Involvement of NtInvVR1 in the increase of reducing sugars during the curing of virginia tobacco

Flue-cured leaves of the Virginia tobacco (Nicotiana tabacum L.) market type are characterized by their high concentrations of sugars, namely sucrose (Suc), glucose (Glc), and fructose (Fru). During leaf curing, the so-called yellowing phase is associated with a dark-senescence process generating Suc and the main reducing sugars (RS), Glc and Fru. Invertase enzymes are reported to convert Suc into Glc and Fru throughout leaf senescence, but also fruit maturation. In this study, we actualize the complete family of genes encoding invertases (Invs) in N. tabacum. Two genes coding for vacuolar Invs, NtInvVR1_S and NtInvVR1_T, originating from the tobacco ancestors N. sylvestris and N. tomentosiformis, were identified to be the highest expressed Inv genes during the first 48 h of flue-curing (yellowing phase). Silencing both genes in the Virginia tobacco significantly reduced the accumulation of Glc and Fru by more than 90%, while Suc levels increased approximately 140% in a greenhouse environment. These data suggested that NtInvVR1_S and NtInvVR1_T play a key role in the hydrolysis of Suc, contributing to the accumulation of RS in cured leaves. NtInvVR1-RNAi plants were also cultivated in the field, showing a significant reduction in Glc and Fru of about 40% at the expense of Suc.