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.