Genetic Regulation Of Floral Development In Populus Tomentosa

Populus tomentosa, native to China, occupies a large area in the northern, central and on both sides of the Yellow River, and plays a very important role in maintaining ecological stability in these areas. In this thesis, Affymetrix GeneChip technology was used to identify differently expressed genes between female and male flowers. To eliminate the confounding effects of genetic background of dioecious plants, three andromonoecious poplar, an exceptional model system, were used in transcriptome analysis. Subsequently, to confirm that microarray results and the expression patterns of candidate genes with significant transcriptional changes, nine time points of floral buds that cover all stages of floral development were collected from normal poplar (5females and5males) for qRT-PCR analysis. Moreover, four phytohormone (GA, CTK, IAA and ABA) and DNA methylation level were detected in flower development progress. These experiments aimed to test the hypothesis that there are significant differences in the transcriptome, phytohormone content and DNA methylation level in female and male flower development. We expect that the knowledge about the interactions of differently expressed gene, phytohormone signaling, and DNA methylation level will help us understand molecular underpinnings of sexual differences in dioecious plant floral development. To identity sex-related physiological changes and gene expression differences during temperature transitions, morphological characteristics (dry matter, length, and width), antioxidant defense systems (SOD, POD, and CAT), biochemical characteristics (MDA), calcium ion analysis, soluble sugars, soluble protein and also related gene expression were compared between male and female individuals during floral bud development. The main results of research were as follow:1. The genetic diversity of natural population of P.tomentosa was studied at the phenotypic level, and these studies indicated that variation of flower phenotypic traits of P. tomentosa is significant different among provenances. The average of phenotypic variation coeffcients of male flower is11.62%. Coefficient of variation for the beginning of bloom (19.25%) was the highest and the later stage of inflorescence abscission (5.86%) was the lowest. The average of phenotypic variation coeffcients of female flower is13.3%. Coefficient of variation for the earlier stage of beginning of pollination (37.24%) was the highest and the later stage of boom (5.21%) was the lowest. The male and female flower phenotypic differentiation coefficient ranged from8.36%to20.07%and2.52%to26.43%, respectively. The average of phenotypic differentiation coefficient is0.1487; variation (85.13%) among provenances is higher than variation (14.87%) among clones within provenances, which indicates that clones within provenance, is the major source of genetic diversity of P. tomentosa.2. Dioecious plants have evolved sex-specific floral development mechanisms. However, precise gene expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. Comparative transcriptome and physiological analysis, allowed us to characterize sex-specific floral development of female and male flowers. Transcriptome analysis showed that significantly differentially expressed genes between the sexes, including genes related to floral development, phytohormone synthesis and metabolism, and DNA methylation regulation. Correlation analysis revealed a significant correlation between phytohormone signaling and gene expression, identifying specific phytohormone-responsive genes and their promoter cis-regulatory elements. Two genes related to DNA methylation, MET1and DDM1which located in the sex determination region of Chromosome XIX, expressed differently between female and male flowers. A time-course analysis of MET1and DDM1expression and DNA methylation levels revealed that MET1and DDM1expression result in different DNA methylation levels between female and male flower. Understanding the interactions of phytohormone signaling, DNA methylation and target gene expression should lead to a better understanding of sexual differences in floral development. The long-term goal of this study is to provide a series of candidate genes for poplar sexual dimorphism studies in further.3. Although the molecular basis of poplar sex-specific flower development remains largely unknown, increasing evidence indicates an essential role for microRNAs (miRNAs). The specific miRNA types and precise miRNA expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. This system, combined with high-throughput sequencing and computational analysis, allowed us to characterize sex-specific miRNAomes from female and male flowers. Comparative miRNAome analysis combined with quantitative real-time PCR revealed the expression patterns of27miRNAs in poplar flower and showed that the targets of these miRNAs are involved in flower organogenesis, Ca2+transport, phytohormone synthesis and metabolism and DNA methylation. This paper describes a complex regulatory network consisting of these miRNAs expressed in sex-specific flower development in a dioecious plant. The conserved and novel miRNA locations were annotated in the P. trichocarpa genome. Among these, miRNA Pto-F70and4targets are located in the sex-determination regions of chromosome XIX. Furthermore, two novel miRNAs, Pto-F47and Pto-F68, were shown for the first time to be regulatory factors in phytohormone interactions. To our knowledge, this report is the first systematic investigation of sex-specific flower-related miRNAs and their targets in poplar, and it deepens our understanding of the important regulatory functions of miRNAs in female and male flower development in this dioecious plant.4. The andromonoecious poplar is an exceptional model system for studying sex-specific flower development in dioecious plants. There is increasing evidence that epigenetic regulation, particularly DNA methylation, is an important regulatory factor during flower development. Here, methylation sensitive amplified polymorphism (MSAP) was used to screen for sex-specific DNA methylation alterations in the andromonoecious poplar. The sequences of27sex-specific amplified fragments were obtained from DNA prepared from sex-specific flower tissues. PtGT2, PtPAL3, and PtCER4, which are homologous to MF26, MF29, and MF35, respectively, were cloned as candidate genes. Expression analysis and DNA methylation pattern profiling of the three candidate genes revealed that gene expression upregulation was always associated with gene body methylation. The results suggested that DNA methylation sites have the potential to regulate the genes’transcript levels. These three genes were shown to play important roles during different phases of flower development. This study will help to provide candidates for future experiments aimed at understanding the mechanism whereby DNA methylation regulates gene expression in poplar.5. The productivity, distribution and population structure of poplar is affected by temperature transitions. Poplar floral buds develop in a fluctuating environment and the molecular basis of temperature-dependent flowering regulation has been extensively studied, but little is known about how sex-specific floral bud development responds to temperature transitions. Here, morphological observations indicated those floral bud growth rates were affected by maximum and minimum temperature of natural environment at the later stages of enlargement (stage4) and later stage of dormancy (stage8) respectively. Physiological, biochemical and gene expression analysis in floral development process and temperature treatment (heat and chilling stress) were investigated. The result show that male floral buds suffered from greater negative effects than did female under temperature treatment. Water culture experiments revealed that temperature treatment significantly increased superoxide dismutase (SOD), Peroxidase (POD) and Catalase (CAT) activities and transcription of related gene in female floral buds, whereas malonaldehyde (MDA) significantly increased only in male. Soluble sugar and protein were increased both in female and male floral buds and higher in male floral buds. Temperature treatment also caused Ca2+content and transcription of gene related with calcium transports significantly increased in female flowers. These results revealed that sex-specific floral development response to seasonal temperature transitions suggesting that female floral buds posses a better environment adaption than do males in P. tomentosa.6. The molecular basis of SUPERMAN (SUP) regulation during floral development in monoecious plants has been extensively studied, but little is known of SUP gene family in dioecious woody plants. In this thesis, we systematically examined the diversification of the SUP gene family in Populus by integrating genomic organization, expression, and phylogeny data. SUP family members showed sex-specific expression throughout flower development. Transcript profiling of rare gynomonoecious poplar flowers revealed that a significant reduction in PtoSUPI mRNA might be important for stamen development in gynomonoecious poplar flowers. We found that the coding regions of Populus SUP genes are very highly conserved and that synonymous sites in exon regions have undergone strong purifying selection during SUP evolution in Populus. These results indicate that SUP genes play an important role in floral development of dioecious plants. Expression analysis of SUP suggested possible regulatory mechanisms for gynomonoecious poplar flower development. These findings provide an important insight into the mechanisms of the evolution of SUP function and may help enable future engineered regulation of flower development in trees for breeding requirements.