number of staminate flowers greatly exceeds that of the pistillate flowers and the average ratio of female to male flowers is 1: Therefore, it is important to investigate the flower development and sex determination processes in the tung tree for further alteration of the sex ratio and to increase in the yield.
However, few studies are available on floral development in the tung tree Abbott, ; McCann, The mechanisms underlying the transition from a bisexual to a unisexual flower and floral organ development are clear in some species, such as Phoenix dactylifera Daher et al. In this paper, we report a detailed morphogenetic and transcriptomic analysis of floral development in V.
The male flowers are always unisexual, but the female flowers present bisexual characteristics with sterile stamen staminode restricted to the anther during the early development stages, and while cell death occurs in later development stages.
Comparative transcriptome sequencing of the male and female flowers was carried out to better understand the molecular regulatory mechanism governing unisexual flower development in V.
The terminal buds were randomly collected every week at various growth stages, from April to April However, the samples were collected every 3—4 days at the beginning of the reproductive phase in July and every 2 weeks during the phase of reproductive dormancy from November to January Then the samples were dehydrated by sequentially immersing through graded ethanol: The following day, the dehydrated materials were incubated in the ethanol gradually with xylene as follows: Samples were longitudinally and transversely sectioned into pieces with 8 um thickness.
The sections were deparaffinized 15 min in xylene two times and washed in water. After that, sections were dehydrated for 2 min each through the following ethanol series: Subsequently, the slides were dipped 5 s in a mixture of methyl salicylate and xylene 1: Finally, the sections were mounted and covered with glass coverslip. Scanning electron microscopy The fixed and dissected materials were dehydrated by passing them through ethanol gradually with an acetone series: All materials were sputter-coated with gold Hitachi E, Japan and examined with an S scanning electron microscope running at an accelerating voltage of 3 kV Hitachi, Japan.
Data were analyzed statistically using the Origin 8. Sections were rehydrated through a graded ethanol series: Afterward, sections were immersed in 0. Then, sections were incubated in a 0. The samples were observed and photographed using a BX53 microscope Olympus, Japan. Slides were examined and photographed under a BX53 microscope using blue and green excitation to visualize the green TUNEL fluorescence and the red PI fluorescence, respectively.
RNA extraction and sequencing Male and female flower buds at developmental stage 7 were identified under the SZX10 stereo microscope Olympus, Japan and separately collected from the same plant.
Three biological replicates were performed for each sample and 30 flowers counted as one sample. All sequencing data of male and female flower buds in V. Annotation and gene ontology enrichment analysis To annotate the unigenes, the BlastX program was used to search against the Arabidopsis thaliana protein sequence data set TAIR 10, http: To further investigate the functions of DEGs, GO enrichment analysis was performed using OmicShare tools, a free online platform for data analysis http: Gene numbers were calculated for every term and significantly enriched GO terms in the DEGs compared to the genome background were defined with a hypergeometric test.
This analysis may be able to recognize the main biological functions of the DEGs.