RNAi construct of a P450 gene CYP82D109 blocks an early step in the biosynthesis of hemigossypolone and gossypol in transgenic cotton plants

Authors: Wagner, Tanya; Liu, Jinggao; WILLIAMS, HOWARD - Texas A&M University; Puckhaber, Lorraine; Bell, Alois - Al; Stipanovic, Robert - Bob

Submitted to: Phytochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/9/2015
Publication Date: 3/17/2015
Citation: Wagner, T.A., Liu, J., Williams, H., Puckhaber, L.S., Bell, A.A., Stipanovic, R.D. 2015. RNAi construct of a P450 gene CYP82D109 blocks an early step in the biosynthesis of hemigossypolone and gossypol in transgenic cotton plants. Phytochemistry. 115:59-69.

Interpretive Summary: Cotton leaves produce chemicals that protect the plant from being eaten by insects and grazing animals and limit the growth of disease-causing micro-organisms. Some of these protective chemicals are always present in the plant, whereas others are induced when the plant senses the attack. The network of enzymes necessary to make these chemicals is largely unknown. The identification of these enzymes could ultimately lead to their manipulation to produce more resistant cotton plants. A new enzyme in this chemical pathway has been identified. When cotton plants are genetically modified to make less of this enzyme, one group of chemicals is no longer produced, nor is the synthesis of these chemicals induced. Instead, a new chemical intermediate is detected and the second group of protective chemicals induced. The identification of this enzyme has significantly increased our understanding of the early steps in the synthesis of these chemicals. The genetically modified cotton plants also demonstrated that the two groups of chemicals in cotton are linked, and that a block in the synthesis of one can lead to increased amounts of the second group. Scientists may be able to use the modified cotton plants to identify other pathway components and generate plants that more quickly make certain protective chemicals as required by the insect or pathogen pressure.

Technical Abstract: Naturally occurring terpenoid aldehydes from cotton, such as hemigossypol, gossypol, hemigossypolone, and the heliocides, are important components of disease and herbivory resistance in cotton. These terpenoids are predominately found in the glands. Differential screening identified a P450 cDNA clone (CYP82D109) from a Gossypium hirsutum cultivar that hybridized to mRNA from glanded cotton but not glandless cotton. Both the D genome cotton G. raimondii and A genome cotton G. arboreum possessed three additional paralogs of the gene. We transformed G. hirsutum with an RNAi construct specific to this gene family and generated eight transgenic plants stemming from at least five independent transformation events. HPLC analysis showed that RNAi plants compared to wild-type (WT) plants had a 90% reduction in hemigossypolone and heliocides levels, and a 70% reduction in gossypol levels in the terminal leaves. Analysis of volatile terpenes by GC-MS revealed a novel terpene (MW: 218) from RNAi leaf extracts. 1H and 13C NMR analyses showed this compound was delta-cadinen-2-one. Double bond rearrangement of this compound gives 7-hydroxycalamenene, a lacinilene C pathway intermediate. Delta-Cadinen-2-one could be derived from delta-cadinene via a yet to be identified intermediate, 2-hydroxy-delta-cadinene. The RNAi construct of CYP82D109 apparently is blocking the synthesis of desoxyhemigossypol, and there is likely cross-regulation between the lacinilene C and gossypol pathways at the transcriptional level. Lacinilene C and cadalene precursors are not constitutively expressed in cotton leaves, and blocking the gossypol pathway by the RNAi construct resulted in a greater induction of the lacinilene C pathway when challenged by pathogens.