Pecan Flowering Mechanism

Process of flowering is very vital in lifetime of plants, it is also the turning point that plants development from vegetation to generation, and from sporophyte to gametophyte. Many researchers have worked on the mechanism of flowering for around a century.Hickory(Carya cathayensis Sarg., Carya Nutt., Juglandaceae) is one of important economic trees, which is also one of very important oil tree plants and has become one of the resource of income for indigene in Zhejiang Province of China.Hickory has a long vegetative period, and it needs nearly 10 years for the first fruit set. The flowers of hickory are monoecism. Its staminate flowers are catkin, and female flowers spica. Flowering (staminate flowers and female flowers) of hickory is different in space and time. Because of the importance of flowering for female flowers, which decides to the yield, more attention should be paid to the flowering mechanism.Olefin slices about different period of time of flowering were observed through scanning electron microscope for the scale of studying the development of staminate flowers and female flowers. The technique of cDNA-AFLP and genechip were exerted to study the network control system of flowering. The important homologous genes on flowering, FT, AG, AP1 and so on, were cloned through the technique of PCR; The mechanism of flowering was studied from the point of view of anatomy and molecular biology. And also flowering control was studied with the use of the means of pulling branches and spraying plant growth regulators. The results were as follows:(1) The formation and development of female and male flower of hickory was mastered in anatomy. Androecium anlages form in the top of male flower anlages in the last ten-day of March. Stamens come into being and farther differentiate in the middle ten-day of April. In the last ten-day of April, pollen sacs forms from anther and microspore mother cells forms into tetrad spores with miosis. Then, tetrad spores disaggregate and produce one-cell microspores. In the first ten-day of May, two-cell microspores come into being. The development of female flower mainly accomplish from the last ten-day of March to the middle ten-day of May. The course can divided into five periods: the period of undifferentiation, pre-differentiation, inflorescence differentiation, involucrum formation, and gynoecium formation. In the early days of differentiation, the growing point goes semi-circle. In the period of inflorescence differentiation, three flowerets anlages come into being. In the period of involucrum formation, one involucrum and three bracts forms around the floweret. In the period of gynoecium formation, the flowerets continue to develop, and the embryo-sac comes into being in this period. The ovule type of hickory is orthogropous with single integument and thick nucellus. The female gametophyte is an embryo-sac with eight-nucleolus or seven-cell.(2) 278 TDFs during flowering in hickory were obtained by cDNA-AFLP technology. The 278 genes were composed of 65 genes of unknown function and 213 genes of known function. The 213 genes of known functions were involved in cell signal transduction, cell growth, senescence and death, cell differentiation and plant mature, plant hormone synthensis, stress and resistance, material transport, photosynthesis, material and energy metabolism, mineral assimilation and organic synthesis, photomorphogenesis, which play a direct or indirect role in hickory flowering process. The expression patterns of these genes were confirmed by Real-time RT PCR analysis. Both are basically consistent, which confirms the reliability of the results of cDNA-AFLP. There are 213(76.6%) annotated TDFs and 65(23.4%) non-annotated ones. Of annotated TDFs, the number or percentage of Biological Process, Cellular Component ,and Molecular Function are 158(54.7%), 211(73.0%), 173(59.9%), respectively. Of biological process, the turn of percentage in descending order is oxidation reduction, translation, proteolysis, photosynthesis, response to UV-B, negative regulation, transport, etc. Of cellular component, the turn of percentage in descending order is chloroplast, integral to membrane, thylakoid, mitochondrion, plasma membrane, etc. Of molecular function, the turn of percentage in descending order is ATP binding, protein binding, serine-type endopeptidase, etc.(3) The expression patterns of total 232 genes during hickory flowering process were obtained by microarry, which accounted for about 1% of total genes. 196 genes with at least 2-fold changes were obtained, which accounted for about 0.9% of total genes. Among them, there were 106 upregulated genes and 90 downregulated genes. The genes during flowering in hickory were invloved in cell wall synthesis, cell cycle regulation, cell signal transduction, stress response, photosynthetic metabolism, lipid metabolism, protein metabolism, carbohydrate metabolism, transcription regulation, apoptosis, hormone synthesis and transport, nucleic acid metabolism, material transport, amino acid metabolism, etc., which confirmed in molecular level the modern flowering theory of cell biology.(4) We designed the special primers according to the conserved sequence of the known FT, AG and AP1 homologues and cloned four FT, two AG and one AP1 fragments, respectively. The length of the four FT sequences are 382bp, 82bp, 80bp and 152bp respectively. The 152bp sequence (GenBank accession number: FJ858260.1) named CcFT, which included the third and fourth exons. The length of two AG homologues fragments are 131bp and 122bp (GenBank accession number: FJ858261.1) named CcAG. One AP1 homologue fragments (GenBank accession number: EU155118) were cloned and named CcAP1, which included two introns (86bp and 291bp).(5) The gene expression of CcLFY in different position in hichory are different. The highest expression was noticed in blossom buds, followed by young stems and young leaves, and the lowest expression was in roots. In the flowering process, CcLFY expression generally was a low expression in early March, a peak in March 18, followed by a decline as time progressed.(6) Both hygromycin assays and PCR amplification demonstrated that CcLFY was integrated into T0 genomes. The Morphological observation showed that the transgenic tobacco plants that survived under natural condition were tall, early flowering and seed grain larger than the wild-type controls. (7) 876,664 reads are derived by 454 sequencing from two samples labeled as A and B in hickory. There are 431,759 and 444,905 reads in sample A and B, respectively. After sequence aligning, there are 25,339 and 26,935 contigs in sample A and B. And 15,085 and 16,387 contigs are derived fromsample A and B, respectively. The number of specific contigs in sample A and B are 6,138 and 7,346. The number of common contigs in the two samples are 19,201 and 19,589, respectively. And all contigs number in both samples is 32,685. Seven sets, A, B, A+B, AU B, AI B, B , A , are annotated. For example, there are 31,179(95.4%) annotated contigs and 1,506(4.6%) non-annotated ones in set AU B. For annotated contigs, there are 3 type named Biological Process, Cellular Component ,and Molecular Function which contig number or percentage are 22,515(68.9%), 21,788(66.7%), 24,037(73.5%), respectively. The rest 6 sets are similar to AU B. Pathway analysis are implemented for B , A , AU B, AI B sets. There are 5~8% contigs have pathways, in which Ncotinate and nicotinamide metabolism, oxidative phosphorylation, steroid biosynthesis have higher percentages than others, comparing to KEGG Arabidopsis thaliana database. And there are around 8~13% contigs have pathways, in which purine and pyrimidine metabolism have higher percentages than others, comparing to KEGG Populus trichocarpadatabase. Several known flowering candidate genes are derived, such as GI, AP1, AP2, EMF1, TFL2, EMF2, FLC, PAF1, DET1, FRI, PLE, PI, HD1, AGL24, VRN1, SOB3, CRY1, FCA, FKF1, PIE, PIE1, AGL75. It's suggested that there are several flowering routes in hickory. Eventually, 792 (3.1%) and 783 SSRs (2.9%) are derived from sample A and B, respectively.