3.1 Expression of plastid-targeted Fld improved potato drought
tolerance
Potato plants were transformed with the fld -containing plasmid
described by Tognetti et al. (2006) using published procedures
(Rocha-Sosa et al. 1989; see Materials and Methods). Several
independent Stpfld lines were obtained, expressing different Fld
levels as revealed by SDS-PAGE and immunoblotting (Supplementary Figure
S1b). The flavoprotein was largely recovered from leaf extracts as a
mature-sized product (Supplementary Figure S1b), suggesting plastid
import and processing (Tognetti et al. 2006). Traces of Fld
precursor and processing intermediates were detected in highly
expressing Stpfld 252 and Stpfld 239 lines (Supplementary
Figure S1b), as already observed in tobacco (Tognetti et al.2006).
To evaluate the drought tolerance conferred by Fld introduction, 30-days
old WT and Stpfld plants cultured under growth chamber conditions
(see Materials and Methods) were exposed to hydric stress by
interrupting irrigation. Visual symptoms of stress were observed in WT
leaves after ~9 days of water withdrawal, and by 14 days
wilting extended to both leaves and stems (Figure 1a), correlating with
significant decreases in leaf water contents (Supplementary Figure S3).
Under the same conditions, Fld-expressing plants looked healthy (Figure
1a) and retained leaf turgor (Supplementary Figure S3).
As indicated, photosynthesis is one of the most sensitive targets of
water deficit (Urban et al. 2017). Measurements of chlorophylla fluorescence on WT leaves revealed a fast decrease in the
maximum quantum efficiency of photosystem II (PSII), as represented by
the F v’/F m’ ratio, evident
after only 2-3 days of stress (Figure 1b). This parameter is customarily
used to monitor photodamage to PSII (Baker 2008). The quantum yield of
PSII (ФPSII), which provides an estimation of electron
flow through this photosystem, also declined steadily with the days of
treatment (Figure 1c). Finally, dissipation of the excess of energy that
cannot be used for photochemistry, a process monitored by the
non-photochemical quenching of chlorophyll fluorescence (NPQt),
increased as stress became more severe (Figure 1d). The results indicate
that photosynthetic impairment caused by the drought regime preceded
visible tissue dehydration and increased as wilting progressed (Figure
1b-d). These detrimental effects were largely prevented by Fld presence
in chloroplasts of the transgenic plants, as indicated by differential
preservation of PSII integrity and electron flow, and comparatively
lower values of NPQt (Figure 1b-d). Only minor differences, without
statistical significance, were observed in well-watered plants of all
lines during the timespan of the assay (Figure 1b-d).
ROS build-up is also a common feature of environmental stress conditions
(Czarnocka & Karpiński 2018; Gómez et al. 2019), and the
protective effect of Fld has been linked to its role as a general
antioxidant specific for chloroplasts (Zurbriggen et al. 2008;
Pierella Karlusich et al. 2014; Rossi et al. 2017). To
visualize ROS accumulation in water-deprived plants, leaves were
infiltrated with the ROS-sensitive fluorescent probe
2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) for detection by
confocal laser scanning microscopy (Materials and Methods). Results are
illustrated in Figure 2 for leaves of Stpfld 252 plants, a line
displaying high levels of Fld expression (Supplementary Figure 1b) and
drought tolerance (Figure 1). Under the conditions employed, DCFDA
fluorescence was nearly undetectable in watered plants from both
genotypes (Figure 2a). At 14 days of water withdrawal, WT leaves showed
significant increases in ROS-associated fluorescence (Figure 2b).
Actually, ROS build-up was already evident after only 3 days of
treatment, indicating that increased production of these reactive
species was an early manifestation of the plant stress response (Figure
2b). Presence of plastid Fld largely prevented this rise (Figure 2b).
About 60% of total ROS was associated to chloroplasts in both
conditions and genotypes (Figure 2c).