Materials and methods

Plant material and growth conditions

Nicotiana tabacum L. cv Havana 425 wild type (WT) and transgenic plants in which the sucrose synthase 2 (NtSUS2 ) gene expression was suppressed under the control of the KST1 promoter (X79779). The transformation was carried out by cloning the SUS3gene from Solanum tuberosum (StSUS3 ) (STU24088) into the pBinK plasmid vector, which was derived from pBinAR-Kan (Höfgen & Willmitzer, 1990) but had the CaMV-35S promoter replaced by the KST1 promoter. A 1567 bp fragment was obtained by the digestion ofSUS3 gene using the Kpn I restriction enzyme (Antunes et al., 2012), which was cloned in the antisense direction in the pBinK vector, between the KST1 promoter and the OCS terminator (Figure 1A). This construct was inserted into Agrobacterium tumefasciens(Strain GV 3101) by electroporation and cultivated in suspension with leaf discs from tobacco in order to achieve plant transformation. The transformation was confirmed by PCR of the NPTII marker gene that confers kanamycin resistance.
Transgenic seeds of the lines L3 and L13 of T3generation were obtained and germinated in vitro . These lines were chosen based on preliminary results of transpiration, leaf temperature and gas exchange analysis, as previously reported (Antunes et al., 2017; Daloso et al., 2016b). After sterilization the seeds were germinated in Petri dishes containing Murashige and Skoog (MS) medium (Murashige & Skoog, 1962) with addition of 50 µM of Kanamycin® (KAN) for the transgenic lines and cultivated in vitro for 30 days under growth chamber conditions (16h of photoperiod, 100 µmol photons m-2 s-1, 25 ± 1 °C and relative humidity 53 ± 5%. Seedlings showing KAN resistance were transferred to 0.1 L pots with substrate composed by a mixture of vermiculite, sand and soil (1:1:1) and kept well-watered for 15 days under greenhouse conditions with natural 12 h photoperiod (maximum of 500 µmol photons m-2 s-1, 30 ± 4 °C and relative humidity 62 ± 10%). Five different experiments were performed in plants growing in soil or hydroponic system under greenhouse condition or in soil under growth chamber condition. The hydroponic experiments were carried out using Hoagland and Arnon nutritive solution in 2.5 L pots (Hoagland & Arnon, 1950). These plants were used for isolation of guard cell-enriched epidermal fragments for metabolomics analysis. Plants growing on soil were cultivated in 2.5 L (for growth chamber) or 5.0 L (for greenhouse) pots containing the same substrate mentioned above. These plants were irrigated with Hoagland and Arnon nutritive solution three times per week. All plants were cultivated during 45 or 60 days until the beginning of each experiment. A water deficit experiment was performed by suspending irrigation on 45 day-old plants cultivated under growth chamber conditions.