Functional Genomics Of Theobroma Cacao Fatty Acid Biosynthesis: convergence Of Fatty Acid Desaturation, Embryo Development, And Defense Signaling Responses

Open Access
Zhang, Yufan
Graduate Program:
Plant Biology
Doctor of Philosophy
Document Type:
Date of Defense:
November 17, 2014
Committee Members:
  • Mark Guiltinan, Dissertation Advisor
  • Mark Guiltinan, Committee Chair
  • Siela Maximova, Committee Member
  • Sarah Mary Assmann, Committee Member
  • Ryan John Elias, Committee Member
  • James Landis Rosenberger, Committee Member
  • cacao
  • fatty acid biosynthesis
  • fatty acid desaturation
  • embryo development
  • defense responses
Theobroma cacao L. (chocolate tree) is an important cash crop for 40-50 million farmers and their families in its tropical growing regions worldwide. Cocoa butter and cocoa powder extracted from cacao seeds provide the main raw ingredients for chocolate manufacturing, supporting a $80 billion global business. A unique fatty acid composition of cocoa butter makes its melting temperature close to the human body temperature, which is not only of particular importance for industrial uses, but also a valuable quality trait targeted by breeding programs. My Ph.D. dissertation focused mainly on the fatty acid biosynthesis pathway in cacao seeds. I identified a key desaturase gene TcSAD1 from a large stearoyl-acyl carrier protein-desaturase gene family in cacao that plays a crucial role in converting stearic acid (18:0, saturated fatty acid) into oleic acid (18:1, unsaturated fatty acid). The expression of TcSAD1 was highly correlated with the change of fatty acid composition during cacao seed development. The activity of TcSAD1 rescued all the Arabidopsis ssi2 (a fatty acid desaturase) related mutant phenotypes, further supporting its in vivo functions. The discovery of the critical function of TcSAD1 offers a new strategy for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs, to help pyramid genes for quality traits such as cocoa butter content. Moreover, because of the significance of fatty acid biosynthesis and lipid accumulation during cacao seed development, to further explore the regulatory mechanism, I functionally characterized of a master regulator, TcLEC2 gene, which controls both zygotic and somatic embryo development of cacao. Transient overexpression of TcLEC2 induced the expression of a variety of seed specific genes in cacao leaves. Furthermore, functions of TcLEC2 were explored during somatic embryogenesis, which is an in vitro propagation system for cacao. My results suggested that the activity of TcLEC2 determines the embryogenic capacity of the cacao tissue explants and correlated with embryogenic capacity of cultured cells. Transgenic embryos overexpressing TcLEC2 produced a significantly higher number of embryos compared to non-transgenic embryos; however, most of these transgenic somatic embryos exhibited abnormal phenotypes, and the development normally ceased at globular stage. This discovery may have future applications in increasing the efficiency of cacao mass propagation programs. Notably, in addition to major storage compounds in cacao seeds, fatty acids also function as signals involved in defense responses. I found that the endogenous level of 18:1 was modulated by exogenous glycerol application. Glycerol application on cacao leaves increased the level of glycerol-3-phosphate and lowered the level of 18:1 through an acylation reaction, which further triggered the defense responses. 100mM glycerol was sufficient to induce the accumulation of ROS, activate the expression of a variety of pathogen-related genes, and confer enhanced resistance against fungal pathogen Phytophthora capsici. My results demonstrated the potential of foliar glycerol application to become an environmentally safe means to induce the plant defense responses and fight important plant diseases in the field. Together, my Ph.D. dissertation makes major contributions to three important research areas in cacao: (1) identification of the key gene regulating fatty acid composition in cocoa butter, (2) improvement of large-scale propagation system (somatic embryogenesis) of cacao, (3) enhancement of cacao foliar disease resistance. This thesis not only provides useful knowledge of the regulatory mechanisms of important quality traits at the molecular and genetic levels, but also demonstrates the potential of taking advantage of cacao genomic resources to accelerate cacao basic research and breeding programs.