Mild reductions in guard cell NtSUS2 expression substantially reduce whole plant transpiration (WPT) under well-watered and fluctuating environmental conditions
We next used a gravimetric methodology to determine WPT in plants grown under well-watered (WW) and non-controlled greenhouse conditions, which varies substantially in terms of light, temperature and humidity throughout the days (Figure 4A). Under these conditions, both L3 and L13 showed significantly lower WPT (up to 44%) compared to WT throughout the eleven days of the experiment (Figures 4B,C). At day 5, plants reached a peak of WPT (Figure 4B), which was associated to a sequence of sunny days (Figure 4A). Following the interruption of irrigation at day 5, a strong reduction in WPT in WT plants was observed at day 6, whilst no changes in WPT of the transgenic lines were observed (Figure 4B). On this day, the percentage of WPT was invariant across the genotypes (Figure 4C). When the cumulative WPT across the experiment were compared, L3 and L13 were found to transpire approximately 32% less than the WT (Figure 4D), meaning that they respectively conserved 416 and 433 g H2O plant-1 during the experiment.
The leaf area was initially smaller in L3 and L13 plants compared to WT. However, from day 5 to 11 of the experiment, only the leaf area of line L13 remained smaller than WT (Figure S3). WPT per leaf area unit (g H2O m-2 d-1) was also lower in L3 (28%) and L13 (22%) than WT (Figures 4E,F), which lead to a lower cumulative water loss per leaf area in these lines (Figure 4G). No changes in leaf stomatal density (SD) and water loss from detached leaves among the genotypes was observed (Figures S4A,B). At the end of the experiment, plants were harvested and growth parameters determined. Although relative growth rate (RGR), total leaf number, specific leaf area and stem length were not significantly altered in the transgenic lines, both L3 and L13 exhibited a reduced total biomass (Table 1). This was associated to a reduced carbon allocation toward the roots, given that reduced roots DW and % of DW roots were observed in these lines (Table 1). By contrast, line L3 showed higher % DW leaf, % DW shoot and shoot-to-root and leaf-to-root ratios than WT (Table 1). It is noteworthy that the leaves are the harvestable part of tobacco plants and the harvest index of L3 and L13 reached 0.58 and 0.54 g g-1, but only L3 was significantly higher than WT (0.52 g g-1). Line L3 also have higher yWUE than WT, whilst no difference in season-long WUE (slWUE) under WW condition was observed (Table 1). Taken all WUE parameters together, reduced expression of the guard cell NtSUS2 did not alter both steady-stateiWUE and slWUE, while L3 showed higheryWUE and increased iWUE in both transgenic lines during dark-to-light transition was observed.