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).