Polyamine and Its Metabolite H2O2 Play a Key Role in the Conversion of Embryogenic Callus into Somatic Embryos in Upland Cotton (Gossypium hirsutum L.).

Wen-Han Cheng,Jie Sun,Fan-Long Wang,Yu-Qiang Sun,Hua-Guo Zhu,Qian-Hao Zhu,Xin-Qi Cheng

doi:10.3389/fpls.2015.01063

Abstract

The objective of this study was to increase understanding about the mechanism by which polyamines (PAs) promote the conversion of embryogenic calli (EC) into somatic embryos in cotton (Gossypium hirsutum L.). We measured the levels of endogenous PAs and H2O2, quantified the expression levels of genes involved in the PAs pathway at various stages of cotton somatic embryogenesis (SE), and investigated the effects of exogenous PAs and H2O2 on differentiation and development of EC. 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 callus growth and embryo formation, but also alleviated the effects of D-arginine and 1, 8-diamino-octane, which are inhibitors of PA 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.

Highlights

Plant regeneration through somatic embryogenesis (SE) has greatly aided gene transformation in many plant species, including cotton (G. hirsutum L.)
To investigate temporal changes of free and conjugated PAs during cotton SE, the concentrations of three common PAs (Put, Spd, and Spm) were analyzed in hypocotylderived explants and in samples collected at different stages of cotton SE (Supplementary Figure S3)
Embryogenic callus showed a significant decrease in Put, a significant increase in Spd, and no changed in Spm (Figures 1A–C)

Summary

Introduction

Plant regeneration through somatic embryogenesis (SE) has greatly aided gene transformation in many plant species, including cotton (G. hirsutum L.). Plantlets have been regenerated via SE using various combinations of plant growth-regulators, such as 2, 4-dichlorophenoxyacetic acid (2, 4-D), indole-3-butyric acid (IBA), and naphthalene acetic acid. Transcriptomic and proteomic studies have shown that genes and proteins involved in stress response, hormone metabolism, respiration, and photosynthesis play a role in increasing plantlet regeneration via SE in cotton (Ge et al, 2014). LEC2 plays critical roles during embryo development and is essential for induction of SE in Arabidopsis (Gaj et al, 2005). Understanding the molecular mechanisms of genes involved in SE and their interactions with other factors, such as hormones, can help with the initiation of embryogenic cultures and the enhancement of embryo yield and quality

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