Desenvolvimento de variedade de cana-de-açúcar (Saccharum spp.) geneticamente modificada para controle pós-transcricional da enzima polifenol oxidase (PPO)

Abstract

Sugarcane is the main raw material to produce sugar and around 80% of the sugar produced worldwide comes from this agricultural crop. Post-processing enzymatic browning of sugarcane juice caused by Polyphenol Oxidase (PPO) can impair sugar production, requiring more clarification steps to produce white sugar. The PPO enzymatic action causes the loss of health-beneficial components, such as phenolic compounds. These compounds add desirable nutritional properties to some types of sugar, such as demerara and brown sugar, since phenolic compounds are substrates for this enzyme. This industrial problem can be overcome through genetic improvement, creating new transgenic varieties using techniques like RNAi, aiming to control the transcription of PPO genes. This study evaluated the PPO metabolic profile and enzymatic activity of four sugarcane varieties: RB92579, RB07818, RB951541, and SP791011. In three sections of the culm (upper third, middle third, and lower third) the metabolic profile was evaluated with the enzyme in its activated form and in the presence of an enzyme inhibitor to detect which compounds were consumed. The RB92579 variety showed juice darkening greater than 70% darker, and also differed from the other varieties qualitatively and quantitatively in its composition, showing a higher concentration of phenolic compounds, PPO substrates. RB07818, RB951541, and SP791011 varieties did not show significant differences in their metabolic profile. Enzymatic activity varied according to the metabolic profile and analyzed tissue, and it was possible to assess in all varieties that the upper third had higher PPO enzymatic activity. RB07818, RB951541, and SP791011 have lighter juice and lower concentration of compounds that are PPO substrates, also the browning is not majorly connected to the enzymatic action of this enzyme. Once the enzymatic activity of all varieties was characterized, a search was carried out in the SP80- 3280 variedade genome for PPO gene candidates. Twenty-six candidates were detected, and the one with the highest expression was selected as a target for transcriptional control based on RNAi technique. Two varieties were transformed: i) SP80-3280 due to its sequenced genome and well-established transformation protocol, and ii) RB92579 which shows high PPO activity. Twenty-nine transformed events were obtained for the SP80-3280 variety, while the RB92579 transformed plants did not regenerate satisfactorily, showing slow and stagnant shoot development and root absence. This phenomenon can be attributed to the PPO control expression since the transformation controls developed as expected morphologically; however, further analyses need to be carried out for verification. The obtained events for the SP80-3280 variety showed resistance to the selector agent, glufosinate ammonium, and possible post-transcriptional control of PPO genes. This study made it possible to understand factors that influence PPO action in different sugarcane varieties, in addition to advancing the development of plant biotechnology techniques, such as RNAi, for the control of enzymatic browning, which can reduce production costs and preserve the nutritional quality of sugar.