Research On Physiological And Molecular Mechanisms Of Somatic Embryogenesis In Cotton(Gossypium Hirsutum L.)

Object: Plant regeneration via somatic embryogenesis(SE) is the key step for genetic improvement of cotton(Gossypium hirsutum L.) through genetic engineering mediated by Agrobacteria, and SE is also treated as a model for understanding of molecular events occurring during plant embryo development. Somatic embryogenesis was usual y considered includ ing twice cell reprograms involved in cel ular dedifferentiation and redifferentiation. However, SE in cotton is often hampered by recalcitrance, long culture time, and high frequency of abnormal embryos. Low efficiency of regeneration is another major issue that limits the utilization of Agrobacterium-mediated transformation through SE in cotton. The molecular mechanis ms underlying SE in cotton is still unclear.Method: RNA-Sequencing and isobaric tags for relative and absolute quantitation(iTRAQ) based comparative proteome was used to analyze the differentially expressed genes and proteins during somatic embryogenesis of cotton cultivar Xinluzao 33 from non-embryogenic callus(NEC), embryogenic cal us(EC) and somatic embryos(SEs). The genes differentia l ly expressed(DEGs) and differentia lly expressed proteins(DEPs) amongst NEC, EC and SEs were identified, annotated and classified in GO, COG, NR, SWISS and KEGG. q RT-PCR confirmed the sequencing data. Endogenous levels of plant hormones, polyamines, ROS, H2O2 and NO were determinated, the effect of certain chemicals and pathways were investiga ted. Complementary physiological experiments and ELISA assay were conducted for confir ming the mechanisms of polyamines on cotton somatic embryogenesis.Results:(1)A total of 101, 670 unigenes were de novo assembled. More DEGs were found between SEs and EC than between EC and NEC. A significant number of DEGs were related to hormone homeostasis, stress and ROS responses, and metabolism of polyamines. Quantitative real-time PCR analysis further confirmed the authenticity and accuracy of the transcriptome expression results. To confirm the expression dynamics of selected DEGs involved in various pathways, experiments were set up to investigate the effects of hormones(Indole-3-acetic acid, IAA; Kinetin, KT), polyamines, H2O2 and stresses on SE. Our results showed that exogenous application of IAA and KT positively regulated the development of EC and SEs, and that PAs and H2O2 promoted the conversion of EC into SEs. Furthermore, we found that low and moderate stress is beneficial for proliferation of EC. Together, our global analysis of transcriptomic dynamics reveals that hormone homeostasis, polyamines, and stress response synergistically regulating SE in cotton.(2)In the proteome profile, 5892 proteins were identified and almost of them classified into catalytic activity, binding activity, transporter activity and structural molecular activit y covered 93.4% in molecular function level. Distribution analysis of differentially expressed proteins(DEPs) suggested 572 and 211 proteins were up-regulated, 452 and 647 proteins were down-regulated during EC and SE differentiation respectively. KEGG(Kyoto Encyclopedia of Genes and Genomes) analysis indicated genetics information transmission, plant hormone transduction, glycolysis, fatty acid biosynthesis and metabolism, galactose metabolism were involved in somatic embryogenesis. Quantitative real-time PCR analysis further confirmed the authenticity and accuracy of the proteomic expression results. Proteome profile analys is facilitated identification of interesting genes and provided insights into the molecular mechanisms underlying somatic embryogenesis in Gossypium hirsutum L.(3)HPLC method for determination of cotton cultures polyamine was established through relevant and recovery analysis. Putrescine(Put), spermidine(Spd) and spermine(Spm) significantly increased from the EC stage to the early phase of embryo differentiation. The levels of Put then decreased until the somatic embryo stage whereas Spd and Spm remained nearly the same. The expression profiles of GhADC genes were consistent with changes in Put during cotton SE. The H2O2 concentrations began to increase significantly at the EC stage, during which time both GhPAO1 and GhPAO4 expressions were highest and PAO activity was significantly increased. Exogenous Put, Spd, Spm and H2O2 not only enhanced embryogenic cal us growth and embryo formation, but also al eviated the effects of D-arginine and 1, 8-diamino-octane, which are inhibitors of polyamine synthesis and PAO activity. Overall, the results suggest that both PAs and their metabolic product H2O2 are essential for the conversion of EC into somatic embryos in cotton.